Category Archives: Cryonics

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Preparing a cryonics patient for cryostorage can involve three distinct stages of alteration of body fluids:

(1) patient cooldown/cardiopulmonary support

(2) blood washout/replacement for patient transport

(3) cryoprotectant perfusion

During patient cooldown/cardiopulmonary support, a cryonics emergency response team or health care personnel may inject a number of medicaments to minimize ischemic injury and facilitate cryopreservation. The first and most important of these medicaments would be heparin, to prevent blood clotting. (For more details on the initial cooldown process, see Emergency Preparedness for a Local Cryonics Group).

Once the patient is cooled, the blood can be washed-out and replaced with a solution intended to keep organs/tissues alive while the patient is being transported to a cryonics facility. At the cryonics facility the organ/tissue preservation solution is replaced with the cryopreservation solution intended to prevent ice formation when the patient is further cooled to temperatures of 120C (glass transition temperature) or 196C (liquid nitrogen temperature) for long-term storage.

For both organ/tissue preservation & cryoprotection it is necessary to replace the fluid contents of blood vessels & tissue cells with other fluids. The process of injecting & circulating fluids through blood vessels is called perfusion. The passive process by which fluids enter & exit both blood vessels & cells is called diffusion.

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Body fluids can be described as solutes dissolved in a solvent, where the solvent is water and the solutes are substances like sodium chloride (NaCl, table salt), glucose or protein. Both water and solute molecules tend to move randomly in fluid with energy and velocity that is directly proportional to temperature. When there is a difference in concentration between water or solute molecules in one area of the fluid compartment compared to the rest of the compartment, random motion of the molecules will eventually result in a uniform distribution of all types of molecules throughout the compartment. In thermodynamics this is termed a decrease in potential energy (Gibbs free energy, not heat energy) due to an increase in entropy at constant temperature leading to equilibrium.

The movement of molecules from an area of high concentration to an area of low concentration is called diffusion. The rate of diffusion (J) can be quantified by Fick's law of diffusion:

dc J = DA---- dx J = rate of diffusion (moles/time) D = Diffusion coefficient A = Area across which diffusion occurs dc/dx = concentration gradient (instantaneous concentration difference divided by instantaneous distance)

Fick's First Law states that the rate of diffusion down a concentration gradient is proportional to the instantaneous magnitude of the concentration gradient (which changes as diffusion proceeds). For movement of molecules from a region of higher concentration to a region of lower concentration dc/dx will be negative, so multiplying by DA gives a positive value to J. Diffusion coefficient is higher for higher temperature and for smaller molecules.

Diffusion can occur not only within a fluid compartment, but across partitions that separate fluid compartments. The relevant partitions for animals are cell membranes and capillary walls. Cell membranes are lipid bilayers that allow for free diffusion of lipid soluble substances like oxygen, nitrogen, carbon dioxide and alcohol, while blocking movement of ions and polar molecules. But cell membranes also contain channels made of protein. Protein channels for water allow for very rapid diffusion of water across the membranes. Protein channels for potassium(K+), sodium(Na+) and other ions allow for more restricted diffusion across cell membranes. There is also facilitated diffusion (active transport) of many types of molecules across membranes.

For a normal 70kilogram (154pound) adult the total body fluid is about 60% of the body weight. Almost all of this fluid can be described as extracellular or intracellular (excluding only cerebrospinal fluid, synovial fluid and a few other small fluid compartments). Extracellular fluid can be further subdivided into plasma (noncellular part of blood) and interstitial fluid (fluid between cells that is not in blood vessels). Cell membranes separate intracellular fluid from extracellular fluid, whereas capillary walls separate plasma from interstitial fluid. The relative percentages of these fluids can be summarized as:

Intracellular fluid 67% Extracellular fluid Interstitial fluid 26% Plasma 7%

Note that blood volume includes both plasma & blood cells such that adding the intracellular fluid volume of blood cells to plasma volume makes blood 12% of total body fluid.

Osmosis refers to diffusion of water (solvent) across a membrane that is semi-permeable, ie, permeable to water, but not to all solutes in the solution. If membrane-impermeable solutes are added to one side of the membrane, but not to the other side, water will be less concentrated on the solute side of the membrane. This concentration gradient will cause water to diffuse across the semi-permeable membrane into the side with the solutes unless pressure is applied to prevent the diffusion of water. The amount of pressure required to prevent any diffusion of water across the semi-permeable membrane is called the osmotic pressure of the solution with respect to the membrane.

Osmotic pressure (like vapor pressure lowering and freezing-point depression) is a colligative property, meaning that the number of particles in solution is more important than the type of particles. One molecule of albumin (molecular weight 70,000) contributes as much to osmotic pressure as one molecule of glucose or one sodium ion. At equilibrium all molecules in a solution have achieved the same average kinetic energy, meaning that molecules with a smaller mass have higher average velocity. Thus, a one molar solution of NaCl will result in twice the osmotic pressure as a one molar solution solution of glucose because Na+ and Cl ions exert osmotic pressure as independent particles.

Solute concentrations are generally expressed in terms of molarity (moles of solute per liter of solution). The osmolarity of a solution is the product of the molarity of the solute and the number of dissolved particles produced by the solute. A one molar (1.0M, one mole per liter) solution of CaCl2 is a three osmolar (three osmoles per liter) solution because of the Ca2+ ion plus the two Cl ions produced when CaCl2 is added to water. Osmolarity, the number of solute particles per liter has been mostly replaced in practice by osmolality, the number of solute particles per kilogram. (For dilute solutions the values of the two are very close.) For describing solute concentrations in body fluids it is more convenient to use thousandths of osmoles, milli-osmoles (mOsm). Total solute osmolality of intracellular fluid, interstitial fluid or plasma is roughly 300mOsm/kgH2O. About half of the osmolality of intracellular fluid is due to potassium ions and associated anions, whereas about 80% of the osmolality of interstitial fluid and plasma is due to sodium and chloride ions.

As stated above, both osmotic pressure and freezing point depresssion are colligative properties. All colligative properties are convertible. One osmole of any solute will lower the freezing point of water by 1.858C. For this reason, a 0.9% NaCl solution is 0.154molar or about 308mOsm/kgH2O, and will lower the freezing point of water by about 0.572C.

The osmolality of a solution is an absolute quantity that can be calculated or measured. The tonicity of a solution is a relative concept that is associated with osmotic pressure and the ability of solutes to cross a semi-permeable membrane. Thus, tonicitiy of a solution is relative to the particular solutes and relative to a particular membrane specifically relative to whether the solutes do or do not cross the membrane. Cell membranes are the membranes of greatest biological significance. Whether a cell shrinks or swells in a solution is determined by the tonicity of the solution, not necessarily the osmolality. Only when all the solutes do not cross the semi-permeable membrane does osmolality provide a quantitative measure of tonicity. It is common to speak as if tonicity and osmolality are equivalent because body fluid solutes are often impermeable. Each mOsm/kgH2O of fluid contributes about 19mmHg to the osmotic pressure.

A solution is said to be isotonic if cells neither shrink nor swell in that solution. Both 0.9%NaCl (physiological saline) and 5%glucose (in the absence of insulin) are isotonic solutions (roughly 300mOsm/kgH2O of impermeable solute). (In the presence of insulin, 5%glucose is a hypotonic solution because insulin causes glucose to cross cell membranes.) Hypertonic solutions cause cells to shrink as water rushes out of cells into the solute, whereas cells placed in hypotonic solutions cause the cells to swell as water from the solution rushes into the cells.

An exact calculation of the osmolality of plasma gives 308mOsm/kgH2O, but the freezing point depression of plasma (0.54C) indicates an osmolality of 286mOsm/kgH2O. Interaction of ions reduces the effective osmolality. Sodium ions (Na+) and accompanying anions (mostly Cl & HCO3) account for all but about 20mOsm/kgH2O of plasma osmolality. Plasma sodium concentration is normally controlled by plasma water content (thirst, etc.)[BMJ; Reynolds,RM; 332:702-705 (2006)]. Normal serum Na+ concentration is in the 135 to 145millimole per liter range, with 135mmol/L being the threshold for hyponatremia. Intracellular sodium concentration is typically about 20mmol/L about one-seventh the extracellular concentration. Glucose and urea account for about 5mOsm/kgH2O. Osmolality of plasma is generally approximated by doubling the sodium ions (to include all associated anions), adding this to glucose & urea molecules, and ignoring all other molecules as being negligible. Protein contributes to less than 1% of the osmolality of plasma. (Cells contain about four times the concentration of proteins as plasma contains.)

Although ethanol increases the osmolality of a solution, it does not increase the tonicity because (like water) ethanol crosses cell membranes. A clinical hyperosmolar state without hypertonicity can occur with an increase in extracellular ethanol (which diffuses into cells)[ MINERVA ANESTESIOLOGICA; Offenstadt,G; 72(6):353-356 (2006)]. Glycerol also readily crosses cell membranes, but it does so thousands of times more slowly than water which means that glycerol is "transiently hypertonic" (only isotonic at equilibrium). Ethylene glycol crosses red blood cell membranes about six times faster than glycerol (and sperm cell membranes four times faster than glycerol). Actually. even for water there is a finite time for hydraulic conductivity across cell membranes.

Cells placed in a "transiently hypertonic" solution (containing solutes that slowly cross a membrane) will initially shrink rapidly as water leaves the cell, and gradually re-swell as the solute slowly enters the cell (the "shrink/swell cycle"). As shown in the diagram for mouse oocytes at 10C, water leaves the cell in the first 100seconds, whereas 1.5Molar ethylene glycol (black squares) or DMSO (DiMethylSulfOxide, white squares) take 1,750seconds to restore the volume to 85% of the original cell volume[CRYOBIOLOGY; Paynter,SJ; 38:169-176 (1999)]. Even if a cell does not burst or collapse due to osmotic imbalance, a sudden change in osmotic balance can injure cells. Nonetheless, cells are somewhat tolerant of hypotonic solutions. Granulocytes are particularly sensitive to osmotic stress, but granulocyte survival is not significantly affected by hypertonic solutions until the osmolality of impermeant solutes approaches twice physiological values (about 600mOsm/kgH2O)[AMERICAN JOURNAL OF PHYSIOLOGY; Armitage WJ; 247(5Pt1):C373-381 (1984)].

PC3 cells show almost no decline of survival upon exposure to 5,000mOsm/kgH2O NaCl for 60minutes at 0C, and show nearly 85% cell survival on rehydration. Nearly 85% survive 9,000mOsm/kgH2O NaCl for 60minutes at 0C, but less than 20% survive rehydration. But although at 23C most cells survive exposure to 5,000mOsm/kgH2O NaCl for 60minutes, only about a third of cells survive rehydration. At 23C and 9,000mOsm/kgH2O NaCl only about half of cells survive 60 minutes and no cells survive rehydration, indicating the protective effect of low temperature against osmotic stress. Water flux at 23C was the same for 9,000mOsm/kgH2O as for 5,000mOsm/kgH2O, and hypertonic cell survival was not affected by the rate of concentration increase[CRYOBIOLOGY; Zawlodzka,S; 50(1):58-70 (2005)].

Hyperosmotic stress damages not only cell membranes, but damages cytoskeleton, inhibits DNA replication & translation, depolarizes mitochondria, and causes damage to DNA & protein. Heat shock proteins and organic osmolytes (like sorbitol & taurine) are synthesized as protection against hyperosmotic stress. Highly proliferative cells (like PC3) suffer from osmotic stress more than less proliferative cells because the latter can mobilize cellular defenses more readily due to fewer cells undergoing mitosis at the time of osmotic stress[PHYSIOLOGICAL REVIEWS; Burg,MB; 87(4):1441-1474 (2007)].

An important distinction to remember in replacing body fluids is the distinction between two kinds of swelling (edema): cell swelling and tissue swelling. Cell swelling occurs when there is a lower concentration of dissolved membrane-impermeable solutes outside cells than inside cells. To prevent either shrinkage or swelling of a cell there must be an osmotic balance of molecules & ions between the liquids outside the cell & inside the cell. Capillary walls are semipermeable membranes that are permeable to most of the small molecules & ions that will not cross cell membranes, but are impermeant to large molecules referred to as colloid (proteins). The colloid osmotic pressure on capillary walls due to proteins is called oncotic pressure. For normal human plasma oncotic pressure is about 28mmHg, 9mmHg of which is due to the Donnan effect which causes small anions to diffuse more readily than small cations because the small cations are attracted-to (but not bound-to) the anionic proteins. About 60% of total plasma protein is albumin (30 to 50 grams per liter), the rest being globulins. But albumin accounts for 75-80% of total intravascular oncotic pressure. Tissue swelling occurs when fluids leak out of blood vessels into the interstitial space (the space between cells in tissues). Injury to blood vessels can result in tissue swelling, but tissue swelling can also result from water leaking out of vessels when there is nothing (like albumin) to prevent the leakage.

Both forms of edema (cell & tissue swelling) can impede perfusion considerably, and is frequently a problem in cryonics patients who have suffered ischemic or other forms of blood vessel damage. Maintaining osmotic balance of the fluids outside & inside cells is as important as maintaining oncotic balance, ie, balance of fluids inside & outside of blood vessels.

Much of the isotonicity of the intracellular and extracellular fluids is maintained by the sodium pump in cell membranes, which exports 3sodium ions for every 2potassium ions imported into cells. Proteins in cells are more osmotically active than interstitial fluid proteins. Because of the Donnan effect the sodium pump is required to prevent cell swelling. When ischemia deprives the sodium pump of energy, cells swell from excessive intracellular sodium (because sodium attracts water more than potassium does) resulting in edema. Inflammation can also cause cell swelling due to increased membrane permeability to sodium and other ions. Interstitial edema can occur when ischemia or inflammation increases capillary permeability leading to leakage of larger plasma solutes into the interstitial space.

[For further details on the sodium pump see MEMBRANE POTENTIAL, K/Na-RATIOS AND VIABILITY]

Near the hypothalamus of the brain are osmoreceptors (outside the blood-brain barrier) that monitor blood osmolality, which is normally in the range of 280-295mOsm/kgH2O. A 2% increase in plasma osmotic pressure can provoke thirst. An increase in plasma osmolality can indicate excessive loss of blood volume. To compensate, the posterior pituitary (neurohypophysis) secretes the hormone 8arginine vasopressin (AVP), which is two hormones in one hence the two names vasopressin and anti-diuretic hormone. AVP action on the V1 receptors on blood vessels causes vasoconstriction (vasopressin). AVP action on the V2 receptors of the kidney causes water retention (anti-diuretic hormone). Deficiency in AVP secretion can lead to diabetes incipidus, so called because the excessively excreted urine is tasteless (incipid), in contrast to the sweet (glucose-laden) urine of diabetes mellitus. Cortisol opposes AVP action on excretion, leading to dehydration and excessive urination of fluid. Reduced blood flow to the kidney stimulates release of renin, which catalyzes the production of angiotensin. Like AVP, angiotensin causes vasoconstriction and kidney fluid retention.

Rats subjected to experimental focal ischemia have shown reduced edema when treated with an AVP antagonist[STROKE; Shuaib,A; 33(12):3033-3037 (2002)]. Hypertonic saline(7.5%) has been shown to halve plasma AVP levels in experimental rats, whereas mannitol(20%) had no effect[JOURNAL OF APPLIED PHYSIOLOGY; Chang,Y; 100(5):1445-1451 (2006)]. Increases in plasma osmolality due to urea or glycerol have no effect on plasma AVP levels[JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY; Verbalis,JG; 18(12):3056-3059 (2007)]. The effect of hypertonic saline on osmotic edema due to AVP in a cryonics patient would likely be negligible because of negligible hormone release and transport. So some of the advantage of hypertonic saline over mannitol seen in clinical trials would not occur in cryonics cases.

The net movement of fluid across capillary membranes due to hydrostatic and oncotic forces can be described by the Starling equation. The Starling equation gives net fluid flow across capillary walls as a result of the excess of capillary hydrostatic pressure over interstitial fluid hydrostatic pressure, and capillary oncotic pressure over interstitial fluid oncotic pressure modified by the water permeability of the capillary. For a normal (animate) person, the hydrostatic pressure (blood pressure) at the arterial end of a capillary is about 35mmHg. The hydrostatic pressure drops in a linear fashion across the length of the capillary until it is about 15mmHg at the venule end. The net oncotic pressure within the capillary is about 25mmHg across the entire length of the capillary. Thus, for the first half of the capillary there is a net loss of fluid into the interstitial space until the hydrostatic pressure has dropped to 25mmHg. For the second half of the capillary there is a net gain of fluid into the capillary from the interstitial space. The flow of fluid into the interstitial space in the first half of the capillary is associated with the delivery of oxygen & nutrient to the tissues, whereas the flow of fluid from the interstitial space into the second half of the capillary is associated with the removal of carbon dioxide and other waste products.

Actually, there is a tiny (tiny relative to the total diffusion back and forth across the capillary wall) net flow of fluid from the capillaries to the interstitial fluid which is returned to the blood vessels by the lymphatic system. The lymphatic vessels contain one-way valves and rely on skeletal muscle movement to propel the lymphatic fluid. Infectious blockage of lymph flow can produce edema. A person sitting for long periods (as during a long trip) or standing a long time without moving may experience swollen ankles due to the lack of muscle activity. Swollen ankles is also a frequent symptom of the edema resulting from congestive heart failure. Venous pressure is elevated by the reduced ability of the heart to pull blood from the venous system, whereas vasoconstriction can better compensate to maintain pressure on the arterial side. Reduced albumin production by the liver as a result of cirrhosis or other liver diseases can reduce plasma osmolality such that the reduced oncotic pressure results in edema typically swollen ankles, pulmonary edema and abdomenal edema (ascites).

The Starling forces are different for the blood-brain barrier (BBB) than they are for other capillaries of the body because of the reduced permeability to water (lower hydraulic conductivity) and the greatly reduced permeability to electrolytes. The osmotic pressure of the plasma and interstitial fluid effectively become the oncotic pressures.

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A critical distinction is made in fluid mechanics between laminar flow and turbulent flow in a pipe. For laminar flow elements of a liquid follow straight streamlines, where the velocity of a streamline is highest in the center of the vessel and slowest close to the walls. Turbulent flow is characterized by eddies & chaotic motion which can substantially increase resistance and reduce flow rate. The Reynolds number is an empirically determined dimensionless quantity which is used to predict whether flow will be laminar or turbulent with 2000 being the approximate lower limit for turbulent flow. Transient localized turbulence can be induced at a Reynolds number as low as 1600, but temporally peristant turbulence forms above 2040[SCIENCE; Eckhart,B; 333:165 (2011)].

Turbulent flow could potentially be a problem in cryonics if it reduced perfusion rate or increased the amount of pressure required to maintain a perfusion rate. It is doubtful that turbulent flow ever plays a role in cryonics perfusion, however. Even for a subject at body temperature (37C) Reynolds numbers in excess of 2000 are only seen in the very largest blood vessels: the aorta and the vena cava.

The formula for Reynolds number is: v D Re = ------ = fluid density (rho) v = fluid velocity D = vessel diameter = fluid viscosity

The fact that diameter (D) is in the numerator indicates that only high diameter vessels have high Reynolds number. Velocity (v), also in the numerator, is highest in the aorta & arteries. But the use of cryoprotectants and the increase in viscosity () with declining temperature essentially guarantee that turbulent flow will not occur in a cryonics patient.

More serious for cryonics is the Hagen-Poisseuille Law, which describes the relationship between flow-rate and driving-pressure: pressure X (radius)4 Flow Rate = ---------------------- length X viscosity

Typically in cryonics the flow rate will be one or two liters per minute when the pressure is around 80mmHg. But because flow rate varies inversely with viscosity and varies directly with pressure, pressure must be increased to maintain flow rates when cryoprotectant viscosity increases with lowering temperature. This poses a serious problem because blood vessels become more fragile with lowering temperature. If blood vessels burst the perfusion can fail.

At 20C glycerol is about 25% more dense (=rho, in the numerator) than water. But the role of viscosity is far more dramatic, with high viscosity in the denominator reducing Reynolds number considerably. The viscosity of water approximately doubles from 37C to 10C, but the viscosity of glycerol increases by a factor of ten (roughly 4Poise to 40Poise). At 37C glycerol is nearly 600 times more viscous than water, but at 10C it is about 2,600 times more viscous.

Although turbulence is not a concern in cryonics, the increase in viscosity of cryoprotectant with lowering temperature certainly is. Fortunately, the newer vitrification mixtures are less viscous than glycerol.

The most common strategy in cryonics has been to cool the patient from 37C to 10C as rapidly as possible and to perfuse with cryoprotectant at 10C. Lowering body temperature reduces metabolism considerably, thereby lessening the amount of oxygen & nutrient required to keep tissues alive. Cryoprotectant toxicity drops as temperature declines. But the very dramatic more-than-exponential increase in cryoprotectant viscosity with lowering temperature poses a significant problem for effective perfusion. When open circuit perfusion is used, a higher temperature may be preferable because the opportunity for diffusion time into cells is so limited (about 2hours 1hour for the head, 1hour for the body) although ischemic damage is difficult to quantify.

With closed circuit perfusion, the perfusion times are longer up to 5hours. If a good carrier solution is used for the cryoprotectant the tissues may receive adequate nutrient. This, along with the oxygen carrying-capacity of water at low temperature, may limit ischemic damage while allowing time for cells to become fully loaded with cryoprotectant. If ischemic damage can be safely prevented in perfusion, the only critical issues for temperature selection are the relative benefits of reduced cryoprotectant toxicity at lower temperatures as against increased chilling injury. The fact that the more-than-exponential increase in viscosity with lowering temperature will increase perfusion time will not be problematic if the risk of ischemia is minimized.

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Typically a cryonics patient deanimates at a considerable distance from a cryonics facility and must be transported before cryoprotectants can be perfused. Blood could be washed-out and replaced with an isotonic (ie, osmotically the same as saline) solution, such as Ringer's solution. The patient is then transported to the cryonics facility at water-ice temperature. Freezing must be avoided because ice crystals would damage cells & blood vessels to such an extent as to prevent effective cryoprotectant perfusion. Water-ice temperature will not freeze tissues because tissues are salty (salt lowers the freezing point below 0C).

As body temperature approaches 10C, metabolic rate has slowed greatly and the oxygen-carrying capacity of blood hemoglobin is no longer required. Cool water, in fact, may carry adequate dissolved oxygen at low temperatures. (Water near freezing temperature can hold nearly three times as much dissolved oxygen as water near boiling temperature. Oxygen is about five times more soluble in water than nitrogen.) The tendency of blood to agglutinate and clog blood vessels becomes a serious problem at low temperature so the blood should be replaced if this does not cause other problems (such as delay and reperfusion injury.)

Replacing blood with a saline-like solution for patient transport, however, does not do a good job of maintaining tissue viability or preventing edema and would likely cause reperfusion injury. For this reason an organ preservation solution such as Viaspan, rather than Ringer's solution, has been used for cryopatient transport. Blood is not simply an isotonic solution carrying blood cells. Blood contains albumin, which attracts water and keeps the water from leaving blood vessels and going into tissues (maintains oncotic balance). Tissues which are swollen by water (edematous tissues) resist cryoprotectant perfusion. One of the most important ingredients in Viaspan preventing edema is HydroxyEthyl Starch (HES), which attracts water in much the way albumin attracts water acting as an oncotic agent by keeping water in the blood vessels. Viaspan contains potassium lactobionate to help maintain osmotic balance. Because HES is difficult to obtain and can cause microcirculatory disturbances, PolyEthylene Glycol (PEG) has been used in organ preservation solutions as a replacement for HES with good results[THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS; Faure,J; 302(3):861-870 (2002) and LIVER TRANSPLANTATION; Bessems,M; 11(11):1379-1388 (2005)].

The same benefit might not apply to cryonics patients, however, because of the prevalence of endothelial damage due to ischemia. Larger "holes" in the vasculature can mean that a larger molecular weight molecule is required for oncotic support. HES molecular weight is about 500,000, whereas the molecular weight for PEG used in organ replacement solutions is more like 20,000. Albumin (which has a molecular weight of about 70,000) provides most of the oncotic support in normal physiology. A PEG with molecular weight of 500,000 would be far too viscous and will form a gel. HES has the benefit of being large enough to always provide oncotic support while being much less viscous than PEG of equivalent molecular weight.

Viaspan (DuPont Merck Pharmaceuticals) contains other ingredients to maintain tissue viability, such as glucose, glutathione, etc. (the full formula can be found on the Viaspan website). Viaspan is FDA approved for preservation of liver, kidney & pancreas, but is used off-label for heart & lung transplants. Viaspan is being challenged in the marketplace for all these applications by the Hypothermosol (Cryomedical Sciences, BioLife Technologies) line of preservation solutions.

Rather than use these expensive commercial products, Alcor and Suspended Animation, Inc. use a preservation solution developed by Jerry Leaf & Mike Darwin called MHP-2. MHP-2 is so-called because it is a Perfusate (P) which contains mannitol (M) as an extracellular osmotic agent and HEPES (H), a buffer to prevent acidosis which is effective at low temperature. MHP-2 also contains ingredients to maintain tissue viability and hydroxyethyl starch as an oncotic agent to prevent edema. Lactobionate permeates cells less than mannitol and can thus maintain osmotic balance for longer periods of time, but mannitol is much less expensive. Mannitol also has an additional effect in the brain. Because of the unique tightness of brain capillary endothelial cell junctions ("blood brain barrier"), little mannitol leaves blood vessels to pass into the brain. This means that mannitol can act like an oncotic agent for the brain. If the blood brain barrier is intact, mannitol will suck water out of the extravascular space. The brain is the only place that mannitol can do this, and that is why a mannitol is effective for inhibiting edema of the brain but only if there is not extensive ischemic damage to the blood brain barrier. (Mannitol has yet another benefit in that it scavenges hydroxyl radical [CHEM.-BIOL. INTERACTIONS 72:229-255 (1989)]).

(For the formula of MHP-2 see TableII of CryoMsg4474 or TableVII of CryoMsg2874 which also contains the formula for Viaspan in TableV.)

The initial perfusate can also contain other ingredients to assist in reducing damage to the cryonics patient. Anticoagulants can reduce clotting problems, and antibiotics can reduce bacterial damage. Damaging effects of ischemia can be reduced with antioxidants, antiacidifiers, an iron chelator and a calcium channel blocker.

Both Alcor and Suspended Animation, Inc. use an Air Transportable Perfusion(ATP) system of equipment which allows them to do blood washout in locations remote from any cryonics facilty by using equipment that can easily be carried on an airplane. There is a video demonstration of an ATP on YouTube.

[For further details on organ preservation solution see ORGAN TRANSPLANTATION SOLUTION]

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Cryoprotectants are used in cryonics to reduce freezing damage by prevention of ice formation (see Vitrification in Cryonics ). Cells are much more permeable to water than they are to cryoprotectant. Platelets & granulocytes, for example, are 4,000 times more permeable to water than they are to glycerol[CRYOBIOLOGY; Armitage,WJ; 23(2):116-125 (1986)]. When a cell is exposed to high-strength cryoprotectant, osmosis causes water to rush out of the cells, causing the cells to shrink. Only very gradually does the cryoprotectant cross cell membranes to enter the cell (the "shrink/swell cycle"). For isolated cells, the halftime (time to halve the difference between a given glycerol concentration in a granulocyte and the maximum possible concentration) is 1.3minutes[EXPERIMENTAL HEMATOLOGY; Dooley,DC; 10(5):423-434 (1982)] but tissues & organs would require more time because their cells are less accessible. Even after equilibration, however, the concentration of glycerol inside neutrophilic granulocytes never rises above 78% of the concentration outside the cells.

As shown in the diagram for mature human oocytes placed in a 1.5molar DMSO solution, the shrink/swell cycle is highly temperature dependent, happening with slower speed of recovery and with greater volume change at lower temperatures[HUMAN REPRODUCTION; Paynter,SJ; 14(9):2338-2342 (1999)]. This creates tough choices in cryonics, because cryoprotectants are more toxic at higher temperatures.

Proliferation of cultured kidney cells declines linearly with increasing osmolality due to urea & NaCl above 300mOsm/kgH2O, but the effect of added glycerol on cell growth is much less[AMERICAN JOURNAL OF PHYSIOLOGY; Michea,L; 278(2):F209-F218 (2000)]. Kidney cells which invivo can tolerate osmolalities of around 300mOsm/kgH2O do not survive over 300mOsm/kgH2O invitro, possibly because of more rapid proliferation[PHYSIOLOGICAL REVIEWS; Burg,MB; 87(4):1441-1474 (2007)].

Cells subjected to high levels of cryoprotectants can be damaged by osmotic stress. Quantifying osmotic damage has been a challenge for experimentalists who must distinguish between electrolyte damage, cryoprotectant toxicity, cell volume effects and osmotic stress. Concerning the last two, osmotic damage due to cell shrinkage may be distinguished from osmotic damage as a result of the speed at which the cryoprotectant crosses the cell membrane, ie, by the membrane permeability to the cryoprotectant. Cryoprotectants with lower permeabilities can cause more osmotic stress than cryoprotectants with high permeability.

Membrane permeabilities of a variety of nonelectrolytes (including cryoprotectants) have been studied on a number of cell types, including human blood cells[THE JOURNAL OF GENERAL PHYSIOLOGY; Naccache,P; 62(6):714-736 (1973)]. Critical factors determining membrane permeability are lipid solubility of the substance (which increases permeability) and hydrogen bonding (which decreases permeability). In general, permeability decreases as the molecular size of the substance increases. In contrast to blood cells, human sperm is more than three times more permeable to glycerol than to DMSO[BIOLOGY OF REPRODUCTION; Gilmore,JA; 53(5):985-995 (1995)]. For both blood cells and sperm cells permeability to ethylene glycol is very high compared to the other common cryoprotectants. Yet for mature human oocytes propylene glycol has the highest permeability and ethylene glycol has the lowest permeability of the most commonly used oocyte cryoprotectants[HUMAN REPRODUCTION; Van den Abbeel,E; 22(7):1959-1972 (2007)]. In contrast to human oocytes, however, for mouse oocytes ethylene glycol(EG) permeability is comparable to that of DMSO, propylene glycol(PG), and acetamide(AA), but not glycerol(Gly)[JOURNAL OF REPRODUCTION AND DEVELOPMENT; Pedro,PB; 51(2):235-246 (2005)].

Water and cryoprotectants both cross cell membranes more slowly at lower temperatures. Cryoprotectants slow the passage of water across cell membranes. Glycerol, DMSO and ethylene glycol all reduce the rate at which water crosses human sperm cell membranes by more than half[BIOLOGY OF REPRODUCTION; Gilmore,JA; 53(5):985-995 (1995)].

Aside from the choice of cryoprotectants, a major concern is the way cryoprotectant is administered. For example, glycerol (the standard cryoprotectant used in cryonics for many years) can either be administered full-strength or it can be introduced in gradually increasing concentrations. Under optimum conditions, glycerol results in 80% vitrification and 20% ice formation. Glycerol has been replaced by better cryoprotectants that can vitrify without any ice formation, but I will typically use glycerol as my example cryoprotectant. A patient should probably not be perfused with a 100% solution of glycerol or other cryoprotectant because of the possibility of osmotic damage. It is prudent to begin perfusion with low concentrations of cryoprotectant because water can diffuse out of cells thousands of times more rapidly than cryoprotectant diffuses into cells. Using gradually increasing concentrations of cryoprotectant (ramping) prevents the osmotic damage this differential could cause.

Human granulocytes (which are more vulnerable to osmotic stress or shrinkage than most other cell types) can experience up to 600mOsm/kgH2O hypertonic solution (which shrinks cells to 68% of normal cell volume) for 5minutes at 0C with no more than 10% of the cells losing membrane integrity. But at about 750mOsm/kgH2O (NaCl) or 950mOsm/kgH2O (sucrose) less than half of granulocytes display intact membranes when returned to isotonic solution. Nonetheless, the cells did not display lysis if retained in hyperosmotic medium. In fact, granulocytes could tolerate up to 1400mOsm/kgH2O if not subsequently diluted to less than 600mOsm/kgH2O[AMERICAN JOURNAL OF PHYSIOLOGY; Armitage WJ; 247(5Pt1):C373-381 (1984)]. A subsequent confirming study showed that rehydration of PC3 cells shrunken by NaCl solution creates more osmotic damage than the initial dehydration[CRYOBIOLOGY; Zawlodzka,S; 50(1):58-70 (2005)]. Cell survival after rehydration was higher at 0C than at 23C.

Although toxic effects of 2M (17%w/w) glycerol on granulocytes are quite evident at 22C, almost no toxic effect is seen at 0C[CRYOBIOLOGY; Frim,J; 20(6):657-676 (1983)]. For no mammalian cells other than granulocytes is 2Molar glycerol toxic. Nonetheless, abrupt addition of only 0.5Molar glycerol at 0C resulted in only 40% of granulocytes surviving when slowly diluted to isotonic solution and warmed to 37C. Only 20% of granulocytes survived this treatment when 1Molar or 2Molar glycerol were added (there was no difference in survival between the two concentrations). But if sucrose or NaCl was added to keep the granulocytes shrunken to 60% of normal cell volume, almost all granulocytes survived when incubated to 37C. Insofar as the transient shrinkage of granulocytes due to glycerol is not less than 85% of normal cell volume, it seems unlikely that cell shrinkage can account for the damage[AMERICAN JOURNAL OF PHYSIOLOGY; Armitage WJ; 247(5Pt1):C382-389 (1984)].

Human spermatazoa tolerate much higher osmolality than granulocytes. Sperm cells can experience up to 1000mOsm/kgH2O hypertonic solution for 5minutes at 0C with no more than 10% of the cells losing membrane integrity. At about 1500mOsm/kgH2O (NaCl, white circles) or 2500mOsm/kgH2O (sucrose, black circles) less than half of sperm cells display intact membranes when returned to isotonic conditions. But 80% of sperm cells showed intact cell membrane after exposure to 2500mOsm/kgH2O at 0C if maintained at hypertonicity rather than restored to isotonic solution (NaCl & sucrose, triangles). Sperm cells gradually returned to isotonic solution following exposure to 1.5Molar glycerol at 22C showed only 3% lysis, whereas 20% of sperm cells lysed if the return to isotonic was sudden. No lysis was seen for sperm not returned to isotonic medium. At nearly 5000mOsm/kgH2O glycerol (about 4.5Molar) 17% of sperm cells showed lysis (had loss of membrane integrity) at 0C and 10% had lysis at 8C if not returned to isotonic media[BIOLOGY OF REPRODUCTION; Gao,DY; 49(1):112-123 (1993)]. For cryonics purposes it would be best to maintain cells in a hypertonic condition to maximize potential viability during cryogenic storage.

Cells from mouse kidney (IMCD, Inner Medullary Collecting Duct) can be killed by NaCl or urea that is 700mOsm/kgH2O, but the death is apoptotic and takes up to 24hours. The IMCD cells can tolerate up to 900mOsm/kgH2O of urea and NaCl in combination because of activation of complementary cellular defenses (including heat-shock protein)[ AMERICAN JOURNAL OF PHYSIOLOGY; Santos,BC; 274(6):F1167-F1173 1998)].

Nearly half of mouse fibroblasts displayed cell membrane lysis after restoration to isotonicity following exposure to the equivalent of 3600mOsm/kgH2O of osmotic stress from rapid addition of 4Molar (30%w/w) DMSO at 0C. Few cells were damaged by slow addition of the DMSO[BIOPHYSICAL JOURNAL; Muldrew,K; 57(3):525-532 (1990)].

Human corneal epithelial cells could tolerate 4.3M (37%w/w) glycerol with only 2% cell loss at 4C if the cells were subjected to gradually increasing (ramped) concentration (doubling osmolality in about 13minutes), but for stepped increases of 0.5M every 5minutes above 2M (17%w/w) to 3.5M (30%w/w) glycerol at 0C there was a 27% cell loss. For the same ramped method with DMSO there was a 6% cell loss at 2M (15%w/w) and a 15% cell loss at 3M (23%w/w). The same stepped method for DMSO resulted in a 1.5% cell loss for cells stepped from 2M to 3.5M (27%w/w) and a 22% cell loss for cells stepped from 2M to 4.3M (33%w/w). In all cases cell viability was assessed after washout and three days of incubation at 37C[CRYOBIOLOGY; Bourne,WM; 31(1):1-9 (1994)]. (Conversion of glycerol molarity to %w/w was approximated by multiplying by 8.6 and for DMSO was approximated by multiplying by 7.6)

In the context of cryonics it should be remembered that cells are not being returned to body temperature and need not be returned to isotonicity before cryoopreservation. There would be little time for apoptosis, and most cells would be far better preserved at low temperature and in hyperosmolar solution. Future technologies may be able to prevent apoptosis and have better methods for restoring irreplaceable cells to normal temperatures and osmolalities. For neurons, even abrupt stepped perfusion with cryoprotectant is likely to effectively result in ramped perfusion when allowances are made for the diffusion times required across blood vessels (blood brain barrier) and interstitial space. A more worrisome effect from the point of view of cryonic cryoprotectant perfusion is the effect of the cryoprotectants on vessel endothelial cells notably the effect on edema and vascular compliance.

Cell shrinkage may directly damage the cell (and cell membrane) due to structural resistance from the cell cytoskeleton and high compression of other cell constituents[HUMAN REPRODUCTION; Gao,DY; 10(5):1109-1122 (1995)]. Aside from membrane damage, other forms of cellular damage occur due to hypertonic environments, including cross-linking of intracellular proteins subsequent to cell dehydration. Bull sperm lose motility (often only temporarily) in a less hypertonic medium than one causing membrane damage[JOURNAL OF DAIRY SCIENCE; Liu,Z; 81(7):1868-1873 (1998)]. Osmotic stress can depress mitochondrial membrane potential in a manner that is mostly reversible after restoration to isotonic conditions[PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); Desai,BN; 99(7):4319-4324 (2002)]. Human oocytes subjected to 600mOsm/kgH2O sucrose showed 44% of metaphaseII spindles having abnormalities[HUMAN REPRODUCTION; Mullen,SF; 19(5):1148-1154 (2004)]. Hypertonic solutions can trigger apoptosis[AMERICAN JOURNAL OF PHYSIOLOGY; Copp,J; 288(2):C403-C415 (2005)].

Despite these other types of damage due to hyperosmolality, the greatest risks in cryoprotectant perfusion in cryonics are those associated with membrane damage and edema due to cell swelling. The evidence that maintaining hypertonicity is more protective of cells than returning to isotonic conditions, and the desire to minimize edema during perfusion seem to make it advisable in cryonics to perfuse in hypertonic conditions.

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Once the patient is at the cryonics facility the transport solution can be replaced with a cryoprotectant solution. A perfusion temperature of 10C gives the best tradeoff of avoiding the high viscosity of lower temperatures and at the same time limiting the ischemic tissue degradation, chilling injury, and cryoprotectant toxicity that would be seen at higher temperatures. (Cryonicists usually worry more about ischemic damage than cryoprotectant toxicity due to a belief that ischemic damage has a greater likelihood of being irreversible irreparable by future molecular-repair technology.)

Cryoprotectants should be sterilized to prevent the growth of bacteria. Sterilization of cryoprotectants by heating can cause the formation of carbon-carbon double-bonds, which are evident by a yellowing of the cryoprotectant. Only a few such double-bonds can produce the yellow appearance, so the fact of yellowing is not evidence that the cryoprotectant is no longer serviceable. But a preferable method of cryoprotectant sterilization is filtration through a 0.2micron filter.

Rapid addition of cryoprotectant causes endothelial cells to shrink thereby breaking the junctions between the cells[CRYOBIOLOGY; Pollock,GA,; 23(6):500-511 (1986)]. On the other hand, endothelial cell shrinkage by hypertonic perfusate can increase capillary volume, thereby increasing blood flow as long as excessive vascular damage does not occur. Blood and clots are often observed to be dislodged during cryoprotectant perfusion in cryonics cases. For cryonics purposes some vascular damage may actually be an advantage insofar as it increases diffusion and vascular repair may be an easy task for future science. In fact, the breakdown of the blood-brain barrier in the 1.8-2.2 molar glycerol range is essential for perfusion of the brain as long as damaging tissue edema (swelling) can be avoided. Aquaporin (water channel) expression in the blood-brain barrier could be a safer means of allowing cryoprotectants into the brain[CRYOBIOLOGY; Yamaji,Y; 53(2):258-267 (2006)].

Closed-circuit perfusion (with perfusion solution following a circuit both inside & outside the patient's body) is contrasted with the open-circuit perfusion used by funeral directors for embalming. In the open-circuit perfusion of embalming, fluid is pumped into a large artery of the corpse and forces-out blood from a large vein and this blood is discarded.

A closed-circuit perfusion, as illustrated in the diagram, can be set up at low cost for gradual introduction of cryoprotectant into cryonics patients. As shown in the diagram, the perfusion circuit bypasses the heart. Perfusate enters the patient through a cannula in the femoral (leg) artery and exits from a cannula in the femoral vein on the same leg. Flowing upwards (opposite from the usual direction) from the femoral artery and up through the descending aorta, the perfusate enters the arch of the aorta (where blood normally exits the heart), but is blocked from entering the heart. Instead, the perfusate flows (in the usual direction) through the distribution arteries of the aorta, notably to the head and brain. Returning in the veins (in the usual direction), the perfusate nontheless again bypasses the heart and flows downward (opposite from the usual direction) to the femoral vein where it exits. A better alternative to the femoral circuit, however, is to surgically open the chest to cannulate the heart aorta (for input) and atrium (for output).

Although it is not shown in the diagram, there will be a pump in the circuit to maintain pressure and fluid movement. A roller pump, rather than an embalmer's pump, should be used. A roller pump achieves pumping action by the use of rollers on the exterior of flexible tubing that forces fluids through the tube without contaminating those fluids. Embalmer's pumps may use pressures much higher than those suitable for cryonics, resulting in blood vessel damage. Embalmer's pumps are also easily contaminated (and hard to clean), unless a filter is used. Contamination doesn't matter much in embalming, but in cryonics contaminants entering the patient through the pump can damage blood vessels, interfering with perfusion. If an embalmer's pump is used for cryonics purposes, ensure that the pressure can be lowered to a suitable level and that it is cleaned and sterilized. The main advantage of roller pumps, however, is the fact that they provide a closed circuit, whereas embalmer's pumps are open-circuit. Roller pumps are generally calibrated in litres per minute. Depending on the viscosity of the solution, a flow rate of 0.5 to 1.5litres per minute will be necessary to achieve the desired perfusion pressure of approximately 80mmHg to 120mmHg (physiological pressures).

Gaseous and particulate microemboli can produce ischemia in capillaries and arterioles. A study of patients having routine cardiopulmonary bypass surgery showed that 16% fewer patients had neuropsychological deficits eight weeks after the surgery when a 40micrometer arterial line filer had been used[STROKE; Pugsley,W; 25(7):1393-1399 (1994)]. Both roller pumps (peristaltic pumps) and centrifugal pumps can generate particles up to 25micrometers in diameter through spallation, although centrifugal pumps generate fewer particles[PERFUSION; Merkle,F; 18(suppl1):81-88 (2003)]. Filtration of perfusate with a 0.2micrometer filter prior to perfusion is a recommended way of removing potential microemboli, including bacteria. At room temperature 20micrometer diameter air bubbles take 1to6seconds to dissolve in water, although high flow rates and turbulence can increase microbubble formation[SEMINARS IN DIALYSIS; Barak,M; 21(3):232-238 (2008)]. De-airing of tubing before perfusion considerably reduces the possibility of microbubbles entering the patient[THE THORACIC AND CARDIOVASCULAR SURGEON; Stock,UA; 54(1):39-41 (2006)].

Mean Arterial Pressure (MAP) for an normal adult is regarded as being in the range of 50 to 150mmHg, and Cerebral Perfusion Pressure (CPP) is in the same range[BRITISH JOURNAL OF ANAETHESIA; Steiner,LA; 91(1):26-38 (2006)]. Vascular pressure normally drops to about 40mmHg in the arterioles, to below 30mmHg entering the capillaries, and is down to 3 to 6mmHg (Central Venous Pressure, CVP) when returning to the right atrium of the heart. Perfusing a cryonics patient at about 120mmHg should open capillaries adequately for good cryoprotectant tissue saturation without damaging fragile blood vessels.

Outside the patient, some of the drainage is discarded, but most is returned to a circulating (stirred) reservoir connected to a concentrated reservoir of cryoprotectant. The circulating reservoir is initially carrier solution which gradually becomes increasingly concentrated with cryoprotectant as the stirring and recirculation proceed. The circulating reservoir can be stirred from the bottom by a magnetic stir bar on a stir table and/or from the top by an eggbeater-type stirring device. The stirring will draw cryoprotectant from the cryoprotectant reservoir, and pumping of the perfusate should also actively draw liquid from the cryoprotectant reservoir. Gradually a higher and higher concentration of cryoprotectant is included in the perfusate and the osmotic shock of full-strength cryoprotectant is avoided.

The carrier solution for the cryoprotectant should perform similar tissue preservation functions as is performed by the transport solution, and should be carefully mixed with the cryoprotectant so as to avoid deviations from isotonicity which could result in dehydration or swelling & bursting of cells. The carrier solution will help keep cells alive during cryoprotectant perfusion.

An excellent carrier solution for cryonics purposes would be RPS-2 (Renal Preservation Solution number2), which was developed by Dr. Gregory Fahy in 1981 as a result of studies on kidney slices. More recently Dr. Fahy used RPS-2 as the carrier solution in cryopreserving hippocampal slices an indication that it is well-suited for brain tissue as well as for kidney. RPS-2 not only helps maintain hippocampal slice viability, it reduces the amount of cryoprotectant needed because it has cryoprotectant (colligative) properties of its own. The formulation of RPS-2 is: K2HPO4, 7.2mM; reduced glutathione, 5mM; adenine HCl, 1mM; dextrose, 180mM; KCl, 28.2mM; NaHCO3, 10mM; plus calcium & magnesium[CRYOBIOLOGY; Fahy,GM; 27(5):492-510 (1990)]. LM5 (Lactose-Mannitol5) is a carrier solution for use in vitrification solutions that include ice blockers. LM5 does not contain dextrose, which is believed to interfere with ice blockers.

The cryoprotectant reservoir will not in general contain pure cryoprotectant (although in principle it could), but rather a "terminal concentration" solution of cryoprotectant that is equal or slightly above the final target concentration. As perfusion proceeds and drainage to discard proceeds, the level of both reservoirs drops in tandem until both reservoirs are nearly empty, at which point the circuit concentration will have reached the cryoprotectant reservoir concentration. Provided that the two reservoirs are the same size and same vertical elevation, the gradient will be linear over time (if the drainage rate to discard was constant).

For cryoprotectant to perfuse into cells there must be constant exposure to cryoprotectant surrounding the cells and there must be pressure to maintain that exposure. In a living animal the heart maintains blood pressure that forces blood through the capillaries and forces nutrients into cells. A dead animal with no blood pressure and which is being perfused with cryoprotectant also requires pressure for the capillaries to remain open and for cryoprotectant to be maintained at high concentrations around cells.

Alcor found that closed-circuit perfusion must be maintained for 5-7 hours for full equilibration of glycerol, because the diffusion rate of water out of cells is thousands of times the rate at which glycerol enters cells. Of course, it would be possible to pump glycerol into a patient for 5-7 hours with open-circuit perfusion, but only by using thousands of dollars worth of glycerol. The newer vitrification cryoprotectants used by Alcor are vastly more expensive than glycerol. When using expensive cryoprotectants it makes far more sense to recirculate in a closed circuit. Closed-circuit perfusion also has the benefit of allowing for ongoing monitoring of physiological changes occurring in the patient's body during the perfusion process. Open-circuit with an inexpensive cryoprotectant has the advantage of avoiding recirculation of toxins.

Cryoprotectants, particularly glycerol, are viscous and cryoprotectants in high concentration are particularly viscous. The introduction of air bubbles into cryoprotectant solutions during pouring and mixing should be avoided because air emboli that enter the cryonics patient can block perfusion. Elimination of air bubbles from viscous cryoprotectant solutions is extremely difficult. Prevention is more effective than cure. Cryonicist Mike Darwin wrote about this problem and possible solutions in a 1994 CryoNet message.

Improper mixing of perfusate containing high levels of cryoprotectant can result in a phenomenon that appears to be high viscosity, but in reality is edema. If, for example, isotonic carrier solution is mixed half-and-half with cryoprotectant solution an open circuit perfusion may have to be halted when no further perfusate will go into the patient. The problem is caused not by viscosity, but by the fact that the isotonic solution became hypotonic due to dilution with cryoprotectant causing the cells to swell and forcing perfusion to end. In closed-circuit perfusion, the cryoprotectant concentrate reservoir contains cryoprotectant at about 125% the terminal concentration in a vehicle of isotonic carrier solution so that when reservoir concentrate is mixed with isotonic carrier there is no change in tonicity.

Newer cryoprotectants are less viscous than glycerol, so perfusions can be done in less time. After 15 minutes of perfusion with carrier solution, cryoprotectant concentration linearly increases at a rate of 50millimolar per minute until full concentration is reached in about two hours (a protocol developed on the basis of minimizing osmotic damage when perfusing kidneys). Perfusion is increased for an additional hour or two until the cryoprotectant has fully diffused into cells (as indicated by similarity of afflux and efflux cryoprotectant concentrations).

Only after a few hours of closed-circuit perfusion is the concentration of cryoprotectant exiting the cryonics patient equal to the concentration of cryoprotectant entering the patient. Only an extended period of sustained pressure will keep capillaries open, and otherwise facilitate diffusion of cryoprotectant into cells. And the exiting cryoprotectant concentration will equal the entering cryoprotectant concentration only when the tissues are fully loaded with cryoprotectant. A refractometer is used to verify that terminal cryoprotectant concentration has been reached in the brain.

(A refractometer measures the index of refraction of a liquid, ie, the ratio of the speed of light in the liquid and the speed of light in a vacuum (or air). Light changes speed when it strikes the boundary of two media, thus causing a change in angle if it strikes the new medium at an angle. Because the refractive index is a ratio of two quantities having the same units, it is unitless. Sodium vapor in an electric arc produces an excitation between the 3s and 3p orbitals resulting in yellow-orange light of 589nm what Joseph Fraunhofer called the "Dline". Insofar as the sodium "Dline" was the first convenient source of monochromatic light, it became the standard for refractometry. The refractive index of a liquid is thus a high-precision 5-digit number between 1.3000 and 1.7000 at a specific temperature, measured at the sodium Dline wavelength. For example, the refractive index of glycerol at 25C nD25 is 1.4730.)

Closed-circuit perfusion may be necessary for removal of water as well as loading of cryoprotectant if it is true that open-circuit perfusion cannot remove water effectively.

One could imagine that the additional time spent doing closed-circuit (rather than open-circuit) perfusion means increased damage due to above-zero temperature. But most cells are still alive and metabolizing very slowly at 10C. Viaspan, RPS-2 and other organ preservation solutions are designed to keep tissues alive for extended periods at near-zero temperatures certainly for the time required for closed-circuit perfusion. Ramping (slowly increasing concentration) of cryoprotectant should be done in such a way that the ion and mannitol or lactobionate concentration remains unchanged in the perfusate. Ramping is not an osmotically neutral process, however, because cryoprotectant is expected to dehydrate tissues.

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Perfusion & Diffusion in Cryonics Protocol - BEN BEST

We see it all the time in movies. A person gets frozen or put in cryosleep and then unfrozen at a later date with no aging taking place, or other ill effects.

Sometimes this happens on purpose, like to someone with an incurable disease hoping a cure exists in the future, or sometimes by accident, like someone getting frozen in a glacier.

The science behind it does exist and the application of the practice is called cryonics. Its a technique used to store a persons body at an extremely low temperature with the hope of one day reviving them. This technique is being performed today, but the technology behind it is still in its infancy.

Someone preserved this way is said to be in cryonic suspension. The hope is that, if someone has died from a disease or condition that is currently incurable, they can be frozen and then revived in the future when a cure has been discovered.

Its currently illegal to perform cryonic suspension on someone who is still alive. Those who wish to be cryogenically frozen must first be pronounced legally dead which means their heart has stopped beating. Though, if theyre dead, how can they ever be revived?

According to companies who perform the procedure, legally dead is not the same as totally dead. Total death, they claim, is the point at which all brain function ceases. They claim that the difference is based on the fact that some cellular brain function remains even after the heart has stopped beating. Cryonics preserves some of that cell function so that, at least theoretically, the person can be brought back to life at a later date.

After your heart stops beating and you are pronounced legally dead, the company you signed with takes over. An emergency response team from the facility immediately gets to work. They stabilize your body by supplying your brain with enough oxygen and blood to preserve minimal function until you can be transported to the suspension facility. Your body is packed in ice and injected with an anticoagulant to prevent your blood from clotting during the trip. A medical team is on standby awaiting the arrival of your body at the cryonics facility.

After you reach the cryonics facility, the actual freezing can begin.

They could, and while youd certainly be frozen, most of the cells in your body would shatter and die.

As water freezes, it expands. Since cells are made up of mostly water, freezing expands the stuff inside which destroys their cell walls and they die. The cryonics companies need to remove and/or replace this water. They replace it with something called a cryoprotectant. Much like the antifreeze in an automobile. This glycerol based mixture protects your organ tissues by hindering the formation of ice crystals. This process is called vitrification and allows cells to live in a sort of suspended animation.

After the vitrification, your body is cooled with dry ice until it reaches -202 Fahrenheit. After this pre-cooling, its finally time to insert your body into the individual container that will be placed into a metal tank filled with liquid nitrogen. This will cool the body down to a temperature of around -320 degrees Fahrenheit.

The procedure isnt cheap. It can cost up to $200,000 to have your whole body preserved. For the more frugal optimist, a mere $60,000 will preserve your brain with an option known as neurosuspension. They hope the technology in the future will allow them to clone or regenerate the rest of the body.

Many critics say the companies that perform cryonics are simply ripping off customers with the dream of immortality and they wont deliver. It doesnt help that the scientists who perform cryonics say they havent successfully revived anyone, and dont expect to be able to do so anytime soon. The largest hurdle is that, if the warming process isnt done at exactly the right speed and temperature, the cells could form ice crystals and shatter.

Despite the fact that no human placed in a cryonic suspension has yet been revived, some living organisms can be, and have been, brought back from a dead or near-dead state. CPR and Defibrillators can bring accident and heart attack victims back from the dead daily.

Neurosurgeons often cool patients bodies so they can operate on aneurysms without damaging or rupturing the nearby blood vessels. Human embryos that are frozen in fertility clinics, defrosted and implanted in a mothers uterus grow into perfectly normal human beings. Some frogs and other amphibians have a protein manufactured by their cells that act as a natural antifreeze which can protect them if theyre frozen completely solid.

Cryobiologists are hopeful that nanotechnology will make revival possible someday. Nanotechnology can use microscopic machines to manipulate single atoms to build or repair virtually anything, including human cells and tissues. They hope one day, nanotechnology will repair not only the cellular damage caused by the freezing process, but also the damage caused by aging and disease.

Some cryobiologists have predicted that the first cryonic revival might occur as early as year 2045.

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Can A Human Be Frozen And Brought Back To Life? - Zidbits

Cryonics is an effort to save lives by using temperatures so cold that a person beyond help by today's medicine might be preserved for decades or centuries until a future medical technology can restore that person to full health.

Cryonics sounds like science fiction, but is based on modern science. It's an experiment in the most literal sense of the word. The question you have to ask yourself is this: would you rather be in the experimental group, or the control group?

Cryonics is justified by three facts that are not well known:

1) Life can be stopped and restarted if its basic structure is preserved.

Human embryos are routinely preserved for years at temperatures that completely stop the chemistry of life. Adult humans have survived cooling to temperatures that stop the heart, brain, and all other organs from functioning for up to an hour. These and many other lessons of biology teach us that life is a particular structure of matter. Life can be stopped and restarted if cell structure and chemistry are preserved sufficiently well.

2) Vitrification (not freezing) can preserve biological structure very well.

Adding high concentrations of chemicals called cryoprotectants to cells permits tissue to be cooled to very low temperatures with little or no ice formation. The state of no ice formation at temperatures below -120C is called vitrification. It is now possible to physically vitrify organs as large as the human brain, achieving excellent structural preservation without freezing.

3) Methods for repairing structure at the molecular level can now be foreseen.

The emerging science of nanotechnology will eventually lead to devices capable of extensive tissue repair and regeneration, including repair of individual cells one molecule at a time. This future nanomedicine could theoretically recover any preserved person in which the basic brain structures encoding memory and personality remain intact.

So...

Then cryonics should work, even though it cannot be demonstrated to work today. That is the scientific justification for cryonics. It is a justification that grows stronger with every new advance in preservation technology.

Death occurs when the chemistry of life becomes so disorganized that normal operation cannot be restored. (Death is not when life turns off. People can and have survived being "turned off".) How much chemical disorder can be survived depends on medical technology. A hundred years ago, cardiac arrest was irreversible. People were called dead when their heart stopped beating. Today death is believed to occur 4 to 6 minutes after the heart stops beating because after several minutes it is difficult to resuscitate the brain. However, with new experimental treatments, more than 10 minutes of warm cardiac arrest can now be survived without brain injury. Future technologies for molecular repair may extend the frontiers of resuscitation beyond 60 minutes or more, making today's beliefs about when death occurs obsolete.

Ultimately, real death occurs when cell structure and chemistry become so disorganized that no technology could restore the original state. This is called the information-theoretic criterion for death. Any other definition of death is arbitrary and subject to continual revision as technology changes. That is certainly the case for death pronounced on the basis of absent "vital signs" today, which is not real death at all.

The object of cryonics is to prevent death by preserving sufficient cell structure and chemistry so that recovery (including recovery of memory and personality) remains possible by foreseeable technology. If indeed cryonics patients are recoverable in the future, then clearly they were never really dead in the first place. Today's physicians will simply have been wrong about when death occurs, as they have been so many times in the past. The argument that cryonics cannot work because cryonics patients are dead is a circular argument.

More than one hundred people have been cryopreserved since the first case in 1967. More than one thousand people have made legal and financial arrangements for cryonics with one of several organizations, usually by means of affordable life insurance. Alcor is the largest organization, and distinguished among cryonics organizations by its advanced technology and advocacy of a medical approach to cryonics.

Alcor procedures ideally begin within moments of cardiac arrest. Blood circulation and breathing are artificially restored, and a series of medications are administered to protect the brain from lack of oxygen. Rapid cooling also begins, which further protects the brain. The goal is to keep the brain alive by present-day criteria for as long as possible into the procedure. It is not always possible to respond so rapidly and aggressively, but that is Alcor's ideal, and it has been achieved in many cases.

In 2001 Alcor adapted published breakthroughs in the field of organ preservation to achieve what we believe is ice-free preservation (vitrification) of the human brain. This is a method of stabilizing the physical basis of the human mind for practically unlimited periods of time. The procedure involves partly replacing water in cells with a mixture of chemicals that prevent ice formation. Kidneys have fully recovered after exposure to the same chemicals in published studies.

Alcor's future goals include expanding ice-free cryopreservation (vitrification) beyond the brain to include the entire human body, and reducing the biochemical alterations of the process to move closer to demonstrable reversibility. Based on the remarkable progress being made in conventional organ banking research, we believe that demonstrably reversible preservation of the human brain is a medical objective that could be achieved in the natural lifetime of most people living today.

To learn more, please read our list of Frequently Asked Questions and the many articles in the Alcor Library.

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Alcor: About Cryonics

by Ben Best CONTENTS: LINKS TO SECTIONS

Although most living organisms are composed of large amounts of water, it is not inevitable that freezing these organisms results in ice-formation. Among amphibians and insects that can tolerate freezing, there is wide variation in the amount of freezing they can tolerate.

Species of frogs can spend days or weeks "with as much as 65 percent of their total body water as ice". Some amphibians achieve their protection due to the glycerol manufactured by their livers. Glycerol is "antifreeze", it reduces ice formation and lowers freezing point. Glycerol (glycerin), like ethylene glycol (automobile anti-freeze) is a cryoprotectant. The sugar glucose is also a cryoprotectant and arctic frogs have a special form of insulin that accelerates glucose release and absorption into cells as temperatures approach freezing. A cryoprotectant can make water harden like glass with no crystal formation a process called vitrification. Freezing-damage to cells is due to the formation of ice-crystals.

Insects most often used sugars for cryoprotectant. They may also refrain from eating (not such a hardship because their metabolism slows at low temperature) and utilize tough waxy coverings to keep nucleating substances out of their body when temperature drops. Adult arctic beetles (Pterostichus brevicornis) normally endure temperatures below 35C. These beetles have been frozen in the laboratory to 87C for 5 hours without apparent injury, ie, they demonstrated "directed, coordinated activity such as walking, feeding, and avoidance response, and no paralysis or erratic behavior..." [SCIENCE166:106-7 (1-OCT-69)]. (A replication of this experiment would be of value to confirm or challenge the results.) This would seem to indicate that neurological tissue can, in principle, recover in a functional way from vitrification. The glycerol, sugars, and other cryoprotectants which are produced naturally in these organisms, are not found in levels that adequately explain (with current knowledge of cryobiology) the remarkable freezing-tolerance.

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Water is not very viscous, therefore it can be vitrified only by an extremely rapid "flash-freezing" of a small sample about 3millionC per second to 135C. Under such rapid cooling, water molecules don't have time to arrange themselves into a crystalline lattice structure. Viscosity increases very little when water is cooled, but at freezing temperature a sudden phase transition occurs to an ice crystal. Molten silica (silicon dioxide, SiO2, liquid glass), by contrast, is very viscous. This viscosity is the result of the tendency of silica to form amorphous networks of polymers rather than to arrange in an orderly crystal lattice.

Quartz (rock-crystal) & sand are examples of SiO2 as pure crystal. SiO2 which has been made to exist in noncrystalline form is called vitreous silica (fused silica). Oxides can be added to prevent crystallization and promote vitrification. About 90% of all manufactured glass (called soda-lime glass) contains about 12% each of soda (Na2O or Na2CO3) and lime (CaO) added to the SiO2. The soda is analogous to cryoprotectants in preventing crystallization (the lime is added to prevent the glass from dissolving in water).

By cooling silica very slowly it is possible to form rock crystal, having very high density and low volume. By cooling faster, resistance to crystallization due to viscosity & the absence of nucleators causes silica to pass below its freezing temperature (supercool) and vitrify at some glass transition temperature (Tg). Viscosity increases rapidly to solidification near Tg, but over a small temperature range rather than at a precise temperature (in contrast to crystallization or fusion, which occurs at a precise temperature). The change that happens at Tg is simply a rapid increase in viscosity, not a change of state. Viscosity becomes very high near Tg when cooling from above, which means that Tg is better characterized as a "rubber/glass transition" than a "liquid/glass transition". Moreover, Tg is a function of cooling-rate. A faster cooling-rate results in Tg at a higher temperature leading to a solid that has a high volume (lower density), is more amorphous and less viscous. A slower cooling-rate results in Tg at a lower temperature leading to a solid that has a low volume (higher density), is less amorphous and is more viscous. In practice, Tg occurs within a narrow temperature range because changing cooling rate an order of magnitude (ie, by a factor of ten) only changes Tg by 35C.

But volume continues to decrease and viscosity continues to increase below Tg. The change at Tg is quantitative, not qualitative (in contrast to crystallization). Because cooling occurs from outside to inside, overly rapid cooling creates stress when the warmer core needs to contract more than the cooler surface. This is the reason why slow cooling reduces cracking. At Tg there is a sudden increase in viscosity and heat capacity (usually many orders of magnitude), but there is no comparable sudden decrease in volume. In fact, Tg is characterized as a temperature-range where the rate of decrease of volume decreases, although volume does continue to decrease (and viscosity continues to increase) linearly below Tg. Tg could be a temperature critical to cracking because the sudden increase in viscosity would be likely to affect heat conduction as well as stress. [For further discussion of Tg, stress and cooling rates, see my essays Physical Parameters of Cooling in Cryonics and Lessons for Cryonics from Metallurgy and Ceramics.]

Sugar, like silica, can form a crystal (rock candy) or a glass (cotton candy) depending on the rate of cooling. Like molten glass, liquid sugar is very viscous and prone to formation of amorphous polymers. In silica the polymerization bonds tend to be of a "mixed" covalent-ionic type, whereas for sugar the polymerization is assisted by weaker forces (van der Waals or hydrogen bonding). In neither case do these bonds have the defined bond-lengths and bond-angles of covalent bonds. Glycerol/water in the human body is more like sugar than like silica. But the situation is complicated by the presence of many salts, proteins, fats, etc.

Vitrifying liquids have been classified as "strong" or "fragile". The term fragile is confusing because it does not refer to the tendency to break under mechanical stress, but rather to a highly rapid rise in viscosity as temperature approaches Tg from above. Substances which are called fragile tend to have more ionic bond types (or hydrogen bonds), whereas substances which are strong (and show a modest decline in viscosity above Tg) have more covalent bonding. "Gripping strength" changes more radically for ionic bonding near Tg. Covalent bonds are stronger (less fragile), and groups of molecules held together by covalent bonds are less susceptible to molecular phase changes just above Tg. Glycerol-type cryoprotectants (which cohere mainly by hydrogen bonding) are more "fragile" than vitreous silica (which has covalent coherence), but is less "fragile" than ionic substances.

(For more on the subject of fragility, viscosity and molecular mobility, see Viscosity and Glass Transition.)

If rapid cooling causes vitrification, it seems plausible that rapid application of pressure could do the same thing at above Tg for rapid-cooling. Because Tg is a function of cooling rate, there is no reason why it could not also be a function of pressure-application rate or some combination of the two (plus cryoprotectant). It is known that pressure distorts the iceI lattice from its ideal tetrahedral orientation, and this could be important in preventing nucleation.

Water can be made to vitrify if cooled at a rate of millions of degrees Celcius per second. Water can also vitrify if mixed with salts or cryoprotectants. Salt solutions have their highest Tg at their eutectic concentration, but this would be too concentrated for cryobiological applications. Salt solutions having cations with a high oxidation state (e.g., trivalent cations) and more basic anions (e.g., citrate) are better glass-formers (have a higher Tg) than salt solutions that do not. Nitrates vitrify better than chlorides, and magnesium (Mg2+) vitrify better than salts of zinc (Zn2+)[THE JOURNAL OF CHEMICAL PHYSICS; Angell,CA; 52(1):1058-1068 (1970)].

In practice, vitrification can be assisted by substances other than cryoprotective agents. Carrier solutions can reduce the amount of cryoprotectant needed to vitrify. The carrier solution described in CRYOBIOLOGY27(5):492-510 (1990) is a mixture of salts, dextrose and glutathione, and is based on the so-called RPS-2 solution used for storing rabbit kidneys. A carrier solution will substitute for water, but only in a 25% range. The carrier solution effect is largely colligative ie, molecules getting in the way of water molecules which might otherwise form ice. A good carrier solution will be non-toxic, and by reducing the amount of cryoprotectant needed to vitrify will reduce toxicity from cryoprotectant. [For further discussion of carrier solutions, see my essay Perfusion & Diffusion in Cryonics Protocol.]

When freezing occurs in aqueous mixtures of non-crystalline solutes (such as ice cream), the unfrozen freeze-concentrated solute can display a transition temperature Tg' that has a more prominent thermal signature than Tg. Tg' is the Tg of the unfrozen portion of a sample that contains ice. For ice cream, Tg' is about 32C and the unfrozen water(Wg') is about 35wt%[FOOD CHEMISTRY; Owen Fennema; 3rd Edition; Table11; page76]. (Wg is water content of the sample at Tg, whereas Wg' is the unfrozen water content at Tg' which has been called bound water, as if that term could be applied to the water content of any vitrified sample). For low molecular weight solutes, Tg' (andTg) typically increases with molecular weight. Biopolymers (starch, gluten, collagen, albumins, etc.) of high molecular weight typically have Tg' near 10C.

Aqueous solutions of cryoprotectants can themselves freeze, and have an unfrozen portion that solidifies at a temperature Tg' which is considerably higher than Tg[JOURNAL OF FOOD SCIENCE; Brake,NC; 64(1):10-15 (1999)]. The glass transition temperature of the maximally freeze-concentrated portion of the sample (Tg') should, by definition, exhibit the same high viscosity as Tg[FOOD RESEARCH INTERNATIONAL; Bai,Y; 34(2-3):89-95 (2001)]. The Tg' will be below the eutectic temperature[PURE & APPLIED CHEMISTRY; Goff,HD; 67(11):1801-1808 (1995)]. Mixtures of basic amino acids with hydroxy dicarboxylic acids added to the protein solute can raise Tg' by hydrogen-bond networking[CHEMICAL & PHARMACEUTICAL BULLETIN; Izutsu,K; 57(1):43-48 (2009)]. If ice is formed in a mixture intended to vitrify as in an imperfectly perfused cryonics patient the vitrification mixture remaining in the unfrozen portion will have a higher concentration and, thus, a higher Tg (which will be Tg')[Figure1; CRYOBIOLOGY; Wowk,B; 60(1):11-22 (2010)]. 57(1):43-48 (2009)].

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The other source of assistance for vitrification comes from ice blockers. While cryoprotectants slow ice-crystal growth and formation, ice blockers act specifically against the formation of the ice nuclei which are necessary for freezing to begin. Arctic fish use ice-blocking proteins to keep the freezing temperature of their bodies at or below 2.2C, which is below the freezing temperature of seawater (1.9C). Some plant flavonal glycosides can depress freezing temperature by as much as 9C[CRYOBIOLOGY; Kasuga,J; 60(2):240-243 (2010)].

Although the melting temperature of water is 0C, water that is absolutely pure will not freeze above 40C because water requires nucleating agents to begin crystal growth[PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY OF LONDON; Lundheim,R; 357:937-43 (2002)]. Tapwater has enough nucleating agents that ice trays in refrigerators can freeze water at close to 0C. Water with small amounts of nucleating agents can be supercooled, but once ice crystals begin forming they spread with explosive speed.

The temperature at which pure water will freeze (40C) is called the homogenous nucleation temperature (Th) in contrast to Tm, which is the melting temperature (0C). The temperatures between Th and Tm are the region of heterogenous nucleation where the ice formation is a function of time and nucleating agent concentration (or nucleating ability of the nucleators).

Heterogenous nucleation takes its name from the fact that the nucleus around which ice crystal growth occurs is composed of both water molecules and other kinds of molecules. Conversely, in homogenous nucleation the only molecule found in the crystal nucleus is water. The string used to make rock candy from a cooling super-saturated sugar and water mixture acts as a heterogenous nucleator around which the sugar molecules can crystallize. At temperatures close to 40C in the sky (in polar regions and above 30,000 feet), homogenous nucleation occurs whenever there is a sufficient concentration of water vapor. Silver iodide (AgI) is water-insoluble and has a crystal structure similar enough to ice that it can readily act as a heterogenous nucleator below 5C. Silver iodide is especially suitable for seeding rainclouds because it can be micronized into particles smaller than 10nanometers.

The critical size for a homogenous nucleus to begin ice crystal growth is 45,000 water molecules at 5C, 650water molecules at 20C, and only 70water molecules at 40C[Vali,G; "Principles of Ice Nucleation" in BIOLOGICAL ICE NUCLEATION AND ITS APPLICATIONS, p.5; Richard E.Lee,Jr., et. al., Editors; APS Press (1995)]. Vibrational spectroscopy indicates the onset of ice-like structure for water in gas phase at about 275molecules[SCIENCE; Pradzynski,CC; 337:1529-1532 (2012)]. Below the critical size spontaneous dissolution of the ice nuclei will occur due to solubility. Thus, the temperature of homogenous nucleation is a function of sample volume and of time. But the function is an exponential one, with nucleation decreasing so rapidly above 40C that it is rarely seen more than a few degrees above 40C [CRYOBIOLOGY41(4):257-279 (2000)]. The probability of a volume V of pure water freezing in time t due to homologous nucleation is J(T)xVxt. J(T) is nucleation rate at temperature T, determined by the empirically-derived equation:

J(T)=6.8x1050e3.9T

for J(T) in meters3/second and T in C[Vali,G; Ibid; p.4]. The e3.9T factor means that the probability of nucleation increases by about 50 for each drop of 1C or by over 6million (504) for a 4C temperature drop. Although volume and time are linear components in the probability, the exponential temperature component means that probability rapidly goes from zero to one in the temperature range between 38C and 42C (being very close to 40C for all practical purposes).

Not surprisingly, the probability of heterogenous nucleation in an animal increases with body size (volume). Some species of reptiles with body mass less than 20grams can supercool to temperatures below 5C, but not reptile having body mass greater than 40grams can supercool to as low as 2C[Costanzo,JP & Lee,RE,Jr.; "Supercooling and Ice Nucleation in Vertebrate Ectotherms" in BIOLOGICAL ICE NUCLEATION AND ITS APPLICATIONS, p.229; Richard E.Lee,Jr., et. al., Editors; APS Press (1995)].

Higher pressures lower Th and elevate Tm [CRYOBIOLOGY21(4):407-426 (1984)]. Increasing cryoprotectant concentrations lower both Th and Tm, but the effect is more dramatic on Th than on Tm. As shown by the two-headed arrow in the figure, enough cryoprotectant to lower Tm by 30C will lower Th to the glass transition temperature (Tg) thereby eliminating homogenous nucleation. Using ice-blockers to prevent heterogenous nucleation creates the possibility of eliminating nucleation (ice formation) altogether and achieving vitrification at roughly 55% cryoprotectant concentration.

The double-arrow shown in the diagram serves to focus attention on another phenomenon which needs to be addressed in attempting tissue cryopreservation. Namely, that maximum nucleation occurs just above Tg near the downward-pointing arrow (80C to 120C) and that maximum ice-crystal growth-rate occurs just below Tm near the upward-pointing arrow (80C to 40C). (The location of the arrowheads is not significant.) The significance of these facts is that it is much easier to avoid ice-crystal formation when cooling (vitrifying) than when re-warming (de-vitrifying). The many nuclei formed when cooling in the 80C to 120C range can cause massive ice growth when rewarming in the 80C to 40C range. This is called the devitrification problem. Before the use of ice-blockers it was believed that only radio-frequency rewarming technology could possibly achieve rewarming rates rapid enough to avoid ice-crystal formation upon devitrification. With ice-blockers, however, ice crystal growth is greatly inhibited during rewarming.

Ice crystals can grow along six symmetric axes the aaxes, all six axes in the same plane or the caxis, which is perpendicular to the plane of the six aaxes. Ice crystal growth at higher temperatures typically occurs along the aaxes, which accounts for the familiar hexagonal shape of snowflakes. Caxis growth results in needle-like, spicular ice crystals, which are potentially damaging[THE FASEB JOURNAL; Davis,PC; 4(8):2460-2468 (1990)]. (For more detail on ice formation see The Freezing Process.) Ice blockers can act by three mechanisms: (1)bind-to and inactivate heterogenous nucleating substances, (2)block aaxis growth or, (3)block caxis growth. In anti-freeze proteins, amino acids such as threonine & serine hydrogen-bond to the ice[CRYOBIOLOGY41(4):257-279 (2000)]. Inhibition of aaxis growth by anti-freeze proteins typically is found in arctic fish. Arctic insects, by contrast, typically have anti-freeze proteins that inhibit caxis growth. By binding to the basal plane(caxis) rather than the prism plane(aaxis), insect anti-freeze proteins can depress freezing temperature by 4-5C, whereas fish anti-freeze proteins only depress freezing temperature by not much more than 1C[BIOPHYSICAL JOURNAL; Pertaya,N; 95(1):333-341 (2008)].

Not all ice-blockers are proteins. In fact, 21st Century Medicine (21CM) researchers have discovered that the polymer polyvinyl alcohol (commonly found in adhesives such as Elmer's glue & postage-stamp glue) is an extremely effective ice-blocker if used in the syndiotactic stereochemical form. In the isotactic stereochemical form, the hydroxyl groups are all on the same side of the molecule, whereas in the syndiotactic stereochemical form, the hydroxyl groups are on alternate sides of the molecule. OH OH OH | | | -CH2-C-CH2-C-CH2-C-CH2-C-CH2-C-CH2-C-(POLYVINYL ALCOHOL) | | | OH OH OH

Polyvinyl alcohol in the syndiotactic stereochemical form is an excellent fit size & conformation for attaching to an ice-crystal surface. Every hydroxyl group of the polyvinyl alcohol will hydrogen-bond to a water molecule. The polyvinyl alcohol molecules adhere to ice crystals (preventing growth) at temperatures as high as 30C, above which temperature separation begins to occur and ice-blocking activity diminishes. Polyvinyl alcohol is most effective against caxis growth and most effective in the temperature range of maximum nucleation.

21st Century Medicine researchers have produced a patented co-polymer (mixture of polymers) consisting of 20% vinyl acetate and 80% vinyl alcohol which they now sell as the commercial product Supercool X-1000. It is believed that the vinyl acetate reduces self-association of the vinyl alcohol, making the latter more available for ice-blocking [CRYOBIOLOGY40(3):228-236 (2000)]. A 0.01% solution of X1000 can reduce the amount of glycerol needed to vitrify by 3%. A 1% solution of X1000 can reduce the amount of glycerol needed to vitrify by 5%. Concentrations of X1000 greater than 1% do not provide much additional benefit.

Although these percentage differences may seem small, the benefits from ice-blockers are actually very great. Toxicity increases exponentially as the cryoprotectant concentrations reach the high levels needed to vitrify. Of particular relevance to cryonics, however, is the fact that cryoprotectants become too viscous to perfuse well at high concentrations, whereas ice blockers add little to viscosity. Thus, ice-blocker plus cryoprotectant can produce a solution that can both perfuse and vitrify.

The widespread presence of biological nucleators in the environment causes water to freeze close to 0C rather than at 40C. The most abundant and widely distributed nucleator is protein on the surface of the Pseudomonas syringae bacteria, a kind of bacteria that causes early freezing damage on plants (most commonly found on plant leaves, and other above-ground plant parts)[PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY OF LONDON; Lundheim,R; 357:937-943 (2002) and APPLIED MICROBIOLOGY; Maki,LR; 28(3):456-459 (1974)]. Such proteins must assume a rigid, ice-like conformation larger than 10nanometers and be able to aggregate[JOURNAL OF MOLECULAR BIOLOGY; Kajava,A; 232(3):709-717 (1993)].

21st Century Medicine researchers have succeeded in finding an ice-blocker that specifically binds-to and inactivates heterogenous nucleating agents. (These proteins evolved specifically to cause freezing at the highest possible temperature). The linear polymer polyglycerol(PGL) binds and inactivates these proteins, and is complementary to the action of polyvinyl alcohol(PVA). PGL is ineffective at inhibiting nucleation in small volumes, but is more effective than PVA at suppressing initial ice nucleation events in large volumes. The lowest number of visible ice-nucleation events is achieved with 0.1%PGL and 0.9%PVA[CRYOBIOLOGY 44(1):14-23 (2002)]. (PVA can also bind-to heterogenous nucleating agents.) 21CM now markets this formulation as Supercool Z-1000.

Ice blockers cannot cross cell membranes and do not cross an intact blood-brain barrier, which means that for a cryonics patient in good condition the only portion of the brain containing ice blocker will be the vasculature (about 4% of the brain). Ice blockers are not needed inside of cells because cells contain few nucleators cryoprotectant diffusion into cells is adequate. Ice blockers in the brain vasculature could prevent ice crystals from forming in the blood vessels which could propagate through the blood-brain barrier, especially in areas that are weakly perfused due to poor circulation. Circulation is often very poor in cryonics patients and the blood-brain barrier is frequently damaged. Although poor perfusion and reduced cryoprotectant concentration poses a danger of ice formation, ice blocker concentration will also be reduced in those areas and may not be of benefit.

As a cautionary note, it should be mentioned that use of ice-blocker without sufficient cryoprotectant (or rapid-enough cooling) to cause vitrification can result in ice formation that is more damaging than the ice that would have formed at a higher temperature if no ice-blocker had been used. Ice-formation at higher temperature tends to be extracellular and dehydrates the cells. At lower temperature osmosis is less active. So if ice-blockers simply result in ice formation at a lower temperature, the ice that forms at those temperatures is more likely to be inside the cells, thus causing greater damage.

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In 1949 it was discovered that glycerol can be used to protect bull sperm against freezing injury. A year later, the same techniques were successfully applied to red blood cells. Since that time large industries have developed around the cryopreservation of bull sperm and human blood.

In 1959 the substance DiMethyl SulfOxide (DMSO) was demonstrated to be useful as a cryoprotectant. DMSO passes through cell membranes more readily than glycerol, but it can be more toxic at higher temperatures. In 1972, 8-cell mouse embryos were cryopreserved to liquid nitrogen temperature temperature and rewarmed to obtain live mice, thanks to slow cooling and skillful combination of DMSO with glycerol. Glycerol is introduced first, the embryo is cooled to a low temperature, and then the highly permeant DMSO can be introduced with minimal toxic effect.

In 1983 a human pregnancy was first established by Trounson & Mohr using an 8-cell human embryo, which had been cryopreserved to liquid nitrogen temperature using gradually increasing concentrations of DMSO giving time for equilibration to prevent osmotic damage (PBI 10 minutes, 0.25M DMSO 10 minutes, 0.5M DMSO 10 minutes, 1.0M DMSO 10 minutes, 1.5M DMSO 10 minutes)[FERTILITY AND STERILITY 46(1):1-12 (1986)].

Since 1983 human embryos have been cryopreserved with not only DMSO, but with glycerol and propylene glycol. The best embryo survival rates are with those at the 2-cell to 4-cell stage of development. No one knows exactly how many human embryos are now being cryopreserved worldwide, but it is at least a million. And the number of living children who were once embryos at liquid nitrogen temperature is in the tens of thousands. (For an online review of human embryo cryopreservation technology see Human Oocyte and Embryo Cryopreservation.)

Over 50% of nematode worm (C. elegans) larvae and about 3% of adult nematodes can survive cooling to liquid nitrogen temperature. The required protocol is pre-treatment with 5% DMSO at 0C for 10minutes, cooling from 0C to 100C at 0.2C/minute, being plunged into liquid nitrogen (196C) and ultimately rewarming to 10C at a rate of 27.6C/minute. [CRYOBIOLOGY12(5):497-505 (1975)]. This is particularly noteworthy insofar as nematodes are fully functioning organisms with a digestive system, reproductive organs, muscles and a nervous system consisting of approximately 300 neurons.

In an organ with such high water & fat content as the brain, proper perfusion to protect the very delicate cell-to-cell relationships (synaptic connections) would be expected to be especially difficult to achieve. It has been known since the 1950s, however, that brains have a certain tolerance for ice crystallization. Audrey Smith [PROC.ROYAL SOCIETYB145:427-442 (1956) and BIOLOGICAL EFFECTS OF FREEZING AND SUPERCOOLING, A.U.Smith,Ed., p.304-368] demonstrated that hamsters could be slowly cooled to nearly 1C such that over 60% of brain water is turned to crystalline ice with no gross loss of normal behavior upon rewarming. The mechanism of this effect is based on the fact that intracellular ice crystallization & elevated intracellular salt concentrations causes the greatest damage. When tissues are cooled slowly, extracellular crystallization starts first and water tends to migrate out of cells to freeze in the extracellular space. If the intracellular electrolyte concentrations increase, it is evidently not enough to cause observable neurological damage at the 60% level.

[For more recent research on cryopreservation of brain tissue see The Hippocampal Slice Cryopreservation Project.]

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The cryoprotectant glycerol has long been used in vitrifying human blood & sperm and for many years was used to reduce freezing in human cryonics patients. Glycerol cannot be used to completely vitrify organs or cryonics patients because it is not possible to perfuse organs with high enough concentrations of glycerol to fully vitrify. When perfusion is performed with full-strength glycerol, enough glycerol gets into the tissues to achieve partial vitrification with about 20% ice formation.

Cryopreservation of tissues & organs is much more difficult than cryopreservation of small collections of cells. Time is required for cryoprotectant to permeate an organ and also for temperature to penetrate. Tissue is subject to degradation if there is no blood circulation even if the temperature is very low, yet a very low temperature will slow the rate of cryoprotectant perfusion. Moreover, organs can be damaged by even extremely small amounts of ice formation due to the critical cell-to-cell relationships which must be maintained for proper function. Even so, cryoprotectants have been used to preserve bone marrow, fetal hearts, intestines, parathyroid glands, skin, spleens, thymus glands, etc., which have been slowly cooled to dry ice temperature (79C) all without ice crystal damage.

Crystallization is not an inevitable consequence of glycerol cooling. A 68%v/v (volume/volume) glycerol/water solution will not crystallize at any subzero temperature it simply hardens like glass. But concentrations of glycerol much greater than 55%v/v have been called too viscous & toxic for cryonics use. Shortly before Alcor began using vitrification solutions, cryobiologist Brian Wowk determined that a combination of 58.4%v/v (8Molar) glycerol and 1%X1000 ice-blocker could vitrify a 2liter flask presumably meaning that a brain could be vitrified with 8Molar glycerol and 1%X1000 ice-blocker.

Most tissues can tolerate having over 80% of the water in the form of ice crystals upon slow cooling without noticeable damage upon re-warming. Most organs can tolerate 40% of water as ice crystals without damage upon re-warming. As mentioned above, the brain is an especially ice-crystal tolerant organ, insofar as 60% water as ice-crystals causes little gross damage.

The experiments of I.Suda [NATURE212:268-270 (1966) and BRAIN RESEARCH70:527-531 (1974)] indicate that cat brains cooled to 20C in 15%v/v glycerol (62% brain water as ice) for 777 days and 7.25 years, both show normal-looking EEG patterns upon re-warming although neurological activity is less for the 7.25-year brains. Hemorrhaging and cell loss of these specimens probably could have been prevented using several measures: (1)addition of glucose (nutrient) to the perfusion fluid, (2)careful washing of glycerol from the brains as part to the thawing/reperfusion process and (3)storing the brains at lower temperatures with higher glycerol concentrations.

According to an excellent paper describing vitrification (cryoprotectant) solutions [CRYOBIOLOGY24:196-213 (1987)], the quantity of glycerol (C), in %v/v, required to prevent mechanical injury from ice at any subzero temperature is:

C = 68 - 0.68P

where "P" is the percentage of liquid volume of an organ which can be converted to ice without crystal-damage. This formula is the equation of the line in Figure3 of the paper. It is related to the fact that a mixture of 68% glycerol and 32% water (volume/volume) will vitrify completely.

Using the finding that at least 60% of the brain can be frozen without neurological damage, gives:

C = 68 0.68(60) = 27.2

ie, 27.2%v/v glycerol (3.72Molar) should be sufficient to prevent ice-crystal damage to brains cooled to any subzero temperature (including liquid nitrogen temperature, 196C). In fact, rabbit brains perfused at room temperature with 23%v/v glycerol (3Molar) and cooled to dry ice temperature (79C) show excellent histological preservation under a light microscope[CRYOBIOLOGY21(4):407-426 (1984)]. For years cryonicists believed that "the Smith Criterion" of a minimum of 3.72 Molar glycerol concentration might be adequate to prevent freezing damage in cryonics patients.

[NOTE: Glycerol is 1,2,3-propanetriol and has a molecular weight of 92.09grams/mole and a density of 1.2613grams/cm3 at 20C. Therefore, to convert glycerol Molarity to %v/v multiply by 7.30]

In the December 1991 issue of CRYONICS magazine, a cryobiologist described the results of an experiment with a single rabbit brain perfused at room temperature with 3.72 Molar glycerol, cooled to 130C, cut into slabs, and the resulting slabs stored at 78C for many months before examination under an electron microscope. He states: "...the pattern of ice formation seems to be potentially quite damaging. Everywhere one looks, thick sheets of ice are found stabbing their way through brain tissue with apparent abandon."

But if this is true, how can we explain the complete neurological recovery of Audrey Smith's hamsters, 60% of whose brain water had been ice. When asked this question, the cryobiologist could give no answer. He also wrote, "Biochemically, all functions measured to date have always survived freezing and thawing, even under poor circumstances, again in possible disagreement with the poor electron microscope (EM) results. Hence, the reality of the EM results and the possibility of artifacts in these results have been in question for some time." He goes on to say, "It is almost miraculous how well the tissue organization re-establishes itself in general after thawing, even in areas where gaps are present. However, the likelihood of extensive damage existing below the level of resolution of the light microscope, but all too visible in the electron microscope, appears high."

Although a certain caution should be taken in accepting the results of a single preparation of a rabbit brain by a single experimenter, the cryobiologist's observations are not entirely inconsistent with those of Audrey Smith. As expected, the observed freezing was extracellular, rather than intracellular. But the damage seen not only seems inconsistent with the complete neurological recovery of Audrey Smith's hamsters, it also seems inconsistent with the finding that even without cryoprotectants, 80% of synapses in whole brain tissues cooled to 70C retain the metabolic properties of fresh brain biopsy synapses ["Metabolically Active Synaptosomes can be Prepared from Frozen Rat and Human Brain", JOURNAL OF NEUROCHEMISTRY40:608-614 (1983)]. Could it be that the glycerol cryoprotectant contributes to extracellular damage in some way? In any case, concentrations of glycerol above 3.72 Molar (27.2%v/v) glycerol are not difficult to achieve in cryonics. Cryonicists are typically more concerned with eliminating structural damage than in loss of viability due to cryoprotectant toxicity. At high concentrations glycerol perfuses poorly into cells and osmotically draws water out of cells resulting in dehydration.

Following the ultramicroscopic evidence of intra-cellular damage the cryonics organizations Alcor and later CryoCare began perfusing patients with the highest possible concentrations of glycerol. Cryonics patients became very dehydrated (losing body volume) by this procedure. The high viscosity of glycerol only allowed 55%v/v (7.5Molar) maximum concentration well below the 68%v/v necessary for vitrification. It has been estimated, however, that in combination with ice-blockers, 8Molar glycerol could vitrify if only such a concentration of glycerol could be attained in human patients. (Cryonics patients are now typically perfused with vitrifying cryoprotectants rather than with glycerol.)

Often a cryonics patient has been perfused with glycerol at one location and shipped in dry ice(frozen carbon dioxide) for storage at another location. The solidification temperature(Tg) of glycerol is 90C, which is below the temperature of dry ice(79C). But glycerol is viscous enough at dry ice temperature that little harm results from holding the glycerolized patient in dry ice for a few days. The result can be very damaging, however, if the dry ice is allowed to melt and the patient rewarms. The liquid portion of a glycerolized patient is about 20% water and about 80% glycerol. As can be seen from the diagram at the beginning of the section on ice blockers melting temperature(Tm) declines rapidly with increasing cryoprotectant concentration. In a glycerolized patient considerable melting of ice occurs at 60C and higher. Melting releases debris created by freezing and thereby causes loss of structural information that could potentially be used by future molecular repair technology. Moreover, cryoprotectant toxicity can result in structural damage to the debris and damaged tissues which is far worse on rewarming than what would occur when passing through the same temperatures on cooling. So extreme caution must be taken to prevent a glycerolized patient from rewarming from dry ice temperature.

[For more information about perfusing cryonics patients with cryoprotectant, see my essay Perfusion & Diffusion in Cryonics Protocol.]

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The cell membrane (plasmalemma) is the site of most freezing damage. Therefore good cryoprotectants may not only perform the anti-freeze function of preventing ice formation, but protect cell membranes as well. Cryoprotectant toxicity, however, can potentially affect any organelle or macromolecule with proteins being the most vulnerable.

Sugars are polyhydroxyl aldehydes or ketones (carbon chains with terminal aldehydes or ketones and hydroxyl side-chains). Glyceraldehyde is a very simple sugar. High levels of sugars and sugar alcohols(polyols) are found in many polar plants, insects, fungi, etc. as non-toxic cryoprotectants. The fact that fructose will not crystallize is the reason sucrose is used as table sugar, despite the fact that fructose is cheaper.

Northern frogs use glucose as a cryoprotectant. When temperatures drop the livers of these frogs produce large amounts of glucose which a special form of insulin allows to enter cells in large quantity. The heart and brain does not freeze, but much of the rest of the body does (the frog is two-thirds ice). Upon thawing the frog must rapidly remove the glucose to prevent metabolic injury, but the glucose is saved in a special bladder because the frog cannot risk losing so many precious calories. Gradually, the glucose from the special bladder re-enters the plasma for metabolism or storage in the liver[JOURNAL OF MOLECULAR ENDOCRINOLOGY; Conlon,JM; 21(2):153-159 (1998)].

The two disaccharides (sugars composed of two simple simple sugars) that most protect proteins & cell membranes against chilling, freezing & dehydration are sucrose (fructose,glucose) and trehalose (glucose,glucose). Sucrose is the most common sugar found in freezing-tolerant plants which can increase their sucrose levels ten-fold in response to low temperature. Sucrose and trehalose inhibit the membrane mixing associated with chilling. Both sugars fit well in cell membranes, binding to phospholipid head groups. Trehalose constitutes 20% of the dry weight of organisms able to survive complete dehydration. Trehalose has an abnormally large hydrated radius well over twice as large as other sugars and (unlike other sugars) is totally excluded from the hydration shell of proteins.

Cryoprotection from freezing injury can differ from cryoprotection for vitrification reducing electrolyte toxicity might be more important for the former, but it would not be so important for the latter. Sucrose and ethylene glycol have been used in combination to vitrify human oocytes[HUMAN REPRODUCTION; Kuleshova,L; 14(12):3077-3079 (1999)]. But sugars are more often used as cryoprotectants against freezing and chilling injury rather than for vitrification, with the disaccharide sucrose being more effective than the monosaccharide glucose[CRYOBIOLOGY; Santarius,KA; 20(1):90-99 (1983) and CRYOBIOLOGY; Carpenter,JF; 25(3):244-255 (1988)].

Carbonyl groups (>C=O) such as are found on aldehydes (RCOH) and ketones (RCOR') can reduce certain heavy metal ions (ie, "reduce" the positive charge by adding an electron). Copper ion in the plus two state (Cu2+), for example, can be reduced to the plus one state (Cu+) in the presence of a ketone or aldehyde. (Sugars are ketones or aldehydes, eg, glucose is an aldehyde and fructose is a ketone.) Copper and iron ions in the reduced state can result in production of damaging hydroxyl radicals as a result of the Fenton Reaction.

Because most sugars have free aldehyde or ketone end-groups, they readily bind to the free amine group of lysine or arginine on proteins, a process called glycation. Sugars that participate in this reaction are called reducing sugars. All monosaccharides are reducing sugars because, although the carbonyl group may not be exposed when a ring structure is formed, the carbonyl group is exposed when a hemiacetal ring opens (e.g., in the interconversion of glucose between anomeric forms). Monosaccharides can dissolve in cryoprotectant solutions more readily and vitrify at lower concentrations than disaccharides[CRYOBIOLOGY; Kuleshova,LL; 38(2):119-130 (1999)], but because of their capacity for glycation, monosaccharide exposure to protein should be brief and at low temperature. A 220millimolar D-galactose solution was shown to be nearly as effective a cryoprotectant as 5%DMSO for human embryonic liver cells (and substantially better than D-glucose)[GLYCOBIOLOGY; Chaytor,JL; 22(1):123-133 (2012)], but galactose can be five times more glycating than glucose.

Some disaccharides (such as maltose) are reducing sugars because the link between the composite monosaccharides (the glycosidic bond) does not prevent the composite monosaccharide hemiacetal rings from opening. But trehalose and sucrose are non-reducing sugars because their glycosidic bonds do prevent opening of hemiacetal bonds. In acidic conditions, however, sucrose is far more vulnerable to hydrolysis into its reducing-sugar monosaccharides than is trehalose[CRYOBIOLOGY; Crowe,JH; 43(2):89-105 (2001)]. To the extent that glycation plays as role in enzyme stability and membrane stability associated with freezing damage or chilling injury, trehalose is a superior cryoprotectant to sucrose.

Disaccharides like trehalose and sucrose do not cross cell membranes, however, and thus only protect the inner cell membranes of organisms that synthesize them. Trehalose allows yeast to dehydrate, and can reach up to 35% of the dried weight. Trehalose is a blood sugar for lobsters, but it is not synthesized by vertebrates. Many strategies have been attempted to get trehalose inside of vertebrate cells so that its cryoprotective, protein-protective and membrane-protective properties can be of benefit on the inside as well as on the outside of cells. Plasmids containing the trehalose transporter gene TRET1 from African chironomid larvae[PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); Kikawada,T; 104(28):11585-11590 (2007)] have been transfected into Chinese hamster ovary cells, resulting in a seven-fold increase in trehalose and a 400% increase in growth after dessication[CRYOBIOLOGY; Chakraborty,N; 64(2):91-96 (2012)]. Microinjection has been used to get trehalose into human oocytes which improves cryopreservation[FERTILITY AND STERILITY 77(1):152-158 (2002)]. When combined with 0.5Molar DMSO, 0.5Molar trehalose microinjected into mouse oocytes resulted in excellent cryosurvival and healthy offspring (presumably because trehalose alone would not enter organelles such as mitochondria and endoplasmic reticulum)[BIOLOGY OF REPRODUCTION; Eroglu,A; 80(1):70-78 (2009)]. Transplanted tissue-engineered epidermis that had been cryopreserved with a trehalose/DMSO mixture was indistinguishable from fresh control grafts[BIOMATERIALS; Chen,F; 32(33):8426-8435 (2011)]. Trehalose may also protect macromolecules by being a free radical scavenger[JOURNAL OF BIOLOGICAL CHEMISTRY; Benaroudj,N; 276(26):24261-24267 (2001)].

Nonetheless, non-penetrating cryoprotectants can assist vitrification because most nucleators are extracellular and because dehydration allows for intracellular vitrification by bound water. Extracellular vitrification which involves sugar prevents cell membranes from coming in contact and fusing[ANNUAL REVIEW OF PHYSIOLOGY; Crowe,JH; 60:73-103 (1998)]. Cell membranes are commonly believed to be the part of cells most vulnerable to freezing damage.

Trehalose displaces bound water and protects cell membranes by hydrogen-bonding to proteins and the polar ends of phospholipids more strongly than bound water[ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 245(1):134-143 (1986)]. At the phospholipid bilayer of cell membranes trehalose is able to displace water molecules bound to carbonyls, but sucrose is not[BIOPHYSICAL JOURNAL; Amalfa,F; 78(5):2452-2458 (2000)]. Trehalose interacts more strongly with water than does sucrose, at least partly because sucrose forms intramolecular hydrogen bonds[THE JOURNAL OF PHYSICAL CHEMISTRYB; Lerbret,A; 109(21):11046-11057 (2005)]. Trehalose has a hydration radius that is 2.5times greater than that of sucrose, and 2.5times the concentration of sucrose is required to provide an equivalent amount of protein protection[ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS; Sola-Penna,M; 360(1):10-14 (1998)]. 1.5Molar solutions of trehalose & sucrose contain 62.5% & 87% water by volume, respectively. The greater hydrated volume of trehalose reduces freezable water and increases viscosity[PROTEIN SCIENCE; Jain,NK; 18(1):24-36 (2009)]. Trehalose has been shown to be about twice as effective as sucrose in suppressing ice crystal growth, evidently due to the larger hydration radius[JOURNAL OF CRYSTAL GROWTH; Sei,T; 240(1-2):218-229 (2002)]. At 41.7wt% trehalose concentration crystal growth is about one quarter of what it is at 20.8wt%. At 52.1wt% trehalose crystal growth rate diminishes as crystal size increases.

Trehalose has a higher glass transition temperature (Tg) than sucrose or any other disaccharide studied. As a group, disaccharides have a Tg that is on average about 60C higher than monosaccharides[FRONTIERS IN BIOSCIENCE; Furuki,T; 14:3523-3535 (2009)]. At 5% water content, Tg for trehalose is about 40C whereas Tg for sucrose is about 15C. After being stored at 44C for 45days glucose/sucrose samples lost their amorphous state completely, whereas less than 4% of glucose/trehalose samples had crystallized[BIOCHEMCIA ET BIOPHYSICA ACTA; Sun,WQ; 1425(1):235-244 (1998)].

Disaccharides are examples of the class of cryoprotectants that do not cross cell membranes:non-penetrating cryoprotectants. Non-penetrating cryoprotectants are partly effective because ice forms much more readily outside of cells than inside cells due to the fact that nucleating agents are much more prevalent outside of cells than inside cells. In general, non-penetrating cryoprotectants are much less toxic than penetrating cryoprotectants. Many effective cryoprotectant cocktails combine non-penetrating cryoprotectants with penetrating cryoprotectants, thereby reducing the amount of penetrating cryoprotectant required. Non-penetrating cryoprotectants act partially by inducing cell dehydration, thereby reducing the amount of ice that can form in cells. For that reason, non-penetrating cryoprotectants are used in classical cryopreservation methods involving freezing.

Although the penetrating cryoprotectant glycerol is widely used for erythrocyte cryopreservation, efforts have been made to use the non-penetrating cryoprotectant hydroxyethyl starch(HES) because HES is a harmless plasma expander that would not need to be removed from erythrocytes after warming and prior to transfusion as is required with glycerol. Although one study concluded that the increased hemoglobin (increased hemolysis) associated with unwashed cryopreserved erythrocytes is not harmful[ANESTHESIA & ANALGESIA; Horn,E; 85(4):739-745 (1997)] concerns over possible renal toxicity from the increased hemoglobin have prevented clinical use of HES[TRANSFUSION MEDICINE REVIEWS; Scott,KL; 19(2):127-142 (2005)]. Erythrocytes preserved in liquid nitrogen with the nonpenetrating cryoprotectants trehalose and Dextran40 retained normal shape and enzyme activity, but had a 56% reduction in ATP[CRYOBIOLOGY; Pellerin-Mendes,C; 35(2):173-186 (1997)].

[For information on sugars in chilling & dehydration injury, see Viability, Cryoprotectant Toxicity and Chilling Injury in Cryonics.]

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Glycerol was the first CryoProtectant Agent (CPA) to gain widespread use in cryobiology, for cryopreserving red blood cells and sperm. The value of DMSO (DiMethylSulfOxide) as a CPA was discovered not long thereafter. Other polyols, such as ethylene glycol (automobile anti-freeze) and propylene glycol (formerly used to reduce ice formation in ice cream) were later shown to be effective cryoprotectants. Glycerol is still regarded as superior for cryopreserving spermatazoa from nearly all species, but lactamide results in motility for rabbit spermatazoa that is nearly double that seen for glycerol[JOURNAL OF REPRODUCTION AND DEVELOPMENT; Kashiwazaki,N; 52(4):511-516 (2006)].

Cryoprotectants are assessed by means of a number of parameters, including glass transition temperature (Tg), permeance, viscosity, toxicity, and concentration needed to vitrify (Cv). Cv is the minimum required concentration of the particular CPA which will vitrify, which is an important quantity to keep in mind because concentrations too much above this minimum result in increased toxicity without increased benefit.

Amides are weak cryoprotectants compared to polyols (formamide is too weak to vitrify on its own, but can assist vitrification by other cryoprotectants). Both amides and polyols become stronger cryoprotectants (have lower Cv) by the addition of methyl groups, as can be seen from the structures and Cvs of methylated amides and polyols [fromCRYOBIOLOGY; Fahy,GM; 24(3):196-213 (1987)].

A Nuclear Magnetic Resonance (NMR) study has indicated that methylation increases hydrogen bonding strength of the polar groups[JOURNAL OF PHYSICAL CHEMISTRY; Forsyth,M; 94:6889-6893 (1990)]. Compared to propylene glycol (1,2-propanediol), 1,3-propanediol has a higher concentration needed to vitrify (57% versus 44%) and a higher homogenous nucleation temperature for a 20%w/w solution (60C versus 68C), indicating that it is weaker and even less toxic than ethylene glycol in comparison with propylene glycol[CRYOBIOLOGY; MacFarlane,DR; 27:345-358 (1990)]. Much less inhibition of disaccharidases is seen with 1,3-propanediol compared to propylene glycol[BIOCHEMICAL MEDICINE AND METABOLIC BIOLOGY; 45(2):161-170 (1991)].

Although polyols have long been known to be good cryoprotectants, other CPAs [such as methoxlylated compounds ie, CPAs in which a methyl (-CH3) group is added to an alcohol to make an ether] now being considered are less toxic and more penetrating. Aside from osmotic effects, however, glycerol is probably the CPA which damages cell membranes the least, compared to DiMethyl SulfOxide(DMSO), ethylene glycol, propylene glycol or methoxylated compounds because these other compounds are more likely to have a dissolving effect. Glycerol is, however, biochemically toxic because kidney tissue cannot be subjected to more than 3-4 molar glycerol without loss of viability.

Methoxylated compound toxicity may be similar to ethylene glycol. Human sperm membrane, for example, is 4 times more permeable to ethylene glycol than to glycerol. And the membrane transport of ethylene glycol is less affected by temperature than is glycerol[HUMAN REPRODUCTION; Gilmore,JA; 12(1):112-118 (1997)]. Ethylene glycol is toxic at 38C due to metabolism to oxalic acid by alcohol dehydrogenase in the liver. The oxalic acid can precipitate as calcium oxalate crystals in the brain, heart, kidney, lung and pancreas causing hypocalcemia with the greatest damage being seen in the kidney (see PRINCIPLES OF INTERNAL MEDICINE by Harrison). Methoxylated compounds can have similar toxicity to that of an unesterified glycol (glycol=alcohol with two hydroxyl groups) like ethylene glycol because they are easily hydrolyzed. But glycol ethers can have other toxic effects, such as hemolysis and chromosome damage (see Casarett & Doull's TOXICOLOGY). It is doubtful, however, that many of these toxic effects would be seen during the application of a cryonics protocol with the blood being washed-out thereby preventing liver metabolites from reaching other cells. Moreover, ethylene glycol is of variable toxicity found to be nontoxic for cow embryos, for example[HUMAN REPRODUCTION; Gilmore,JA; 12(1):112-118 (1997)]].

Insofar as the cell membrane (plasmalemma) seems to be the cellular structure that is the most critical target in chilling and freezing injury, stabilization of cell membranes can be a significant aspect of cryoprotectant action. Proline, betaine, sarcosine, glycerol, DMSO, trehalose and sucrose all reduce membrane fusion. Proline, betaine and sarcosine stabilize phosphlipid bilayers by hydrophobic interactions. Hydrophobic interaction with membranes is a less effective means of membrane stabilization than the hydrogen bonding of the other cryoprotectants[CRYOBIOLOGY; Anchordoguy,TJ; 24(4):324-331 (1987)].

The major cryoprotectants can be listed with respect to vitrifying strength, toxicity and viscosity. A listing of cryoprotectants in order of glass-forming ability of 45%(v/v) solutions can be found in[CRYOBIOLOGY; Baudot;A; 40(2):151-158 (2000)]:

propylene glycol > DMSO > DMF > 1,4-butanediol > Ethylene glycol > glycerol > 1,3-propanediol

Studies by 21st Century Medicine (21CM) researchers on kidney slices have indicated that the relative order of toxicity matches the order of glass-forming ability. Formamide is exceptional, being the most toxic CPA while having the weakest glass-forming capability. Mixing CPAs reduces the toxicities of many of the individual agents.

Ordering CPAs by viscosity gives:

glycerol > Propylene glycol > Ethylene glycol > DMSO

A cryoprotectant mixture with high glass-forming ability, low toxicity and low viscosity is the elusive goal of vitrification research.

At the 2005 Society for Cryobiology Conference, 21st Century Medicine announced that it had successfully vitrified a rabbit kidney to solid state, rewarmed the kidney and transplanted it to a rabbit with complete viability[ORGANOGENESIS; Fahy,GM; 5(3):167-175 (2009)]. A rabbit brain has been vitrified with the same cryoprotectant mixture with no ice formation[ANNALS OF THE NEW YORK ACADEMY OF SCIENCES; 1019:559 (2004)]. In both cases the cryoprotectant mixture used was M22[CRYOBIOLOGY 48:157-178 (2004)], a 65%w/v (9.35M) cryoprotectant mixture having a melting temperature of 55C and a Tg of 123.3C. (M22 was so-named because of the intention to introduce this vitrification cocktail to a biological specimen at Minus 22C.) The vitrified kidney was held at 135C for 4minutes.

Most of the cryoprotectants in M22 are penetrating cryoprotectants (cross cell membranes), with the exception of PVPK12 and (although they are not technically cryoprotectants) ice blockers. The cryoprotectantive agents and ice blockers used in M22 are:

22% dimethyl sulfoxide 13% formamide 17% ethylene glycol 3% N-methylformamide 4% 3-methoxy-1,2-propanediol 3% PVP K12 2% Z-1000 ice blocker 1% X-1000 ice blocker

Cryoprotectants are not simply added to water, they are added to an isotonic carrier solution that often can act as an organ preservation solution. The carrier solution used for M22 is LM5, a mixture of glucose, mannitol, lactose, KCl, K2HPO2, GSH, NaHCO3, and adenineHCl[TABLE1; CRYOBIOLOGY; Fahy,GM; 48(2):157-178 (2004)]. Adding ice blockers and reducing the amount of glucose in a previous carrier solution cut the required warming rate to prevent devitrification by more that half. Choice of carrier solution can affect tissue recovery, and the composition of tissue fluid also has an effect[CRYOBIOLOGY; Wusterman,MC; 56(1):62-71 (2008)]. M22 was optimized for kidney slices.

The cryoprotectant solution used by the Cryonics Institute (CIVM1) has 35% ethylene glycol and 35% dimethyl sulfoxide, a pair of cryoprotectants that can reduce toxicity when used in combination[REPRODUCTION, FERTILITY, AND DEVELOPMENT; Gautam,SK; 20(4):490-496 (2008)]. The carrier solution is mRPS2, a mixture of glucose, KCl, HCl, and TRIS buffer. CIVM1 is a more powerful and much less expensive cryoprotectant mixture than M22, but it is more toxic. CIVM1 was optimized on hippocampal slices by cryobiologist Dr.Yuri Pichugin.

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Vitrification in Cryonics - BEN BEST

The year is 1967. A British secret agent has been "frozen," awaiting the day when his arch nemesis will return from his own deep freeze to once again threaten the world. That day finally arrives in 1997. The agent is revived after 30 years on ice, and he saves the world from imminent destruction.

You'll probably recognize this scenario from the hit movie, "Austin Powers: International Man of Mystery" (1997). Cryonics also shows up in films like "Vanilla Sky" (2001), "Sleeper" (1973) and "2001: A Space Odyssey" (1968). But is it pure Hollywood fiction, or can people really be frozen and then thawed to live on years later?

The science behind the idea does exist. It's called cryogenics -- the study of what happens to materials at really low temperatures. Cryonics -- the technique used to store human bodies at extremely low temperatures with the hope of one day reviving them -- is being performed today, but the technology is still in its infancy.

In this article, we'll look at the practice of cryonics, learn how it's done and find out whether humans really can be brought back from the deep freeze.

See original here:

How Cryonics Works | HowStuffWorks

Humans have been ingesting mindand mood-altering substances for millennia, but it has only rather recently become possible to begin to elucidate drug mechanisms of action and to use this information, along with our burgeoning knowledge of neuroscience, to design drugs intended to have a specific effect. And though most people think of pharmaceuticals as medicine, it has become increasingly popular to discuss the possibilities for the use of drugs in enhancement, or improvement of human form or functioning beyond what is necessary to sustain or restore good health (E.T. Juengst; in Parens, 1998, p 29).

Some (transhumansits) believe that enhancement may not only be possible, but that it may even be a moral duty. Others (bioconservatives) fear that enhancement may cause us to lose sight of what it means to be human altogether. It is not the intention of this article to advocate enhancement or to denounce it. Instead, lets review some of the drugs (and/or classes of drugs) that have been identified as the most promisingly cognitive- or mood-enhancing. Many of the drugs we will cover can be read about in further depth in Botox for the brain: enhancement of cognition, mood and pro-social behavior and blunting of unwanted memories (Jongh, R., et al., Neuroscience and Biobehavioral Reviews 32 (2008): 760-776).

Of most importance in considering potentially cognitive enhancer drugs is to keep in mind that, to date, no magic bullets appear to exist. That is, there are no drugs exhibiting such specificity as to have only the primary, desired effect. Indeed, a general principle of trade-offs (particularly in the form of side effects) appears to exist when it comes to drug administration for any purpose, whether treatment or enhancement. Such facts may constitute barriers to the practical use of pharmacological enhancers and should be taken into consideration when discussing the ethics of enhancement.

Some currently available cognitive enhancers include donepezil, modafinil, dopamine agonists, guanfacine, and methylphenidate. There are also efforts underway to develop memory-enhancing drugs, and we will discuss a few of the mechanisms by which they are proposed to act. Besides cognitive enhancement, the enhancement of mood and prosocial behavior in normal individuals are other types of enhancement that may be affected pharmacologically, most usually by antidepressants or oxytocin. Lets briefly cover the evidence for the efficacy of each of these in enhancing cognition and/or mood before embarking on a more general discussion of the general principles of enhancement and ethical concerns.

One of the most widely cited cognitive enhancement drugs is donepezil (Aricept), an acetylcholinesterase inhibitor. In 2002, Yesavage et al. reported the improved retention of training in healthy pilots tested in a flight simulator. In this study, after training in a flight simulator, half of the 18 subjects took 5 mg of donepezil for 30 days and the other half were given a placebo. The subjects returned to the lab to perform two test flights on day 30. The donepezil group was found to perform similarly to the initial test flight, while placebo group performance declined. These results were interpreted as an improvement in the ability to retain a practiced skill. Instead it seems possible that the better performance of the donepezil group could have been due to improved attention or working memory during the test flights on day 30.

Another experiment by Gron et al. (2005) looked at the effects of donepezil (5 mg/day for 30 days) on performance of healthy male subjects on a variety of neuropsychological tests probing attention, executive function, visual and verbal short-term and working memory, semantic memory, and verbal and visual episodic memory. They reported a selective enhancement of episodic memory performance, and suggested that the improved performance in Yesavage et al.s study is not due to enhanced visual attention, but to increased episodic memory performance.

Ultimately, there is scarce evidence that donepezil improves retention of training. Better designed experiments need to be conducted before we can come to any firm conclusions regarding its efficacy as a cognitive-enhancing.

The wake-promoting agent modafinil (Provigil) is another currently availabledrug that is purported to have cognitive enhancing effects. Provigil is indicated for the treatment of excessive daytime sleepiness and is often prescribed to those with narcolepsy, obstructive sleep apnea, and shift work sleep disorder. Its mechanisms of action are unclear, but it is supposed that modafinil increases hypothalamic histamine release, thereby promoting wakefulness by indirect activation of the histaminergic system. However, some suggest that modafinil works by inhibiting GABA release in the cerebral cortex.

In normal, healthy subjects, modafinil (100-200 mg) appears to be an effective countermeasure for sleep loss. In several studies, it sustained alertness and performance of sleep-deprived subjects(up to 54.5 hours) and has also been found to improve subjective attention and alertness, spatial planning, stop signal reaction time, digit-span and visual pattern recognition memory. However, at least one study (Randall et al., 2003) reported increased psychological anxiety and aggressive mood and failed to find an effect on more complex forms of memory, suggesting that modafinil enhances performance only in very specific, simple tasks.

The dopamine agonists d-amphetamine, bromocriptine, and pergolide have all been shown to improve cognition in healthy volunteers, specifically working memory and executive function. Historically, amphetamines have been used by the military during World War II and the Korean War, and more recently as a treatment for ADHD (Adderall). But usage statistics suggest that it is commonly used for enhancement by normal, healthy peopleparticularly college students.

Interestingly, the effect of dopaminergic augmentation appears to have an inverted U-relationship between endogenous dopamine levels and working memory performance. Several studies have provided evidence for this by demonstrating that individuals with a low workingmemory capacity benefit from greater improvements after taking a dopamine receptor agonist, while high-span subjects either do not benefit at all or show a decline in performance.

Guanfacine (Intuniv) is an 2 adrenoceptor agonist, also indicated for treatment of ADHD symptoms in children, but by increasing norepinephrine levels in the brain. In healthy subjects, guanfacine has been shown to improve visuospatial memory (Jakala et al., 1999a, Jakala et al., 1999b), but the beneficial effects were accompanied by sedative and hypotensive effects (i.e., side effects). Other studies have failed to replicate these cognitive enhancing effects, perhaps due to differences in dosages and/or subject selection.

Methylphenidate (Ritalin) is a well-known stimulant that works by blocking the reuptake of dopamine and norepinephrine. In healthy subjects, it has been found to enhance spatial workingmemory performance. Interestingly, as with dopamine agonists, an inverted U-relationship was seen, with subjects with lower baseline working memory capacity showing the greatest improvement after methylphenidate administration.

Future targets for enhancing cognition are generally focused on enhancing plasticity by targeting glutamate receptors (responsible for the induction of long-term potentiation) or by increasing CREB (known to strengthen synapses). Drugs targeting AMPA receptors, NMDA receptors, or the expression of CREB have all shown some promise in cognitive enhancement in animal studies, but little to no experiments have been carried out to determine effectiveness in normal, healthy humans.

Beyond cognitive enhancement, there is also the potentialfor enhancement of mood and pro-social behavior. Antidepressants are the first drugs that come to mind when discussing the pharmacological manipulation of mood, including selective serotonin reuptake inhibitors (SSRIs). Used for the treatment of mood disorders such as depression, SSRIs are not indicated for normal people of stable mood. However, some studies have shown that administration of SSRIs to healthy volunteers resulted in a general decrease of negative affect (such as sadness and anxiety) and an increase in social affiliation in a cooperative task. Such decreases in negative affect also appeared to induce a positive bias in information processing, resulting in decreased perception of fear and anger from facial expression cues.

Another potential use for pharmacological agents in otherwise healthy humans would be to blunt unwanted memories by preventing their consolidation.Thismay be accomplished by post-training disruption of noradrenergic transmission (as with -adrenergic receptor antagonist propranolol). Propranolol has been shown to impair the long-term memory of emotionally arousing stories (but not emotionally neutral stories) by blocking the enhancing effect of arousal on memory (Cahill et al., 1994). In a particularly interesting study making use of patients admitted to the emergency department, post-trauma administration of propranolol reduced physiologic responses during mental imagery of the event 3 months later (Pitman et al., 2002). Further investigations have supported the memory blunting effects of propranolol, possibly by blocking the reconsolidation of traumatic memories.

GENERAL PRINCIPLES

Reviewing these drugs and their effects leads us to some general principles of cognitive and mood enhancement. The first is that many drugs have an inverted U-shaped dose-response curve, where low doses improve and high doses impair performance.This is potentially problematic for the practical use of cognition enhancers in healthy individuals, especially when doses that are most effective in facilitating one behavior simultaneously exert null or detrimental effects on other behaviors.

Second, a drugs effect can be baseline dependent, where low-performing individuals experience greater benefit from the drug while higher-performing individuals do not see such benefits (which might simply reflect a ceiling effect), or may, in fact, see a deterioration in performance (which points to an inverted U-model).In the case of an inverted U-model, low performing individuals are found on the up slope of the inverted U and thus benefit from the drug, while high-performing individuals are located near the peak of the inverted U already and, in effect, experience an overdose of neurotransmitter that leads to a decline in performance.

Trade-offs exist in the realm of cognitive enhancing drugs as well. As mentioned, unwanted side effects are often experienced with drug administration, ranging from mild physiological symptoms such as sweating to more concerning issues like increased agitation, anxiety, and/or depression.

More specific trade-offs may come in the form of impairment of one cognitive abilityat the expense of improving another. Some examples of this include the enhancement of long-term memory but deterioration of working memory with the use of drugs that activate the cAMP/protein kinase A (PKA) signaling pathway. Another tradeoff could occur between the stability versus the flexibility of long-term memory, as in the case of certain cannabinoid receptor antagonists which appear to lead to more robust long-term memories, but which also disrupt the ability of new information to modify those memories. Similarly, a trade-off may exist between stability and flexibility of working memory. Obviously, pharmacological manipulations that increase cognitive stability at the cost of a decreased capacity to flexibly alter behavior are potentially problematic in that one generally does not wish to have difficulty in responding appropriately to change.

Lastly, there is a trade-off involving the relationship between cognition and mood. Many mood-enhancing drugs, such as alcohol and even antidepressants, impair cognitive functioning to varying degrees. Cognition-enhancing drugs may also impair emotional functions. Because cognition and emotion are intricately regulated through interconnected brain pathways, inducing change in one area may have effects in the other. Much more research remains to be performed to elucidate these interactions before we can come to any firm conclusions.

ETHICAL CONCERNS

Again, though it is not the place of this article to advocate or denounce the use of drugs for human enhancement, obviously there are considerable ethical concerns when discussing the administration of drugs to otherwise healthy human beings. First and foremost, safety is of paramount importance. The risks and side-effects, including physical and psychological dependence, as well as long-term effects of drug use should be considered and weighed heavily against any potential benefits.

Societal pressure to take cognitive enhancing drugs is another ethical concern, especially in light of the fact that many may not actually produce benefits to the degree desired or expected. In the same vein, the use of enhancers may give some a competitive advantage, thus leading to concerns regarding fairness and equality (as we already see in the case of physical performance-enhancing drugs such as steroids). Additionally, it may be necessary, but very difficult, to make a distinction between enhancement and therapy in order to define the proper goals of medicine, to determine health-care cost reimbursement, and to discriminate between morally right and morally problematic or suspicious interventions (Parens, 1998). Of particular importance will be determining how to deal with drugs that are already used off-label for enhancement. Should they be provided by physicians under certain conditions? Or should they be regulated in the private commercial domain?

There is an interesting argument that using enhancers might change ones authentic identitythat enhancing mood or behavior will lead to a personality that is not really ones own (i.e., inauthenticity), or even dehumanizationwhile others argue that such drugs can help users to become who the really are, thereby strengthening their identity and authenticity. Lastly, according to the Presidents Council on Bioethics, enhancement may threaten our sense of human dignity and what is naturally human (The Presidents Council, 2003). According to the Council, the use of memory blunters is morally problematic because it might cause a loss of empathy if we would habitually erase our negative experiences, and because it would violate a duty to remember and to bear witness of crimes and atrocities. On the other hand, many people believe that we are morally bound to transcend humans basicbiological limits and to control the human condition. But even they must ask: what is the meaning of trust and relationships if we are able to manipulate them?

These are all questions without easy answers. It may be some time yet before the ethical considerations of human cognitive and mood enhancement really come to a head, given the apparently limited benefits of currently available drugs. But we should not avoid dealing with these issues in the meantime; for there will come a day when significant enhancement, whether via drugs or technological means, will be possible and available. And though various factions may disagree about the morality of enhancement, one thing is for sure: we have a moral obligation to be prepared to handle the consequences of enhancement, both positive and negative.

Originally published as an article (in the Cooler Minds Prevail series) in Cryonics magazine, December, 2013

See the article here:

The Institute for Evidence-Based Cryonics

by Ben Best CONTENTS: LINKS TO SECTIONS BY TOPIC

Preparing a cryonics patient for cryostorage can involve three distinct stages of alteration of body fluids:

(1) patient cooldown/cardiopulmonary support

(2) blood washout/replacement for patient transport

(3) cryoprotectant perfusion

During patient cooldown/cardiopulmonary support, a cryonics emergency response team or health care personnel may inject a number of medicaments to minimize ischemic injury and facilitate cryopreservation. The first and most important of these medicaments would be heparin, to prevent blood clotting. (For more details on the initial cooldown process, see Emergency Preparedness for a Local Cryonics Group).

Once the patient is cooled, the blood can be washed-out and replaced with a solution intended to keep organs/tissues alive while the patient is being transported to a cryonics facility. At the cryonics facility the organ/tissue preservation solution is replaced with the cryopreservation solution intended to prevent ice formation when the patient is further cooled to temperatures of 120C (glass transition temperature) or 196C (liquid nitrogen temperature) for long-term storage.

For both organ/tissue preservation & cryoprotection it is necessary to replace the fluid contents of blood vessels & tissue cells with other fluids. The process of injecting & circulating fluids through blood vessels is called perfusion. The passive process by which fluids enter & exit both blood vessels & cells is called diffusion.

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Body fluids can be described as solutes dissolved in a solvent, where the solvent is water and the solutes are substances like sodium chloride (NaCl, table salt), glucose or protein. Both water and solute molecules tend to move randomly in fluid with energy and velocity that is directly proportional to temperature. When there is a difference in concentration between water or solute molecules in one area of the fluid compartment compared to the rest of the compartment, random motion of the molecules will eventually result in a uniform distribution of all types of molecules throughout the compartment. In thermodynamics this is termed a decrease in potential energy (Gibbs free energy, not heat energy) due to an increase in entropy at constant temperature leading to equilibrium.

The movement of molecules from an area of high concentration to an area of low concentration is called diffusion. The rate of diffusion (J) can be quantified by Fick's law of diffusion:

dc J = DA---- dx J = rate of diffusion (moles/time) D = Diffusion coefficient A = Area across which diffusion occurs dc/dx = concentration gradient (instantaneous concentration difference divided by instantaneous distance)

Fick's First Law states that the rate of diffusion down a concentration gradient is proportional to the instantaneous magnitude of the concentration gradient (which changes as diffusion proceeds). For movement of molecules from a region of higher concentration to a region of lower concentration dc/dx will be negative, so multiplying by DA gives a positive value to J. Diffusion coefficient is higher for higher temperature and for smaller molecules.

Diffusion can occur not only within a fluid compartment, but across partitions that separate fluid compartments. The relevant partitions for animals are cell membranes and capillary walls. Cell membranes are lipid bilayers that allow for free diffusion of lipid soluble substances like oxygen, nitrogen, carbon dioxide and alcohol, while blocking movement of ions and polar molecules. But cell membranes also contain channels made of protein. Protein channels for water allow for very rapid diffusion of water across the membranes. Protein channels for potassium(K+), sodium(Na+) and other ions allow for more restricted diffusion across cell membranes. There is also facilitated diffusion (active transport) of many types of molecules across membranes.

For a normal 70kilogram (154pound) adult the total body fluid is about 60% of the body weight. Almost all of this fluid can be described as extracellular or intracellular (excluding only cerebrospinal fluid, synovial fluid and a few other small fluid compartments). Extracellular fluid can be further subdivided into plasma (noncellular part of blood) and interstitial fluid (fluid between cells that is not in blood vessels). Cell membranes separate intracellular fluid from extracellular fluid, whereas capillary walls separate plasma from interstitial fluid. The relative percentages of these fluids can be summarized as:

Intracellular fluid 67% Extracellular fluid Interstitial fluid 26% Plasma 7%

Note that blood volume includes both plasma & blood cells such that adding the intracellular fluid volume of blood cells to plasma volume makes blood 12% of total body fluid.

Osmosis refers to diffusion of water (solvent) across a membrane that is semi-permeable, ie, permeable to water, but not to all solutes in the solution. If membrane-impermeable solutes are added to one side of the membrane, but not to the other side, water will be less concentrated on the solute side of the membrane. This concentration gradient will cause water to diffuse across the semi-permeable membrane into the side with the solutes unless pressure is applied to prevent the diffusion of water. The amount of pressure required to prevent any diffusion of water across the semi-permeable membrane is called the osmotic pressure of the solution with respect to the membrane.

Osmotic pressure (like vapor pressure lowering and freezing-point depression) is a colligative property, meaning that the number of particles in solution is more important than the type of particles. One molecule of albumin (molecular weight 70,000) contributes as much to osmotic pressure as one molecule of glucose or one sodium ion. At equilibrium all molecules in a solution have achieved the same average kinetic energy, meaning that molecules with a smaller mass have higher average velocity. Thus, a one molar solution of NaCl will result in twice the osmotic pressure as a one molar solution solution of glucose because Na+ and Cl ions exert osmotic pressure as independent particles.

Solute concentrations are generally expressed in terms of molarity (moles of solute per liter of solution). The osmolarity of a solution is the product of the molarity of the solute and the number of dissolved particles produced by the solute. A one molar (1.0M, one mole per liter) solution of CaCl2 is a three osmolar (three osmoles per liter) solution because of the Ca2+ ion plus the two Cl ions produced when CaCl2 is added to water. Osmolarity, the number of solute particles per liter has been mostly replaced in practice by osmolality, the number of solute particles per kilogram. (For dilute solutions the values of the two are very close.) For describing solute concentrations in body fluids it is more convenient to use thousandths of osmoles, milli-osmoles (mOsm). Total solute osmolality of intracellular fluid, interstitial fluid or plasma is roughly 300mOsm/kgH2O. About half of the osmolality of intracellular fluid is due to potassium ions and associated anions, whereas about 80% of the osmolality of interstitial fluid and plasma is due to sodium and chloride ions.

As stated above, both osmotic pressure and freezing point depresssion are colligative properties. All colligative properties are convertible. One osmole of any solute will lower the freezi
ng point of water by 1.858C. For this reason, a 0.9% NaCl solution is 0.154molar or about 308mOsm/kgH2O, and will lower the freezing point of water by about 0.572C.

The osmolality of a solution is an absolute quantity that can be calculated or measured. The tonicity of a solution is a relative concept that is associated with osmotic pressure and the ability of solutes to cross a semi-permeable membrane. Thus, tonicitiy of a solution is relative to the particular solutes and relative to a particular membrane specifically relative to whether the solutes do or do not cross the membrane. Cell membranes are the membranes of greatest biological significance. Whether a cell shrinks or swells in a solution is determined by the tonicity of the solution, not necessarily the osmolality. Only when all the solutes do not cross the semi-permeable membrane does osmolality provide a quantitative measure of tonicity. It is common to speak as if tonicity and osmolality are equivalent because body fluid solutes are often impermeable. Each mOsm/kgH2O of fluid contributes about 19mmHg to the osmotic pressure.

A solution is said to be isotonic if cells neither shrink nor swell in that solution. Both 0.9%NaCl (physiological saline) and 5%glucose (in the absence of insulin) are isotonic solutions (roughly 300mOsm/kgH2O of impermeable solute). (In the presence of insulin, 5%glucose is a hypotonic solution because insulin causes glucose to cross cell membranes.) Hypertonic solutions cause cells to shrink as water rushes out of cells into the solute, whereas cells placed in hypotonic solutions cause the cells to swell as water from the solution rushes into the cells.

An exact calculation of the osmolality of plasma gives 308mOsm/kgH2O, but the freezing point depression of plasma (0.54C) indicates an osmolality of 286mOsm/kgH2O. Interaction of ions reduces the effective osmolality. Sodium ions (Na+) and accompanying anions (mostly Cl & HCO3) account for all but about 20mOsm/kgH2O of plasma osmolality. Plasma sodium concentration is normally controlled by plasma water content (thirst, etc.)[BMJ; Reynolds,RM; 332:702-705 (2006)]. Normal serum Na+ concentration is in the 135 to 145millimole per liter range, with 135mmol/L being the threshold for hyponatremia. Intracellular sodium concentration is typically about 20mmol/L about one-seventh the extracellular concentration. Glucose and urea account for about 5mOsm/kgH2O. Osmolality of plasma is generally approximated by doubling the sodium ions (to include all associated anions), adding this to glucose & urea molecules, and ignoring all other molecules as being negligible. Protein contributes to less than 1% of the osmolality of plasma. (Cells contain about four times the concentration of proteins as plasma contains.)

Although ethanol increases the osmolality of a solution, it does not increase the tonicity because (like water) ethanol crosses cell membranes. A clinical hyperosmolar state without hypertonicity can occur with an increase in extracellular ethanol (which diffuses into cells)[ MINERVA ANESTESIOLOGICA; Offenstadt,G; 72(6):353-356 (2006)]. Glycerol also readily crosses cell membranes, but it does so thousands of times more slowly than water which means that glycerol is "transiently hypertonic" (only isotonic at equilibrium). Ethylene glycol crosses red blood cell membranes about six times faster than glycerol (and sperm cell membranes four times faster than glycerol). Actually. even for water there is a finite time for hydraulic conductivity across cell membranes.

Cells placed in a "transiently hypertonic" solution (containing solutes that slowly cross a membrane) will initially shrink rapidly as water leaves the cell, and gradually re-swell as the solute slowly enters the cell (the "shrink/swell cycle"). As shown in the diagram for mouse oocytes at 10C, water leaves the cell in the first 100seconds, whereas 1.5Molar ethylene glycol (black squares) or DMSO (DiMethylSulfOxide, white squares) take 1,750seconds to restore the volume to 85% of the original cell volume[CRYOBIOLOGY; Paynter,SJ; 38:169-176 (1999)]. Even if a cell does not burst or collapse due to osmotic imbalance, a sudden change in osmotic balance can injure cells. Nonetheless, cells are somewhat tolerant of hypotonic solutions. Granulocytes are particularly sensitive to osmotic stress, but granulocyte survival is not significantly affected by hypertonic solutions until the osmolality of impermeant solutes approaches twice physiological values (about 600mOsm/kgH2O)[AMERICAN JOURNAL OF PHYSIOLOGY; Armitage WJ; 247(5Pt1):C373-381 (1984)].

PC3 cells show almost no decline of survival upon exposure to 5,000mOsm/kgH2O NaCl for 60minutes at 0C, and show nearly 85% cell survival on rehydration. Nearly 85% survive 9,000mOsm/kgH2O NaCl for 60minutes at 0C, but less than 20% survive rehydration. But although at 23C most cells survive exposure to 5,000mOsm/kgH2O NaCl for 60minutes, only about a third of cells survive rehydration. At 23C and 9,000mOsm/kgH2O NaCl only about half of cells survive 60 minutes and no cells survive rehydration, indicating the protective effect of low temperature against osmotic stress. Water flux at 23C was the same for 9,000mOsm/kgH2O as for 5,000mOsm/kgH2O, and hypertonic cell survival was not affected by the rate of concentration increase[CRYOBIOLOGY; Zawlodzka,S; 50(1):58-70 (2005)].

Hyperosmotic stress damages not only cell membranes, but damages cytoskeleton, inhibits DNA replication & translation, depolarizes mitochondria, and causes damage to DNA & protein. Heat shock proteins and organic osmolytes (like sorbitol & taurine) are synthesized as protection against hyperosmotic stress. Highly proliferative cells (like PC3) suffer from osmotic stress more than less proliferative cells because the latter can mobilize cellular defenses more readily due to fewer cells undergoing mitosis at the time of osmotic stress[PHYSIOLOGICAL REVIEWS; Burg,MB; 87(4):1441-1474 (2007)].

An important distinction to remember in replacing body fluids is the distinction between two kinds of swelling (edema): cell swelling and tissue swelling. Cell swelling occurs when there is a lower concentration of dissolved membrane-impermeable solutes outside cells than inside cells. To prevent either shrinkage or swelling of a cell there must be an osmotic balance of molecules & ions between the liquids outside the cell & inside the cell. Capillary walls are semipermeable membranes that are permeable to most of the small molecules & ions that will not cross cell membranes, but are impermeant to large molecules referred to as colloid (proteins). The colloid osmotic pressure on capillary walls due to proteins is called oncotic pressure. For normal human plasma oncotic pressure is about 28mmHg, 9mmHg of which is due to the Donnan effect which causes small anions to diffuse more readily than small cations because the small cations are attracted-to (but not bound-to) the anionic proteins. About 60% of total plasma protein is albumin (30 to 50 grams per liter), the rest being globulins. But albumin accounts for 75-80% of total intravascular oncotic pressure. Tissue swelling occurs when fluids leak out of blood vessels into the interstitial space (the space between cells in tissues). Injury to blood vessels can result in tissue swelling, but tissue swelling can also result from water leaking out of vessels when there is nothing (like albumin) to prevent the leakage.

Both forms of edema (cell & tissue swelling) can impede perfusion considerably, and is frequently a problem in cryonics patients who have suffered ischemic or other forms of blood vessel damage. Maintain
ing osmotic balance of the fluids outside & inside cells is as important as maintaining oncotic balance, ie, balance of fluids inside & outside of blood vessels.

Much of the isotonicity of the intracellular and extracellular fluids is maintained by the sodium pump in cell membranes, which exports 3sodium ions for every 2potassium ions imported into cells. Proteins in cells are more osmotically active than interstitial fluid proteins. Because of the Donnan effect the sodium pump is required to prevent cell swelling. When ischemia deprives the sodium pump of energy, cells swell from excessive intracellular sodium (because sodium attracts water more than potassium does) resulting in edema. Inflammation can also cause cell swelling due to increased membrane permeability to sodium and other ions. Interstitial edema can occur when ischemia or inflammation increases capillary permeability leading to leakage of larger plasma solutes into the interstitial space.

[For further details on the sodium pump see MEMBRANE POTENTIAL, K/Na-RATIOS AND VIABILITY]

Near the hypothalamus of the brain are osmoreceptors (outside the blood-brain barrier) that monitor blood osmolality, which is normally in the range of 280-295mOsm/kgH2O. A 2% increase in plasma osmotic pressure can provoke thirst. An increase in plasma osmolality can indicate excessive loss of blood volume. To compensate, the posterior pituitary (neurohypophysis) secretes the hormone 8arginine vasopressin (AVP), which is two hormones in one hence the two names vasopressin and anti-diuretic hormone. AVP action on the V1 receptors on blood vessels causes vasoconstriction (vasopressin). AVP action on the V2 receptors of the kidney causes water retention (anti-diuretic hormone). Deficiency in AVP secretion can lead to diabetes incipidus, so called because the excessively excreted urine is tasteless (incipid), in contrast to the sweet (glucose-laden) urine of diabetes mellitus. Cortisol opposes AVP action on excretion, leading to dehydration and excessive urination of fluid. Reduced blood flow to the kidney stimulates release of renin, which catalyzes the production of angiotensin. Like AVP, angiotensin causes vasoconstriction and kidney fluid retention.

Rats subjected to experimental focal ischemia have shown reduced edema when treated with an AVP antagonist[STROKE; Shuaib,A; 33(12):3033-3037 (2002)]. Hypertonic saline(7.5%) has been shown to halve plasma AVP levels in experimental rats, whereas mannitol(20%) had no effect[JOURNAL OF APPLIED PHYSIOLOGY; Chang,Y; 100(5):1445-1451 (2006)]. Increases in plasma osmolality due to urea or glycerol have no effect on plasma AVP levels[JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY; Verbalis,JG; 18(12):3056-3059 (2007)]. The effect of hypertonic saline on osmotic edema due to AVP in a cryonics patient would likely be negligible because of negligible hormone release and transport. So some of the advantage of hypertonic saline over mannitol seen in clinical trials would not occur in cryonics cases.

The net movement of fluid across capillary membranes due to hydrostatic and oncotic forces can be described by the Starling equation. The Starling equation gives net fluid flow across capillary walls as a result of the excess of capillary hydrostatic pressure over interstitial fluid hydrostatic pressure, and capillary oncotic pressure over interstitial fluid oncotic pressure modified by the water permeability of the capillary. For a normal (animate) person, the hydrostatic pressure (blood pressure) at the arterial end of a capillary is about 35mmHg. The hydrostatic pressure drops in a linear fashion across the length of the capillary until it is about 15mmHg at the venule end. The net oncotic pressure within the capillary is about 25mmHg across the entire length of the capillary. Thus, for the first half of the capillary there is a net loss of fluid into the interstitial space until the hydrostatic pressure has dropped to 25mmHg. For the second half of the capillary there is a net gain of fluid into the capillary from the interstitial space. The flow of fluid into the interstitial space in the first half of the capillary is associated with the delivery of oxygen & nutrient to the tissues, whereas the flow of fluid from the interstitial space into the second half of the capillary is associated with the removal of carbon dioxide and other waste products.

Actually, there is a tiny (tiny relative to the total diffusion back and forth across the capillary wall) net flow of fluid from the capillaries to the interstitial fluid which is returned to the blood vessels by the lymphatic system. The lymphatic vessels contain one-way valves and rely on skeletal muscle movement to propel the lymphatic fluid. Infectious blockage of lymph flow can produce edema. A person sitting for long periods (as during a long trip) or standing a long time without moving may experience swollen ankles due to the lack of muscle activity. Swollen ankles is also a frequent symptom of the edema resulting from congestive heart failure. Venous pressure is elevated by the reduced ability of the heart to pull blood from the venous system, whereas vasoconstriction can better compensate to maintain pressure on the arterial side. Reduced albumin production by the liver as a result of cirrhosis or other liver diseases can reduce plasma osmolality such that the reduced oncotic pressure results in edema typically swollen ankles, pulmonary edema and abdomenal edema (ascites).

The Starling forces are different for the blood-brain barrier (BBB) than they are for other capillaries of the body because of the reduced permeability to water (lower hydraulic conductivity) and the greatly reduced permeability to electrolytes. The osmotic pressure of the plasma and interstitial fluid effectively become the oncotic pressures.

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A critical distinction is made in fluid mechanics between laminar flow and turbulent flow in a pipe. For laminar flow elements of a liquid follow straight streamlines, where the velocity of a streamline is highest in the center of the vessel and slowest close to the walls. Turbulent flow is characterized by eddies & chaotic motion which can substantially increase resistance and reduce flow rate. The Reynolds number is an empirically determined dimensionless quantity which is used to predict whether flow will be laminar or turbulent with 2000 being the approximate lower limit for turbulent flow. Transient localized turbulence can be induced at a Reynolds number as low as 1600, but temporally peristant turbulence forms above 2040[SCIENCE; Eckhart,B; 333:165 (2011)].

Turbulent flow could potentially be a problem in cryonics if it reduced perfusion rate or increased the amount of pressure required to maintain a perfusion rate. It is doubtful that turbulent flow ever plays a role in cryonics perfusion, however. Even for a subject at body temperature (37C) Reynolds numbers in excess of 2000 are only seen in the very largest blood vessels: the aorta and the vena cava.

The formula for Reynolds number is: v D Re = ------ = fluid density (rho) v = fluid velocity D = vessel diameter = fluid viscosity

The fact that diameter (D) is in the numerator indicates that only high diameter vessels have high Reynolds number. Velocity (v), also in the numerator, is highest in the aorta & arteries. But the use of cryoprotectants and the increase in viscosity () with declining temperature essentially guarantee that turbulent flow will not occur in a cryonics patient.

More serious for cryonics is the Hagen-Poisseuille Law, which describes the
relationship between flow-rate and driving-pressure: pressure X (radius)4 Flow Rate = ---------------------- length X viscosity

Typically in cryonics the flow rate will be one or two liters per minute when the pressure is around 80mmHg. But because flow rate varies inversely with viscosity and varies directly with pressure, pressure must be increased to maintain flow rates when cryoprotectant viscosity increases with lowering temperature. This poses a serious problem because blood vessels become more fragile with lowering temperature. If blood vessels burst the perfusion can fail.

At 20C glycerol is about 25% more dense (=rho, in the numerator) than water. But the role of viscosity is far more dramatic, with high viscosity in the denominator reducing Reynolds number considerably. The viscosity of water approximately doubles from 37C to 10C, but the viscosity of glycerol increases by a factor of ten (roughly 4Poise to 40Poise). At 37C glycerol is nearly 600 times more viscous than water, but at 10C it is about 2,600 times more viscous.

Although turbulence is not a concern in cryonics, the increase in viscosity of cryoprotectant with lowering temperature certainly is. Fortunately, the newer vitrification mixtures are less viscous than glycerol.

The most common strategy in cryonics has been to cool the patient from 37C to 10C as rapidly as possible and to perfuse with cryoprotectant at 10C. Lowering body temperature reduces metabolism considerably, thereby lessening the amount of oxygen & nutrient required to keep tissues alive. Cryoprotectant toxicity drops as temperature declines. But the very dramatic more-than-exponential increase in cryoprotectant viscosity with lowering temperature poses a significant problem for effective perfusion. When open circuit perfusion is used, a higher temperature may be preferable because the opportunity for diffusion time into cells is so limited (about 2hours 1hour for the head, 1hour for the body) although ischemic damage is difficult to quantify.

With closed circuit perfusion, the perfusion times are longer up to 5hours. If a good carrier solution is used for the cryoprotectant the tissues may receive adequate nutrient. This, along with the oxygen carrying-capacity of water at low temperature, may limit ischemic damage while allowing time for cells to become fully loaded with cryoprotectant. If ischemic damage can be safely prevented in perfusion, the only critical issues for temperature selection are the relative benefits of reduced cryoprotectant toxicity at lower temperatures as against increased chilling injury. The fact that the more-than-exponential increase in viscosity with lowering temperature will increase perfusion time will not be problematic if the risk of ischemia is minimized.

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Typically a cryonics patient deanimates at a considerable distance from a cryonics facility and must be transported before cryoprotectants can be perfused. Blood could be washed-out and replaced with an isotonic (ie, osmotically the same as saline) solution, such as Ringer's solution. The patient is then transported to the cryonics facility at water-ice temperature. Freezing must be avoided because ice crystals would damage cells & blood vessels to such an extent as to prevent effective cryoprotectant perfusion. Water-ice temperature will not freeze tissues because tissues are salty (salt lowers the freezing point below 0C).

As body temperature approaches 10C, metabolic rate has slowed greatly and the oxygen-carrying capacity of blood hemoglobin is no longer required. Cool water, in fact, may carry adequate dissolved oxygen at low temperatures. (Water near freezing temperature can hold nearly three times as much dissolved oxygen as water near boiling temperature. Oxygen is about five times more soluble in water than nitrogen.) The tendency of blood to agglutinate and clog blood vessels becomes a serious problem at low temperature so the blood should be replaced if this does not cause other problems (such as delay and reperfusion injury.)

Replacing blood with a saline-like solution for patient transport, however, does not do a good job of maintaining tissue viability or preventing edema and would likely cause reperfusion injury. For this reason an organ preservation solution such as Viaspan, rather than Ringer's solution, has been used for cryopatient transport. Blood is not simply an isotonic solution carrying blood cells. Blood contains albumin, which attracts water and keeps the water from leaving blood vessels and going into tissues (maintains oncotic balance). Tissues which are swollen by water (edematous tissues) resist cryoprotectant perfusion. One of the most important ingredients in Viaspan preventing edema is HydroxyEthyl Starch (HES), which attracts water in much the way albumin attracts water acting as an oncotic agent by keeping water in the blood vessels. Viaspan contains potassium lactobionate to help maintain osmotic balance. Because HES is difficult to obtain and can cause microcirculatory disturbances, PolyEthylene Glycol (PEG) has been used in organ preservation solutions as a replacement for HES with good results[THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS; Faure,J; 302(3):861-870 (2002) and LIVER TRANSPLANTATION; Bessems,M; 11(11):1379-1388 (2005)].

The same benefit might not apply to cryonics patients, however, because of the prevalence of endothelial damage due to ischemia. Larger "holes" in the vasculature can mean that a larger molecular weight molecule is required for oncotic support. HES molecular weight is about 500,000, whereas the molecular weight for PEG used in organ replacement solutions is more like 20,000. Albumin (which has a molecular weight of about 70,000) provides most of the oncotic support in normal physiology. A PEG with molecular weight of 500,000 would be far too viscous and will form a gel. HES has the benefit of being large enough to always provide oncotic support while being much less viscous than PEG of equivalent molecular weight.

Viaspan (DuPont Merck Pharmaceuticals) contains other ingredients to maintain tissue viability, such as glucose, glutathione, etc. (the full formula can be found on the Viaspan website). Viaspan is FDA approved for preservation of liver, kidney & pancreas, but is used off-label for heart & lung transplants. Viaspan is being challenged in the marketplace for all these applications by the Hypothermosol (Cryomedical Sciences, BioLife Technologies) line of preservation solutions.

Rather than use these expensive commercial products, Alcor and Suspended Animation, Inc. use a preservation solution developed by Jerry Leaf & Mike Darwin called MHP-2. MHP-2 is so-called because it is a Perfusate (P) which contains mannitol (M) as an extracellular osmotic agent and HEPES (H), a buffer to prevent acidosis which is effective at low temperature. MHP-2 also contains ingredients to maintain tissue viability and hydroxyethyl starch as an oncotic agent to prevent edema. Lactobionate permeates cells less than mannitol and can thus maintain osmotic balance for longer periods of time, but mannitol is much less expensive. Mannitol also has an additional effect in the brain. Because of the unique tightness of brain capillary endothelial cell junctions ("blood brain barrier"), little mannitol leaves blood vessels to pass into the brain. This means that mannitol can act like an oncotic agent for the brain. If the blood brain barrier is intact, mannitol will suck water out of the extravascular space. The brain is the only place that mannitol can do this, and that is why a mannitol is eff
ective for inhibiting edema of the brain but only if there is not extensive ischemic damage to the blood brain barrier. (Mannitol has yet another benefit in that it scavenges hydroxyl radical [CHEM.-BIOL. INTERACTIONS 72:229-255 (1989)]).

(For the formula of MHP-2 see TableII of CryoMsg4474 or TableVII of CryoMsg2874 which also contains the formula for Viaspan in TableV.)

The initial perfusate can also contain other ingredients to assist in reducing damage to the cryonics patient. Anticoagulants can reduce clotting problems, and antibiotics can reduce bacterial damage. Damaging effects of ischemia can be reduced with antioxidants, antiacidifiers, an iron chelator and a calcium channel blocker.

Both Alcor and Suspended Animation, Inc. use an Air Transportable Perfusion(ATP) system of equipment which allows them to do blood washout in locations remote from any cryonics facilty by using equipment that can easily be carried on an airplane. There is a video demonstration of an ATP on YouTube.

[For further details on organ preservation solution see ORGAN TRANSPLANTATION SOLUTION]

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Cryoprotectants are used in cryonics to reduce freezing damage by prevention of ice formation (see Vitrification in Cryonics ). Cells are much more permeable to water than they are to cryoprotectant. Platelets & granulocytes, for example, are 4,000 times more permeable to water than they are to glycerol[CRYOBIOLOGY; Armitage,WJ; 23(2):116-125 (1986)]. When a cell is exposed to high-strength cryoprotectant, osmosis causes water to rush out of the cells, causing the cells to shrink. Only very gradually does the cryoprotectant cross cell membranes to enter the cell (the "shrink/swell cycle"). For isolated cells, the halftime (time to halve the difference between a given glycerol concentration in a granulocyte and the maximum possible concentration) is 1.3minutes[EXPERIMENTAL HEMATOLOGY; Dooley,DC; 10(5):423-434 (1982)] but tissues & organs would require more time because their cells are less accessible. Even after equilibration, however, the concentration of glycerol inside neutrophilic granulocytes never rises above 78% of the concentration outside the cells.

As shown in the diagram for mature human oocytes placed in a 1.5molar DMSO solution, the shrink/swell cycle is highly temperature dependent, happening with slower speed of recovery and with greater volume change at lower temperatures[HUMAN REPRODUCTION; Paynter,SJ; 14(9):2338-2342 (1999)]. This creates tough choices in cryonics, because cryoprotectants are more toxic at higher temperatures.

Proliferation of cultured kidney cells declines linearly with increasing osmolality due to urea & NaCl above 300mOsm/kgH2O, but the effect of added glycerol on cell growth is much less[AMERICAN JOURNAL OF PHYSIOLOGY; Michea,L; 278(2):F209-F218 (2000)]. Kidney cells which invivo can tolerate osmolalities of around 300mOsm/kgH2O do not survive over 300mOsm/kgH2O invitro, possibly because of more rapid proliferation[PHYSIOLOGICAL REVIEWS; Burg,MB; 87(4):1441-1474 (2007)].

Cells subjected to high levels of cryoprotectants can be damaged by osmotic stress. Quantifying osmotic damage has been a challenge for experimentalists who must distinguish between electrolyte damage, cryoprotectant toxicity, cell volume effects and osmotic stress. Concerning the last two, osmotic damage due to cell shrinkage may be distinguished from osmotic damage as a result of the speed at which the cryoprotectant crosses the cell membrane, ie, by the membrane permeability to the cryoprotectant. Cryoprotectants with lower permeabilities can cause more osmotic stress than cryoprotectants with high permeability.

Membrane permeabilities of a variety of nonelectrolytes (including cryoprotectants) have been studied on a number of cell types, including human blood cells[THE JOURNAL OF GENERAL PHYSIOLOGY; Naccache,P; 62(6):714-736 (1973)]. Critical factors determining membrane permeability are lipid solubility of the substance (which increases permeability) and hydrogen bonding (which decreases permeability). In general, permeability decreases as the molecular size of the substance increases. In contrast to blood cells, human sperm is more than three times more permeable to glycerol than to DMSO[BIOLOGY OF REPRODUCTION; Gilmore,JA; 53(5):985-995 (1995)]. For both blood cells and sperm cells permeability to ethylene glycol is very high compared to the other common cryoprotectants. Yet for mature human oocytes propylene glycol has the highest permeability and ethylene glycol has the lowest permeability of the most commonly used oocyte cryoprotectants[HUMAN REPRODUCTION; Van den Abbeel,E; 22(7):1959-1972 (2007)]. In contrast to human oocytes, however, for mouse oocytes ethylene glycol(EG) permeability is comparable to that of DMSO, propylene glycol(PG), and acetamide(AA), but not glycerol(Gly)[JOURNAL OF REPRODUCTION AND DEVELOPMENT; Pedro,PB; 51(2):235-246 (2005)].

Water and cryoprotectants both cross cell membranes more slowly at lower temperatures. Cryoprotectants slow the passage of water across cell membranes. Glycerol, DMSO and ethylene glycol all reduce the rate at which water crosses human sperm cell membranes by more than half[BIOLOGY OF REPRODUCTION; Gilmore,JA; 53(5):985-995 (1995)].

Aside from the choice of cryoprotectants, a major concern is the way cryoprotectant is administered. For example, glycerol (the standard cryoprotectant used in cryonics for many years) can either be administered full-strength or it can be introduced in gradually increasing concentrations. Under optimum conditions, glycerol results in 80% vitrification and 20% ice formation. Glycerol has been replaced by better cryoprotectants that can vitrify without any ice formation, but I will typically use glycerol as my example cryoprotectant. A patient should probably not be perfused with a 100% solution of glycerol or other cryoprotectant because of the possibility of osmotic damage. It is prudent to begin perfusion with low concentrations of cryoprotectant because water can diffuse out of cells thousands of times more rapidly than cryoprotectant diffuses into cells. Using gradually increasing concentrations of cryoprotectant (ramping) prevents the osmotic damage this differential could cause.

Human granulocytes (which are more vulnerable to osmotic stress or shrinkage than most other cell types) can experience up to 600mOsm/kgH2O hypertonic solution (which shrinks cells to 68% of normal cell volume) for 5minutes at 0C with no more than 10% of the cells losing membrane integrity. But at about 750mOsm/kgH2O (NaCl) or 950mOsm/kgH2O (sucrose) less than half of granulocytes display intact membranes when returned to isotonic solution. Nonetheless, the cells did not display lysis if retained in hyperosmotic medium. In fact, granulocytes could tolerate up to 1400mOsm/kgH2O if not subsequently diluted to less than 600mOsm/kgH2O[AMERICAN JOURNAL OF PHYSIOLOGY; Armitage WJ; 247(5Pt1):C373-381 (1984)]. A subsequent confirming study showed that rehydration of PC3 cells shrunken by NaCl solution creates more osmotic damage than the initial dehydration[CRYOBIOLOGY; Zawlodzka,S; 50(1):58-70 (2005)]. Cell survival after rehydration was higher at 0C than at 23C.

Although toxic effects of 2M (17%w/w) glycerol on granulocytes are quite evident at 22C, almost no toxic effect is seen at 0C[CRYOBIOLOGY; Frim,J; 20(6):657-676 (1983)]. For no mammalian cells other than granulocytes is 2Molar glycerol toxic. Nonetheless, abrupt addition of only 0.5Molar glycerol at 0C resulted in only 40% of granulocytes surviving when slowly diluted to isotonic solution a
nd warmed to 37C. Only 20% of granulocytes survived this treatment when 1Molar or 2Molar glycerol were added (there was no difference in survival between the two concentrations). But if sucrose or NaCl was added to keep the granulocytes shrunken to 60% of normal cell volume, almost all granulocytes survived when incubated to 37C. Insofar as the transient shrinkage of granulocytes due to glycerol is not less than 85% of normal cell volume, it seems unlikely that cell shrinkage can account for the damage[AMERICAN JOURNAL OF PHYSIOLOGY; Armitage WJ; 247(5Pt1):C382-389 (1984)].

Human spermatazoa tolerate much higher osmolality than granulocytes. Sperm cells can experience up to 1000mOsm/kgH2O hypertonic solution for 5minutes at 0C with no more than 10% of the cells losing membrane integrity. At about 1500mOsm/kgH2O (NaCl, white circles) or 2500mOsm/kgH2O (sucrose, black circles) less than half of sperm cells display intact membranes when returned to isotonic conditions. But 80% of sperm cells showed intact cell membrane after exposure to 2500mOsm/kgH2O at 0C if maintained at hypertonicity rather than restored to isotonic solution (NaCl & sucrose, triangles). Sperm cells gradually returned to isotonic solution following exposure to 1.5Molar glycerol at 22C showed only 3% lysis, whereas 20% of sperm cells lysed if the return to isotonic was sudden. No lysis was seen for sperm not returned to isotonic medium. At nearly 5000mOsm/kgH2O glycerol (about 4.5Molar) 17% of sperm cells showed lysis (had loss of membrane integrity) at 0C and 10% had lysis at 8C if not returned to isotonic media[BIOLOGY OF REPRODUCTION; Gao,DY; 49(1):112-123 (1993)]. For cryonics purposes it would be best to maintain cells in a hypertonic condition to maximize potential viability during cryogenic storage.

Cells from mouse kidney (IMCD, Inner Medullary Collecting Duct) can be killed by NaCl or urea that is 700mOsm/kgH2O, but the death is apoptotic and takes up to 24hours. The IMCD cells can tolerate up to 900mOsm/kgH2O of urea and NaCl in combination because of activation of complementary cellular defenses (including heat-shock protein)[ AMERICAN JOURNAL OF PHYSIOLOGY; Santos,BC; 274(6):F1167-F1173 1998)].

Nearly half of mouse fibroblasts displayed cell membrane lysis after restoration to isotonicity following exposure to the equivalent of 3600mOsm/kgH2O of osmotic stress from rapid addition of 4Molar (30%w/w) DMSO at 0C. Few cells were damaged by slow addition of the DMSO[BIOPHYSICAL JOURNAL; Muldrew,K; 57(3):525-532 (1990)].

Human corneal epithelial cells could tolerate 4.3M (37%w/w) glycerol with only 2% cell loss at 4C if the cells were subjected to gradually increasing (ramped) concentration (doubling osmolality in about 13minutes), but for stepped increases of 0.5M every 5minutes above 2M (17%w/w) to 3.5M (30%w/w) glycerol at 0C there was a 27% cell loss. For the same ramped method with DMSO there was a 6% cell loss at 2M (15%w/w) and a 15% cell loss at 3M (23%w/w). The same stepped method for DMSO resulted in a 1.5% cell loss for cells stepped from 2M to 3.5M (27%w/w) and a 22% cell loss for cells stepped from 2M to 4.3M (33%w/w). In all cases cell viability was assessed after washout and three days of incubation at 37C[CRYOBIOLOGY; Bourne,WM; 31(1):1-9 (1994)]. (Conversion of glycerol molarity to %w/w was approximated by multiplying by 8.6 and for DMSO was approximated by multiplying by 7.6)

In the context of cryonics it should be remembered that cells are not being returned to body temperature and need not be returned to isotonicity before cryoopreservation. There would be little time for apoptosis, and most cells would be far better preserved at low temperature and in hyperosmolar solution. Future technologies may be able to prevent apoptosis and have better methods for restoring irreplaceable cells to normal temperatures and osmolalities. For neurons, even abrupt stepped perfusion with cryoprotectant is likely to effectively result in ramped perfusion when allowances are made for the diffusion times required across blood vessels (blood brain barrier) and interstitial space. A more worrisome effect from the point of view of cryonic cryoprotectant perfusion is the effect of the cryoprotectants on vessel endothelial cells notably the effect on edema and vascular compliance.

Cell shrinkage may directly damage the cell (and cell membrane) due to structural resistance from the cell cytoskeleton and high compression of other cell constituents[HUMAN REPRODUCTION; Gao,DY; 10(5):1109-1122 (1995)]. Aside from membrane damage, other forms of cellular damage occur due to hypertonic environments, including cross-linking of intracellular proteins subsequent to cell dehydration. Bull sperm lose motility (often only temporarily) in a less hypertonic medium than one causing membrane damage[JOURNAL OF DAIRY SCIENCE; Liu,Z; 81(7):1868-1873 (1998)]. Osmotic stress can depress mitochondrial membrane potential in a manner that is mostly reversible after restoration to isotonic conditions[PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA); Desai,BN; 99(7):4319-4324 (2002)]. Human oocytes subjected to 600mOsm/kgH2O sucrose showed 44% of metaphaseII spindles having abnormalities[HUMAN REPRODUCTION; Mullen,SF; 19(5):1148-1154 (2004)]. Hypertonic solutions can trigger apoptosis[AMERICAN JOURNAL OF PHYSIOLOGY; Copp,J; 288(2):C403-C415 (2005)].

Despite these other types of damage due to hyperosmolality, the greatest risks in cryoprotectant perfusion in cryonics are those associated with membrane damage and edema due to cell swelling. The evidence that maintaining hypertonicity is more protective of cells than returning to isotonic conditions, and the desire to minimize edema during perfusion seem to make it advisable in cryonics to perfuse in hypertonic conditions.

(return to contents)

Once the patient is at the cryonics facility the transport solution can be replaced with a cryoprotectant solution. A perfusion temperature of 10C gives the best tradeoff of avoiding the high viscosity of lower temperatures and at the same time limiting the ischemic tissue degradation, chilling injury, and cryoprotectant toxicity that would be seen at higher temperatures. (Cryonicists usually worry more about ischemic damage than cryoprotectant toxicity due to a belief that ischemic damage has a greater likelihood of being irreversible irreparable by future molecular-repair technology.)

Cryoprotectants should be sterilized to prevent the growth of bacteria. Sterilization of cryoprotectants by heating can cause the formation of carbon-carbon double-bonds, which are evident by a yellowing of the cryoprotectant. Only a few such double-bonds can produce the yellow appearance, so the fact of yellowing is not evidence that the cryoprotectant is no longer serviceable. But a preferable method of cryoprotectant sterilization is filtration through a 0.2micron filter.

Rapid addition of cryoprotectant causes endothelial cells to shrink thereby breaking the junctions between the cells[CRYOBIOLOGY; Pollock,GA,; 23(6):500-511 (1986)]. On the other hand, endothelial cell shrinkage by hypertonic perfusate can increase capillary volume, thereby increasing blood flow as long as excessive vascular damage does not occur. Blood and clots are often observed to be dislodged during cryoprotectant perfusion in cryonics cases. For cryonics purposes some vascular damage may actually be an advantage insofar as it increases diffusion and vascular repair may be an easy task for future science. In fact, the breakdown of the blood-brain barrier in the 1.8-2.2 molar glycerol range is essential for perfusion of the brain as long as damag
ing tissue edema (swelling) can be avoided. Aquaporin (water channel) expression in the blood-brain barrier could be a safer means of allowing cryoprotectants into the brain[CRYOBIOLOGY; Yamaji,Y; 53(2):258-267 (2006)].

Closed-circuit perfusion (with perfusion solution following a circuit both inside & outside the patient's body) is contrasted with the open-circuit perfusion used by funeral directors for embalming. In the open-circuit perfusion of embalming, fluid is pumped into a large artery of the corpse and forces-out blood from a large vein and this blood is discarded.

A closed-circuit perfusion, as illustrated in the diagram, can be set up at low cost for gradual introduction of cryoprotectant into cryonics patients. As shown in the diagram, the perfusion circuit bypasses the heart. Perfusate enters the patient through a cannula in the femoral (leg) artery and exits from a cannula in the femoral vein on the same leg. Flowing upwards (opposite from the usual direction) from the femoral artery and up through the descending aorta, the perfusate enters the arch of the aorta (where blood normally exits the heart), but is blocked from entering the heart. Instead, the perfusate flows (in the usual direction) through the distribution arteries of the aorta, notably to the head and brain. Returning in the veins (in the usual direction), the perfusate nontheless again bypasses the heart and flows downward (opposite from the usual direction) to the femoral vein where it exits. A better alternative to the femoral circuit, however, is to surgically open the chest to cannulate the heart aorta (for input) and atrium (for output).

Although it is not shown in the diagram, there will be a pump in the circuit to maintain pressure and fluid movement. A roller pump, rather than an embalmer's pump, should be used. A roller pump achieves pumping action by the use of rollers on the exterior of flexible tubing that forces fluids through the tube without contaminating those fluids. Embalmer's pumps may use pressures much higher than those suitable for cryonics, resulting in blood vessel damage. Embalmer's pumps are also easily contaminated (and hard to clean), unless a filter is used. Contamination doesn't matter much in embalming, but in cryonics contaminants entering the patient through the pump can damage blood vessels, interfering with perfusion. If an embalmer's pump is used for cryonics purposes, ensure that the pressure can be lowered to a suitable level and that it is cleaned and sterilized. The main advantage of roller pumps, however, is the fact that they provide a closed circuit, whereas embalmer's pumps are open-circuit. Roller pumps are generally calibrated in litres per minute. Depending on the viscosity of the solution, a flow rate of 0.5 to 1.5litres per minute will be necessary to achieve the desired perfusion pressure of approximately 80mmHg to 120mmHg (physiological pressures).

Gaseous and particulate microemboli can produce ischemia in capillaries and arterioles. A study of patients having routine cardiopulmonary bypass surgery showed that 16% fewer patients had neuropsychological deficits eight weeks after the surgery when a 40micrometer arterial line filer had been used[STROKE; Pugsley,W; 25(7):1393-1399 (1994)]. Both roller pumps (peristaltic pumps) and centrifugal pumps can generate particles up to 25micrometers in diameter through spallation, although centrifugal pumps generate fewer particles[PERFUSION; Merkle,F; 18(suppl1):81-88 (2003)]. Filtration of perfusate with a 0.2micrometer filter prior to perfusion is a recommended way of removing potential microemboli, including bacteria. At room temperature 20micrometer diameter air bubbles take 1to6seconds to dissolve in water, although high flow rates and turbulence can increase microbubble formation[SEMINARS IN DIALYSIS; Barak,M; 21(3):232-238 (2008)]. De-airing of tubing before perfusion considerably reduces the possibility of microbubbles entering the patient[THE THORACIC AND CARDIOVASCULAR SURGEON; Stock,UA; 54(1):39-41 (2006)].

Mean Arterial Pressure (MAP) for an normal adult is regarded as being in the range of 50 to 150mmHg, and Cerebral Perfusion Pressure (CPP) is in the same range[BRITISH JOURNAL OF ANAETHESIA; Steiner,LA; 91(1):26-38 (2006)]. Vascular pressure normally drops to about 40mmHg in the arterioles, to below 30mmHg entering the capillaries, and is down to 3 to 6mmHg (Central Venous Pressure, CVP) when returning to the right atrium of the heart. Perfusing a cryonics patient at about 120mmHg should open capillaries adequately for good cryoprotectant tissue saturation without damaging fragile blood vessels.

Outside the patient, some of the drainage is discarded, but most is returned to a circulating (stirred) reservoir connected to a concentrated reservoir of cryoprotectant. The circulating reservoir is initially carrier solution which gradually becomes increasingly concentrated with cryoprotectant as the stirring and recirculation proceed. The circulating reservoir can be stirred from the bottom by a magnetic stir bar on a stir table and/or from the top by an eggbeater-type stirring device. The stirring will draw cryoprotectant from the cryoprotectant reservoir, and pumping of the perfusate should also actively draw liquid from the cryoprotectant reservoir. Gradually a higher and higher concentration of cryoprotectant is included in the perfusate and the osmotic shock of full-strength cryoprotectant is avoided.

The carrier solution for the cryoprotectant should perform similar tissue preservation functions as is performed by the transport solution, and should be carefully mixed with the cryoprotectant so as to avoid deviations from isotonicity which could result in dehydration or swelling & bursting of cells. The carrier solution will help keep cells alive during cryoprotectant perfusion.

An excellent carrier solution for cryonics purposes would be RPS-2 (Renal Preservation Solution number2), which was developed by Dr. Gregory Fahy in 1981 as a result of studies on kidney slices. More recently Dr. Fahy used RPS-2 as the carrier solution in cryopreserving hippocampal slices an indication that it is well-suited for brain tissue as well as for kidney. RPS-2 not only helps maintain hippocampal slice viability, it reduces the amount of cryoprotectant needed because it has cryoprotectant (colligative) properties of its own. The formulation of RPS-2 is: K2HPO4, 7.2mM; reduced glutathione, 5mM; adenine HCl, 1mM; dextrose, 180mM; KCl, 28.2mM; NaHCO3, 10mM; plus calcium & magnesium[CRYOBIOLOGY; Fahy,GM; 27(5):492-510 (1990)]. LM5 (Lactose-Mannitol5) is a carrier solution for use in vitrification solutions that include ice blockers. LM5 does not contain dextrose, which is believed to interfere with ice blockers.

The cryoprotectant reservoir will not in general contain pure cryoprotectant (although in principle it could), but rather a "terminal concentration" solution of cryoprotectant that is equal or slightly above the final target concentration. As perfusion proceeds and drainage to discard proceeds, the level of both reservoirs drops in tandem until both reservoirs are nearly empty, at which point the circuit concentration will have reached the cryoprotectant reservoir concentration. Provided that the two reservoirs are the same size and same vertical elevation, the gradient will be linear over time (if the drainage rate to discard was constant).

For cryoprotectant to perfuse into cells there must be constant exposure to cryoprotectant surrounding the cells and there must be pressure to maintain that exposure. In a living animal the heart maintains blood pressure that for
ces blood through the capillaries and forces nutrients into cells. A dead animal with no blood pressure and which is being perfused with cryoprotectant also requires pressure for the capillaries to remain open and for cryoprotectant to be maintained at high concentrations around cells.

Alcor found that closed-circuit perfusion must be maintained for 5-7 hours for full equilibration of glycerol, because the diffusion rate of water out of cells is thousands of times the rate at which glycerol enters cells. Of course, it would be possible to pump glycerol into a patient for 5-7 hours with open-circuit perfusion, but only by using thousands of dollars worth of glycerol. The newer vitrification cryoprotectants used by Alcor are vastly more expensive than glycerol. When using expensive cryoprotectants it makes far more sense to recirculate in a closed circuit. Closed-circuit perfusion also has the benefit of allowing for ongoing monitoring of physiological changes occurring in the patient's body during the perfusion process. Open-circuit with an inexpensive cryoprotectant has the advantage of avoiding recirculation of toxins.

Cryoprotectants, particularly glycerol, are viscous and cryoprotectants in high concentration are particularly viscous. The introduction of air bubbles into cryoprotectant solutions during pouring and mixing should be avoided because air emboli that enter the cryonics patient can block perfusion. Elimination of air bubbles from viscous cryoprotectant solutions is extremely difficult. Prevention is more effective than cure. Cryonicist Mike Darwin wrote about this problem and possible solutions in a 1994 CryoNet message.

Improper mixing of perfusate containing high levels of cryoprotectant can result in a phenomenon that appears to be high viscosity, but in reality is edema. If, for example, isotonic carrier solution is mixed half-and-half with cryoprotectant solution an open circuit perfusion may have to be halted when no further perfusate will go into the patient. The problem is caused not by viscosity, but by the fact that the isotonic solution became hypotonic due to dilution with cryoprotectant causing the cells to swell and forcing perfusion to end. In closed-circuit perfusion, the cryoprotectant concentrate reservoir contains cryoprotectant at about 125% the terminal concentration in a vehicle of isotonic carrier solution so that when reservoir concentrate is mixed with isotonic carrier there is no change in tonicity.

Newer cryoprotectants are less viscous than glycerol, so perfusions can be done in less time. After 15 minutes of perfusion with carrier solution, cryoprotectant concentration linearly increases at a rate of 50millimolar per minute until full concentration is reached in about two hours (a protocol developed on the basis of minimizing osmotic damage when perfusing kidneys). Perfusion is increased for an additional hour or two until the cryoprotectant has fully diffused into cells (as indicated by similarity of afflux and efflux cryoprotectant concentrations).

Only after a few hours of closed-circuit perfusion is the concentration of cryoprotectant exiting the cryonics patient equal to the concentration of cryoprotectant entering the patient. Only an extended period of sustained pressure will keep capillaries open, and otherwise facilitate diffusion of cryoprotectant into cells. And the exiting cryoprotectant concentration will equal the entering cryoprotectant concentration only when the tissues are fully loaded with cryoprotectant. A refractometer is used to verify that terminal cryoprotectant concentration has been reached in the brain.

(A refractometer measures the index of refraction of a liquid, ie, the ratio of the speed of light in the liquid and the speed of light in a vacuum (or air). Light changes speed when it strikes the boundary of two media, thus causing a change in angle if it strikes the new medium at an angle. Because the refractive index is a ratio of two quantities having the same units, it is unitless. Sodium vapor in an electric arc produces an excitation between the 3s and 3p orbitals resulting in yellow-orange light of 589nm what Joseph Fraunhofer called the "Dline". Insofar as the sodium "Dline" was the first convenient source of monochromatic light, it became the standard for refractometry. The refractive index of a liquid is thus a high-precision 5-digit number between 1.3000 and 1.7000 at a specific temperature, measured at the sodium Dline wavelength. For example, the refractive index of glycerol at 25C nD25 is 1.4730.)

Closed-circuit perfusion may be necessary for removal of water as well as loading of cryoprotectant if it is true that open-circuit perfusion cannot remove water effectively.

One could imagine that the additional time spent doing closed-circuit (rather than open-circuit) perfusion means increased damage due to above-zero temperature. But most cells are still alive and metabolizing very slowly at 10C. Viaspan, RPS-2 and other organ preservation solutions are designed to keep tissues alive for extended periods at near-zero temperatures certainly for the time required for closed-circuit perfusion. Ramping (slowly increasing concentration) of cryoprotectant should be done in such a way that the ion and mannitol or lactobionate concentration remains unchanged in the perfusate. Ramping is not an osmotically neutral process, however, because cryoprotectant is expected to dehydrate tissues.

Read more from the original source:
Perfusion & Diffusion in Cryonics Protocol - BEN BEST

Cryonics claims it can store a dead human body at low temperatures in such a way that it will be possible to revitalize that body and restore life at some unspecified future date. One hook the cryonics folks use is to give hope that a cure for a disease one dies of today will be found tomorrow, allowing that cure to be applied to the thawed body before or while bringing the dead person back to life. Cryonics might be called resurrection by technology and believers in it might be classified as suffering from the Moses syndrome. The simple fact is once you are dead, you are dead forever. This fact may seem horrifying, but it is not nearly as horrifying as the thought of living forever.

The technology exists to freeze or preserve people and that technology is improving and will probably get better. The technology to revivify a frozen body exists in the imagination. Nanotechnology, for example, is a technology that supporters of cryonics appeal to. Someday, they say, we'll be able to rebuild anything, including diseased or damaged cells in the body, with nanobots. So, no matter what disease destroyed healthy cells in the living body before preservation and no matter what damage was done to the cells of the frozen body during storage, nanotechnology will allow us to bring the dead back to life. This seems like wishful thinking. Nanotechnology might rebuild a mass of dead tissue into a mass of healthy tissue, but without a complete isomorphic model of the brain it will be impossible to return a mushy brain to the exact state it was in before death occurred. (Of course, since this is an exercise in imagination, one can posit that some day we will be able to preserve the brain without any decomposition or transformation at all.) In any case, some other jolt, probably electricity, will be needed to get the heart beating and the brain working again, assuming, of course, that the mush brain has been reconstructed into a healthy brain.

Some preserved by cryonics have the head severed from the body after death. Then, either the head alone is preserved, or both the head and the body are preserved separately. Maybe some future technology will allow the head to be attached to an artificial body. It can be imagined without contradiction, as the philosophers say, so it is not logically impossible that some day our planet will be inhabited by bodiless heads that are connected to machines that allow either actual or virtual experiences of any kind imaginable without requiring the head to leave the room. Of course, when that times comes medical science will have advanced to the point where the aging process can be reversed or maintained in stasis.

A business based on little more than hope for developments that can be imagined by science is quackery. (Cryonics should not be confused with cryogenics, which is a branch of physics that studies the effects of low temperatures on the structure of objects.) There is little reason to believe that the promises of cryonics will ever be fulfilled. Even if a dead body is somehow preserved for a century or two and then repaired, whatever is animated by whatever process will not be the same person who died. The brain is the key to consciousness and to who a person is. There is no reason to believe that a brain preserved by whatever means and restored to whatever state by nanobots will result in a consciousness that is in any way connected to the consciousness of the person who died two centuries earlier.

For those who want to live forever, cloning might be a more realistic possibility but I wouldn't bank on it. First, there is the aging problem. Even if cloning is successful, you won't be able to clone yourself as younger. Of course, you can hope that future technology will have solved the aging problem. Perhaps your body can be cloned repeatedly until science can assist you to overcome aging. However, there is no reason to believe that your clone would be a continuation of you. Your bodies might have identical looking cells, but the only way your minds could be identical is if you had no experience. (It is logically impossible for your bodies to have identical experiences since they occupy different spatial and temporal coordinates.) In that case, you would be as good as dead.

origin of cryonics

Teacher Robert Ettinger (physics and math) brought cryonics into the intellectual mainstream in 1964 with The Prospect of Immortality. Ettinger founded the Cryonics Institute and the related Immortalist Society. He got the idea for cryonics from a story by Neil R. Jones. "The Jameson Satellite" appeared in the July 1931 issue of Amazing Stories. It told the tale of

one Professor Jameson [who] had his corpse sent into earth orbit where (as the author mistakenly thought) it would remain preserved indefinitely at near absolute zero. And so it did, in the story, until millions of years later, when, with humanity extinct, a race of mechanical men with organic brains chanced upon it. They revived and repaired Jameson's brain, installed it in a mechanical body, and he became one of their company.*

Thus was born the idea that we could freeze our bodies, repair them at a later date, and bring them back to life when technology had advanced sufficiently to do the repairs and the reviving.

ethical & other issues

I will leave to others to discuss most of the ethical, legal, political, and economic issues of cryonics. I'll conclude with some comments about the cryonics case of Ted Williams.

Williams died in 2002 at the age of 83. According to his estranged daughter, Barbara Joyce (Bobby-Jo Ferrell) Williams, he left a will in which he expressed his desire to be cremated and have his ashes spread over his favorite fishing grounds in the Florida Keys. His son (Barbara Joyce's half-brother), John Henry Williams, arranged for Williams's body to be processed by Alcor LIfe Extension Foundation. A story in SportsIllustrated.com (SI) stated:

Hall of Famer Ted Williams' head and body are being stored in separate containers at an Arizona cryonics lab that is still trying to collect a $111,000 bill from Williams' son [he had already paid $25,000], according to a story by Tom Verducci in the latest issue of Sports Illustrated.

Alcor still has Williams's head in a canister and his body in a tank, both filled with liquid nitrogen (to keep the remains at a cool -321 degrees Fahrenheit). According to SI, Alcor representatives met with John Henry Williams, but not Ted Williams, about a year before Ted's death. Furthermore, SI reported that the Consent for Cryonic Suspension form submitted to Alcor after Williams had died had a blank line where his signature should have been.

There was a lawsuit by the estranged daughter that fizzled, allegedly for lack of funds, but no legal action by the authorities was taken against John Henry or Alcor. There is a movement still going to right this ship (see the Free Ted Williams website.) Larry Johnson, who worked briefly at Alcor, is leading the crusade to get Congress and a couple of state legislatures to regulate the cryonics industry and have Ted Williams cremated. A video interview with Johnson on "Good Morning America" discussing the disposition of Ted Williams's body at Alcor can be viewed by clicking here. Johnson's book on the subject, Shiver: A Whistleblower's Chilling Expose of Cryonics and the Truth Behind What Happened to Ted Williams, is scheduled to be published in May 2009.

See also Ralian and my comments on cryonics in Mass Media Funk.

further reading

books and
articles

Ettinger, Robert C. W. 1964. The Prospect of Immortality. Doubleday.

Kunzman, Alan, with Paul Nieto. 2004. Mothermelters: The inside story of Cryonics and the Dora Kent Homicide. 1st Books Library. (For Alcor's version of the case, see Our Finest Hours: Notes On the Dora Kent Crisis by Michael Perry, Ph.D.)

Johnson, Larry with Scott Baldyga. 2009. Shiver: A Whistleblower's Chilling Expose of Cryonics and the Truth Behind What Happened to Ted Williams. Morgan James Publishing.

Polidoro, J. P. 2005. Brain Freeze -321 f ~Saving "Reggie" Sanford~. Xlibris Corporation. (A novel about a former baseball player whose body is whisked off to a cryonics facility....)

websites and blogs

Nano Nonsense & Cryonics by Michael Shermer

CryonicsA futile desire for everlasting life - Only on Wednesdays

Is Cryonics Feasible? Stephen Barrett, M.D.

Dora Kent - Wikipedia ("News coverage at the time [1987] was limited, due to the gruesomeness of the case and the Christmas season.")

Cryonics UK

Debates about cryonics with skeptics (condensed from exchanges that occurred in May-June 2006 in the James Randi Educational Forum (JREF).)

Cryonics: The Issues (An Overview) by Ben Best

Can cryogenic cooling miraculously improve car parts, sports equipment, and musical instruments? - The Straight Dope

Last updated 05-Dec-2013

Original post:
cryonics - The Skeptic's Dictionary - Skepdic.com

(and some possible solutions)

When you buy a house, the seller is legally obliged to disclose any known defects. When you review a company's annual report, it tells you every problem that could affect the corporate share value. Since arrangements for cryopreservation may have a much greater impact on your life than home ownership or stock investments, we feel an ethical obligation to disclose problems that affect cryonics in general and Alcor specifically. We also believe that an organization which admits its problems is more likely to address them than an organization which pretends it has none. Thus full disclosure should encourage, rather than discourage, consumer confidence.

As of 2011, Alcor is nearly 40 years old. Our Patient Care Trust Fund is endowed with more than 7 million dollars and is responsible for the long-term care of over 100 cryopatients. In almost every year since its inception Alcor has enjoyed positive membership growth. We are the largest cryonics organization in the world yet in many respects we are still a startup company. We have fewer than a dozen employees in Scottsdale, Arizona and approximately 20 part-time independent contractors in various locations around the USA, mostly dedicated to emergency standby and rescue efforts. We serve fewer than 1,000 members and the protocols that aid our pursuit of the goal of reversible suspended animation continue to be developed. At the present time the technology required for the realization of our goal far exceeds current technical capabilities. Cryonics will not be comparable with mainstream medicine until our patients can be revived using contemporary technology, and we expect to wait for decades to see this vision fulfilled. Nevertheless, we have made important progress by introducing brain vitrification to improve patient tissue structure preservation.

Alcor shares some of the characteristics of startup companies. The organization is understaffed in some important areas and lacks as much capitalization as would be desired to support maximum growth. Limited resources prevent the organization from hiring as many highly qualified and experienced personnel as desired, and sometimes we have to postpone enhancements to equipment and procedures.

Because Alcor must react quickly to circumstances, it cannot always handle multiple tasks simultaneously. We feel a significant impact if, for example, several members experience legal death in quick succession. A heavy caseload generally means that administrative and even technical development work is postponed while member emergencies take precedence.

On the other hand, Alcor staff believe very strongly in the mission of the organization and are extremely dedicated. Alcor transport team members feel that they are saving lives, and behave accordingly. Most of all, everyone at Alcor is concerned with insuring the security of the patients who have been cryopreserved for the indefinite future. The organization's powerful sense of purpose is reinforced by the fact that all Alcor directors and most staff members have made arrangements to be cryopreserved themselves in the future.

Unlike most startups, Alcor is unlikely to fail for financial reasons. Due to the legally independent status of the Patient Care Trust from Alcor, patients can be maintained indefinitely through its portfolio of cash, investments, real estate, and capital equipment. Some wealthy Alcor members have contributed gifts and endowments to help the organization to advance, and in the event of a financial crisis, many of the people who hope ultimately to be cryopreserved would probably provide assistance. In this sense Alcor benefits from its small size, since it maintains an intimate relationship with many members which would be more problematic if our membership was ten times as large.

Inability to Verify Results

When a conventional surgical procedure is successful, usually the patient recovers and is cured. If the same surgical procedure is unsuccessful or a surgeon makes a serious error, the patient may die. These clear outcomes provide prompt feedback for the people involved. A physician may feel deeply satisfied if a life is saved, or may be deeply troubled (and may be sued for malpractice) if errors cause a death that should have been avoidable.

Clear feedback of this type does not exist in cryonics, because the outcome of our procedures will not be known definitively until decades or even a century from now. We have good reason to expect future technologies capable of repairing cellular damage in cryonics patients, but we feel equally certain that if a patient experiences very severe brain damage prior to cryopreservation, repairs may be delayed, may be incomplete, or may be impossible. The dividing line between these positive and negative outcomes cannot be established clearly at this time.

Suppose a patient experiences 30 minutes of warm ischemia (lack of blood flow at near-normal body temperature) after legal death occurs. Will this downtime create damage that is irreversible by any imaginable technology? Probably not. But what if the ischemic interval lasts for an hour or two hours, or a day? We simply don't know where to draw the line between one patient who is potentially viable, and another who is not.

Of course we can refer to experimental work that has evaluated the injury which occurs when cells are deprived of essential nutrients. These studies provide some guidance regarding the likely damage that a patient may experience, but they still cannot tell us with certainty if future science will be able to reverse that damage.

Another problem afflicting cryonics cases is that many uncontrolled variables prevent us from developing objective criteria to compare one case with another. Consider these two examples:

In the first case, will the long transport time negate the advantage of a rapid initial response and replacement of blood with a chilled preservation solution? In the second case, will the initial hours of warm ischemia outweigh the advantage of the rapid transport to Alcor? We can make educated guesses, but we cannot answer these questions definitively. We have no certain way of knowing which case will work out better, because we have no evidence no outcome.

We do have some simple ways to determine if a patient's circulatory system allows good perfusion with cryoprotectant. Personnel in the operating room will notice if blood clots emerge when perfusion begins. The surface of the brain, visible through burr holes which are created to enable observation, should be pearly white in color. The brain should shrink slightly as water is replaced with cryoprotectant. When perfusion is complete the patient's features should have acquired a sallow color indicating that cryoprotectant has diffused through the tissues.

These simple observations are helpful, but still the people who work hard to minimize transport time and maximize the rate of cooling can never enjoy the satisfying payoff that a physician receives when one of his patients recovers and returns to a normal, active life. This lack of positive outcome can cause feelings of frustration and futility, sometimes leading to disillusionment and burnout.

Conversely, if a case goes badly, team members will be protected from negative feedback. A team leader can never say to one of the personnel, "Because of your error, the patient has no chance of recovery."

The lack of a clear outcome also prevents us from refuting people who claim that future science will be able to undo almost any degree of damage. The danger o
f this extreme positive thinking is that it can lead to laziness. Why bother to make heroic efforts to minimize injury, if nanotechnology will fix everything?

Alcor's stated policy firmly rejects this attitude. Team members are very highly motivated to minimize injury because we believe that our members should not bet their lives on unknown capabilities of future science. Alcor generally hosts a debriefing after each case, encouraging all participants to share complaints, frustrations, and suggestions for improvement. Ideally, each case should be a learning experience, and participants should welcome criticism as an opportunity to identify weaknesses and overcome them in the future.

Still the lack of a clear outcome remains one of the biggest weaknesses in cryonics, since it encourages complacency and prevents accountability. The antidote to this problem is a better set of objective criteria to evaluate cases, and Alcor is working in consultation with brain ischemia experts to develop such criteria.

Volunteer Help

During the 1960s the first cryonics organizations were run entirely by volunteers. The field was not sufficiently reputable to attract qualified medical staff, and no one could have paid for professional help anyway.

Today cryonics is making a transition to professionalism, but financial limitations are prolonging the process. Some paramedics are associated with Alcor, and we hope for more in the future. We have an MD medical director, access to three contract surgeons, access to a hospice nurse, and assistance from an ischemia research laboratory in California where staff has extensive experience in relevant procedures such as vascular cannulation and perfusion. Alcor also communicates with a cryobiology laboratory that has made the most important advances in organ preservation during the past decade. Still, most transport team members who work remotely from the facility are volunteers who receive a week or two of training and modest payment for their work.

In the future, as Alcor becomes more financially secure and is able to offer higher salaries, the organization will attract more medical professionals. At this time, the transition is incomplete.

Limited Support from Mainstream Science

In the 1960s scientists in mainstream laboratories investigated techniques to cryopreserve whole organs. By the end of the 1970s most of this work had ended, and the field of cryobiology separated itself very emphatically from cryonics. The Society for Cryobiology has discouraged scientists from doing work that could advance cryonics, and has adopted a bylaw that threatens to expel any member who practices or promotes cryonics. Consequently the few scientists who are willing to do cryonics-related research live in fear of being excluded from the scientific specialty that is most relevant to their work.

The rift between cryonics and cryobiology may have been caused initially by fears among mainstream scientists that cryonics had a "tabloid journalism" flavor incompatible with science. In addition many scientists have been dissatisfied with the idea of applying procedures without a complete and full understanding of their outcome. Generally, in medicine, first a technique is studied, validated, and perfected, and then it is applied clinically. Cryonics has, of necessity, done an end-run around this formal approach by rushing to apply a technique based on theoretical arguments rather than validated clinical effectiveness.

During the past decade our knowledge and procedures have advanced far beyond the crude freezing methods imagined by most cryobiologists, and experts in molecular nanotechnology have voiced strong support. As more papers are published describing technical advances, we expect that cryobiologists and other scientists will revise their negative assessment of cryonics. In the future we believe that the arbitrary barrier between cryonics and cryobiology will gradually dissolve, and cryonics research will be recognized as a legitimate specialty of the field. However, for the time being the dim view taken of cryonics by most cryobiologists remains problematic, impairing Alcor's ability to achieve respectable status among other relevant groups such as prospective members, regulatory officials, and legislators.

Limited Legal and Government Support

Cryonics is not explicitly recognized in the laws of any state in the United States (see The Legal Status of Cryonics Patients). This does not mean that cryonics is illegal or unregulated. In fact, Alcor must comply with state laws controlling the transport and disposition of human remains, and we make arrangements with licensed morticians to insure that these requirements are met. Alcor also complies with federal regulations established by agencies such as OSHA and EPA.

Still, the lack of specific enabling legislation for cryonics can cause problems. In the late 1980s the California Department of Health Services (DHS) asserted that because there was no statutory procedure for becoming a cryonics organization, human remains could not be conveyed to a cryonics organization via the Uniform Anatomical Gift Act (UAGA), and therefore cryonics was illegal. Fortunately, the courts were unimpressed by this argument. In 1992 the legality of cryonics, and the legality of using the UAGA for cryonics, were upheld at the appellate level.

In 1990 the Canadian province of British Columbia enacted a law that specifically banned the sale of cryonics services in that province. In 2002 the Solicitor General (Canadian equivalent of a state Attorney General) issued a written clarification stating that the law only prohibited funeral homes from selling cryonics arrangements. Cryonics could still be performed in the province, even with the paid assistance of funeral homes, provided they were not involved in the direct sale of cryonics. This position is affirmed by the Business Practices and Consumer Protection Authority of British Columbia. Despite these assurances, anxiety about the law remains.

In 2004 a bill was passed by the Arizona House of Representatives to place cryonics and cryonics procedures under the regulation of the state funeral board. In its original form this law would have prevented our use of the UAGA. The bill was ultimately withdrawn, but may be revived at a later date. Very hostile comments were made about cryonics during the floor debate of this bill. We cannot guarantee that any future legislation will be friendly to cryonics or will permit cryonics to continue in Arizona.

Despite these uncertainties, the United States enjoys a strong cultural tradition to honor the wishes of terminal patients. We believe that the freedom to choose cryonics is constitutionally protected, and so far courts have agreed. We are hopeful that we will be able to continue performing cryonics without technical compromise, under state supervision where necessary, for the indefinite future.

Limited Mainstream Medical Support

Cryonics is not an accepted or recognized "therapy" in the general medical community. To the average medical professional, cryonics is at best an unusual anatomical donation. At worst it can be viewed by some physicians as fraud upon their patient. Hospitals have sometimes deliberately delayed pronouncement of legal death, delayed release of patients to Alcor, or forbade the use of cryonics life support equipment or medications within their facilities. On one occasion in 1988 Alcor had to obtain a court order to compel a hospital to release a patient to Alcor promptly at legal death and permit our stabilization proce
dures on their premises.

Relations with hospitals and their staff are not always difficult. Usually when nurses and physicians learn that cryonics is a sincere practice that is overseen by other medical professionals, they will be willing to accommodate a patient's wishes, or at least will not interfere with them. Sometimes medical staff will even assist with cryonics procedures such as administering medications and performing chest compressions if Alcor personnel are not present when legal death occurs.

The lack of formal medical recognition or support for cryonics generally means that cryonics patients remote from Alcor must be moved to a mortuary for blood replacement before transport to Alcor. Ideally these preparatory procedures should be performed within hospitals, not mortuaries. Hospitals presently allow organ procurement personnel to harvest organs from deceased patients (a fairly elaborate procedure) within their walls. We are hopeful that similar privileges will be extended to cryonics more often as the process becomes better understood and accepted, but we cannot predict how quickly this change will occur.

High Incidence of Poor Cases

In more than 50 percent of cryonics cases legal death occurs before Alcor standby personnel can be deployed, and is often followed by hours of warm ischemia. This downtime may cause severe cellular damage.

The threat of autopsy, in which the brain is routinely dissected, is an even greater danger. Any person who suffers legal death under unexpected circumstances, especially involving accidents or foul play, is liable to be autopsied. Alcor strongly urges members living in California, Maryland, New Jersey, New York, and Ohio to sign Religious Objection to Autopsy forms.

Sometimes cryonicists perish under circumstances resulting in complete destruction or disappearance of their remains. Cryonicists have been lost at sea, suffered misadventures abroad, or even disappeared without a trace. Two members of cryonics organizations were lost in the 2001 collapse of the World Trade Center towers. One was a policeman performing rescue operations.

Cryonics is not a panacea or a "cure" for death. The cryonics ideal of immediate cooling and cardiopulmonary support following cardiac arrest cannot be achieved in the majority of cases. We have good reasons to believe that molecular records of memory persist in the brain even after hours of clinical death, but only future physicians using medical technology which we do not yet possess will be able to determine, finally, whether such a person is really still "there."

What can be done?

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Read the rest here:
Problems Associated with Cryonics - Cryonics: Alcor Life ...

CryonicsMagazine, July 2013

[The following is a text adaptation of a PowerPoint presentation given on Sunday, May 12, 2013at the Resuscitation and Reintegration of Cryonics Patients Symposium in Portland, Oregon]

An understanding of probable futurerepair requirements for cryonicspatients could affect current cryostoragetemperature practices. I believe thatmolecular nanotechnology at cryogenictemperatures will probably be required forrepair and revival of all cryonics patientsin cryo-storage now and in the foreseeablefuture. Current nanotechnology is far frombeing adequate for that task. I believe thatwarming cryonics patients to temperatureswhere diffusion-based devices couldoperate would result in dissolutionof structure by hydrolysis and similarmolecular motion before repair could beachieved. I believe that the technologiefor scanning the brain/mind of a cryonicspatient, and reconstructing a patient fromthe scan are much more remote in thefuture than cryogenic nanotechnology.

Cryonicists face a credibility problem.It is important to show that resuscitationtechnology is possible (or not impossible)if cryonicists are to convince ourselvesor convince others that current cryonicspractice is not a waste of money and effort.For some people it is adequate to know thatthe anatomical basis of the mind is beingpreserved well enough even if in a veryfragmented form that some unspecifiedfuture technology could repair and restorememory and personal identity. Otherpeople want more detailed elaboration.

Books have detailed whatnanotechnology robots (nanorobots) willlook-like and be capable-of, including(notably) Nanosystems by K. Eric Drexler(1992) and Nanomedicine by Robert A.Freitas, Jr. (Volume I, 1999; Volume IIA,2003). The online Alcor library containsarticles detailing repair of cryonics patientsby nanorobots at cryogenic temperature,in particular, A CryopreservationRevival Scenario using MolecularNanotechnology by Ralph Merkle andRobert Freitas as well as RealisticScenario for Nanotechnological Repairof the Frozen Human Brain. Despitethe detailed descriptions, calculations, andquantitative analyses that have been given,any technology as remote from presentcapabilities as cryogenic nanotechnology iscertain to be very different from whateveranyone may currently imagine. It is difficultto argue against claims that all suchdescriptions are nothing more than handwaving,blue-sky speculations.

Current medical applications ofnanotechnology are mainly limited to theuse of nanoparticles for drug delivery.1Nanomachines are being built, but they arelittle more than toys including a rotor thatcan propel a molecule2 or microcantileverdeflection of DNA by electrostatic force.3In classical mechanics and kinetictheory of gases, on a molecular level,temperature is defined in terms of theaverage translational kinetic energy ofmolecules, which means that the lowerthe temperature the slower the motion ofthe molecules. According to the ArrheniusEquation, the rate of a chemical reactiondeclines exponentially with temperaturedecline. It would be wrong to concludethat nanomachines would barely be able tomove at cryogenic temperatures, however.Nanomachines operate by mechanicalmovement of constituent atoms, a processthat is temperature-independent. In fact,nanomachines would probably operatemore effectively at cryogenic temperaturebecause there would be far less jostlingof atoms in the molecular structuresupon which nanomachines would operate.Nanomachines would also be less vulnerableto reactions with oxygen at cryogenictemperature, although it would nonethelessbe preferable for cryogenic nanorepair tooccur in an oxygen-free environment.

Although under ideal circumstances iceformation can be prevented in cryonicspatients, circumstances too often result inat least some freezingsuch as inability toperfuse with vitrification solution, or poorperfusion with vitrification solution becauseof ischemia due to delayed treatment.Past cryonics patients were perfusedwith the (anti-freeze) cryoprotectantglycerol, whereas cryonics patients arecurrently perfused with cryoprotectantsolutions that include ethylene glycoland dimethylsulfoxide (DMSO). Unlikewater, which forms crystalline ice whensolidifying upon cooling, cryoprotectantsform an amorphous (non-crystalline,vitreous) solid (a hardened liquid) whensolidifying upon cooling. The hardenedliquid is a glass rather than an ice. Thetemperature at which the solidification(vitrification) occurs is called the glasstransition temperature (Tg).

For M22, the cryoprotectant used byAlcor to vitrify cryonics patients, Tg istypically between 123C and 124C(depending on the cooling rate). Tg isabout the same for the cryoprotectant(VM-1) used for cryonics patients at theCryonics Institute.Although freezing can be reduced oreliminated by perfusing cryonics patientswith vitrification solution before coolingto Tg, eliminating cracking is a moredifficult problem. Cryonics patients arecooled to cryogenic temperatures byexternal cooling. Thermal conductivity isslow in a cryonics patient, which meansthat the outside gets much colder thanthe inside. When the outside of a samplecools more quickly than the inside of thesample, thermal stress results. A vitrifiedpatient subjected to such thermal stresscan crack or fracture. No efforts have beenmade to find additives to M22 that wouldhave a similar effect as boron oxide hason allowing Pyrex glass to reduce thermalstress.

If a vitrified sample is small enough,and if cooling is slow enough, the samplecan be cooled far below Tg down toliquid nitrogen temperature withoutcracking. A rabbit kidney (10 millilitervolume) can be cooled down to liquidnitrogen temperature in two days withoutcracking/fracturing.6 Cryonics patientsare much too large to be cooled to liquidnitrogen temperature over a period ofdays without cracking. The amount oftime required for cooling vitrified cryonicspatients to liquid nitrogen temperaturewithout cracking is unknown, and wouldprobably be much too long.

In 1990 cryobiologist Dr. Gregory Fahypublished results of cracking experimentsthat he performed on samples of thecryoprotectant propylene glycol.4 Tg forpropylene glycol is 108C, but in RPS-2carrier solution the Tg is 107C. In oneexperiment he demonstrated that crackingbegan at lower temperatures for smallersamples, specifically: 143C for 46 mL,116C for 482 mL, and 111C for 1412mL. (The last volume is comparable to thevolume of an adult human brain.) Dr. Fahyalso demonstrated that cracking could bedelayed by cooling at slower cooling rates.But when cracking did occur, the cracksformed at the lower temperatures werefiner and more numerous.

Based on evidence that large cracksformed at higher temperatures by morerapid cooling results in a relief of thermalstress that prevents the fine and morenumerous cracks formed when crackingbegins at lower temperature, the CryonicsInstitute (CI) altered its cooling protocolfor cryonics patients. CI patients arecooled quickly from 118C to 145C,and then cooled slowly to 196C.5In order to minimize or eliminatecracking in cryonics patients, proposalshave been made to store the patients attemperatures lower than Tg (124C), buthigher than liquid nitrogen temperature(196C).6 Such a cryo-storage protocolis described as Intermediate TemperatureStorage (ITS). Alcor currently cares for anumber of ITS patients at 140C, but aconsensus has not yet been reached aboutwhat ITS temperature will be chosen whenthis service is made available to all Alcormembers.

Although Alcors vitrification solutionM22 can prevent ice formation with somesamples and protocols, M22 cannot preventice nuclei from forming at cryogenictemperatures. Ice nuclei are local clustersof water molecules that rotate into anorientation that favors later growth of icecrystals when a solution is warmed. Icenuclei are not damaging, but the fact that icenuclei can form indicates molecular mobilitywhich could be damaging. Specifically,between the temperatures of 100C and135C, ice nuclei can form in M22, withthe maximum ice nucleation rate occurringnear Tg. At 140C the ice nucleation ratefor M22 is undetectable. But nuclei will beprobably formed in cooling to 140C.

Although cryostorage at 140C is anattempt to minimize cracking and minimizenucleation, this ITS neither eliminatescracking nor ice nuclei formation.Cryonics patients slowly cooled from Tgto 140C will surely experience someice nucleation. Alcor places a listeningdevice (crackphone) under the skullof its cryonics patients for the purposeof monitoring cracking events. Myunderstanding is that for most Alcorpatients the crackphone detects crackingat Tg or only slightly below Tg, althoughthere was reportedly one M22-perfusedpatient for which the first fracturing eventoccurred at 134C. The propylene glycolexperiments would support the view ofcracking occurring slightly below Tg, butvitrified biological samples resist crackingbetter than pure cryoprotectant solutions.

With ice formation, cracking could occurat temperatures higher than Tg. AlthoughITS may prevent the formation of crackingthat could occur in cooling below 140C,it does not prevent the cracks that occur incooling from Tg to 140C.I have wondered whether there areforms of damage which would occurin a cryonics patient stored at 140Cthat would not occur during storage at196C. A solid cryogenic state of matterdoes not prevent molecular motion.Molecular motion in a biological sampleheld at cryogenic temperature could resultin damage to that sample.

Ions generated by radiation aremuch more mobile than molecules.An ionic species (probably protons) intrimethylammonium dihydrogen phosphateglass is nine orders of magnitude moremobile than the glass moleculesandsodium ions in sodium disilicate glass aretwelve orders of magnitude more mobilethan the glass molecules.9

Cryobiologist Peter Mazur has statedthat below 130C viscosity is so high(>1013 Poise) that diffusion is insignificantover less than geological time spans. Headds that there is no confirmed case ofcell death ascribed to storage at 196Cfor some 2-15 years and none even whencells are exposed to levels of ionizingradiation some 100 times background forup to 5 yr.10 Frozen 8-cell mouse embryossubjected to the equivalent of 2,000 yearsof background gamma rays during 5 to8 months in liquid nitrogen showed noevident detrimental effect on survival ordevelopment.11

In attempting to evaluate damagingeffects of temperature and radiation, itcould be valuable to analyze chemicalalterations, rather than complete cell deathor viability. Acetylcholinesterase enzymesubjected to X-ray irradiation showsconformational changes at 118C, but noconformational changes when irradiatedat 173C.12 X-ray irradiation of insulinand elastase crystals resulted in four timesas much damage to disulfide bridges at173C compared to 223C.13 Anotherstudy showed a 25% crystal diffractionlifetime extension for D-xylose isomerasecrystals X-ray irradiated at less than 253Ccompared to those irradiated at 173C.14

One study showed that lettuce seedsshow measurable deterioration when storedat liquid nitrogen temperature for periodsof 10 to 20 years. Rotational molecularmobility was quantified. A graphical plotwas generated showing increasing timesfor when 50% of lettuce seeds would failto germinate as a function of decreasingtemperature. Those times were estimated tobe about 500 years for 135C and about3,400 years for 196C.15 Translationalvibrational motion has been given as anexplanation for seed quality deterioration atcryogenic temperatures.16 The mean squarevibrational amplitude of a water moleculeis not even zero at 0 Kelvins (273C), andhas been determined to be 0.0082 squareAngstroms. The mean square vibrationalamplitude is 0.0171 square Angstroms at173C and 0.0339 square Angstroms at73C.17

Realistically, however, 3,400 years ismuch longer than cryonics patients arelikely to be stored. Storage in liquid heliumat 269C or in a shadowed moon craterat 235C18 would certainly be moretrouble than it is worth. Northern woodfrogs spend months in a semi-frozen stateat 3C to 6C, and are able to revivewith full recovery of heartbeat uponre-warming.19 An empirical study of acryoprotectant very similar to M22 (VS55) showed viscosity continuing to increaseexponentially below Tg, just as viscosityincreases exponentially with temperaturedecrease above Tg.20 The exponentialdecrease in viscosity (molecular mobility)that makes ice nucleation cease at 135Cindicates that there is probably littlemolecular mobility at 140C, despite thepossibility of damage from ionic species orvibrational motion. All things considered,however, my personal preference is forstorage in liquid nitrogen, rather than someintermediate temperature above 196C. Iwould also prefer for cryogenic nanorobotrepair to be at liquid nitrogen temperature.

I am by no means a nanotechnologyexpert, but I can give a brief descriptionof my own views of how cryogenicnanotechnology repair of a cryonicspatient would proceed. I must thank RalphMerkle for his assistance in allowing me toconsult with him to formulate and clarifymany of my views.I believe that repair of cryonics patientsat cryogenic temperature would be acombination of nano-mining and nanoarcheology.Nanorobots (nanometer-sizedrobots) would first clear blood vessels ofwater, cryoprotectant, plasma, blood cells,etc. The blood vessels would becomemining shafts that would provide access toall body tissues. Nanometer-sized conveyorbelts or trucks on rails could removeblood vessel contents. Where freezingor ischemia had destroyed blood vessels,artificial shafts would be created. Unlikethe nano-mining that simply removes allblood vessel contents, the creation ofartificial shafts would have the characterof an archeological dig. Care would betaken in removing material to avoiddamaging precious artifacts that mightindicate original structure which could be discovered at any unexpected moment.

Section 13.4 of K. Eric Drexlers bookNanosystems provides diagrams and detailsof a nanorobot manipulator arm. Such adiamondoid component would containabout four million atoms, and could befitted with a variety of tools at the endof the arm. A variety of tips with varyingdegrees of chemical reactivity couldallow for reversible, temporary chemicalbonds that could be used for grabbingand moving molecules. These could rangefrom radicals or carbenes that would formstrong covalent bonds, to boron thatcan form relatively weak and reversiblebonds to nitrogen and oxygen, to simpleO-H groups that can form even weakerhydrogen bonds. Tools for digging neednot be so refined. The manipulator arm isdepicted as being 100 nanometers long and50 nanometers wide, although nanorobotswould need to be larger to includecapability for locomotion, computation,and power. A complete nanorobot couldbe as large as a few thousand nanometersin size. A capillary is between 5,000 to10,000 nanometers in diameter, so thereshould be plenty of room for many suchnanorobots to operate. Ralph Merkleestimates that 3,200 trillion nanorobotsweighing a total of 53 grams could repaira cryonics patient in about 3 years.21,22 Likemany of the calculations associated withnanotechnology, I take these figures with apound of salt. It is certainly true, however,that it could take years to repair a patient,and that there should not be a rush tofinish the job.

Merkle & Freitas have suggested thatnanorobots be powered by electrostaticmotors. Stators and rotors would be electricrather than magnetic. Tiny moving chargedplates are easier to fabricate than tiny coilsand tiny iron cores, but more fundamentally,magnetic properties do not scale well withreduced size (i.e., molecular-scale magneticmotors dont work), whereas electrostaticproperties do scale well with reduced size.Electrostatic actuators are already beingused in microelectromechanical systems(MEMS).23 High density batteries couldprovide power for days, and rechargingstations could be located throughout thepatient. Alternatively, nanotube cablescould bring power to the patient fromthe outside. Such cables could also bea means of transmitting and receivingcomputational data. Nanotube cablescould also be used to reunite fracture faces created by cracking. Scanning and imageprocessing capabilities would need toevaluate what needs to be fixed.

As much as possible I would favorreplacement rather than repair, whichwould greatly simplify the process. Itwould be much easier to replace a kidneythan to repair the diseased kidney ofan elderly patient who died of kidneydisease. Curing disease and rejuvenationwould thus become part of the repair of acryonics patient. Of course, neuro patientswould require an entirely new body. Thebrain would be the major exception toreplacement strategy because the braincould not be replaced without loss ofmemory and personal identity.

Even within the brain, however, it couldbe feasible to replace many componentswithout loss of memory and personalidentity. It could be feasible to replacemany organelles such as mitochondria,lysosomes, etc., and many macromoleculessuch as proteins, carbohydrates, and lipids.DNA could be repaired, and possiblyeven modified to cure genetic disease,but epigenetic expression in neurons maybe critical for reconstruction of synapticstructure. Synaptic connections wouldnot only be restored, but the quantityand quality of neurotransmitter contentsshould be restored. It is not simply a matterthat some neurotransmitters are inhibitoryand others are stimulatory. There are morethan 40 different neurotransmitters used inthe brain, and there must be a good reasonwhy such variety is necessitated.

Part of the repair process could involveremoval of ice nuclei, nearly all of whichwould be extracellular. Re-created bloodvessel contents would include freshcryoprotectant, water, plasma, and bloodcells without the original ice nuclei. Althoughsome repair scenarios favor different typesof repair above cryogenic temperature, Idoubt that this is necessary or desirable.Alternative repair scenarios involvesplitting the brain in half, and halvingthe halves repeatedly at cryogenictemperaturewith digitization at eachstepuntil the brain has been totallydigitized.21,22 Or digitization could bedone by repetitive nano-microtomes atcryogenic temperature. The digital datacould be used for full reconstruction. Somepeople might object that if one individualcould be created from digital data, manysuch individuals could be createdraisingquestions of which are duplicates and which is the original. There is detaileddiscussion of the duplicates problem/paradox in the philosophy section of mywebsiteBENBEST.COM.

Although other repair scenarioscould prove to be feasible, I believethat cryogenic nanotechnology will berequired for all cryonics patients in theforeseeable future until the problem ofcryoprotectant toxicity can be solved.With effective nontoxic cryoprotectants,sufficient cryoprotectant could be usedto prevent ice nuclei formation at alltemperatures, prevent devitrification(freezing) upon rewarming, and eliminateall toxic damage. In such a case, therecould be true reversible cryopreservation(suspended animation).

What is needed to create thenanotechnology required for repair ofcryonics patients? Small machines willneed to build parts for smaller machines,which would in turn build even smallermachines. Many details of machine operation must be perfected at each stage.Current modern technological civilizationbegan with cave people pounding on rocks.Ralph Merkle has said that compared tofuture technology, current technology ispounding on rocks.

References

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2. Kudernac T, Ruangsupapichat N,Parschau M, Maci B, Katsonis N,Harutyunyan SR, Ernst KH, Feringa BL.Electrically driven directional motion of afour-wheeled molecule on a metal surface.Nature. 2011 Nov 9;479(7372):208-11.

3. Zhang J, Lang HP, Yoshikawa G,Gerber C. Optimization of DNAhybridization efficiency by pH-drivennanomechanical bending. Langmuir. 2012Apr 17;28(15):6494-501.

4. Fahy GM, Saur J, Williams RJ. Physicalproblems with the vitrification of largebiological systems. Cryobiology. 1990Oct;27(5):492-510.

5. Best B. The Cryonics Institutes 95thPatient. Long Life. 2009 Sept-Oct; 41(9-10):17-21.

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