Monthly Archives: July 2016

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 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