Daily Archives: December 20, 2015

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

1. Chi AH, Clayton K, Burrow TJ, Lewis R,Luciano D, Alexis F, Dhers S, Elman NM.Intelligent drug-delivery devices based onmicro- and nano-technologies. Ther Deliv.2013 Jan;4(1):77-94.

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.

6. Wowk B. Systems for IntermediateTemperature Storage for FractureReduction and Avoidance. 2011 ThirdQuarter;32(3):7-12.

7. Okamoto M, Nakagata N, Toyoda Y.Cryopreservation and transport of mousespermatozoa at -79 degrees C. Exp Anim.2001 Jan;50(1):83-6.

8. Angell CA. Entropy and Fragility inSupercooling Liquids. Journal of Researchof the National Institute of Standardsand Technology. 1997 March-April;102(2):171-185.

9. Mizunoa F, Belieresa J.-P, KuwatabN, Pradelb A, Ribesb M, Angell CA.Highly decoupled ionic and protonicsolid electrolyte systems, in relation toother relaxing systems and their energylandscapes. 2006 Nov;352(42/49):5147-5155.

10. Mazur P. Freezing of living cells:mechanisms and implications. Am JPhysiol. 1984 Sep;247(3 Pt 1):C125-42.

11. Glenister PH, Whittingham DG,Lyon MF. Further studies on the effectof radiation during the storage of frozen8-cell mouse embryos at -196 degrees C. JReprod Fertil. 1984 Jan;70(1):229-34.

12. Weik M, Ravelli RB, Silman I,Sussman JL, Gros P, Kroon J. Specificprotein dynamics near the solvent glasstransition assayed by radiation-inducedstructural changes. Protein Sci. 2001Oct;10(10):1953-61.

13. Meents A, Gutmann S, Wagner A,Schulze-Briese C. Origin and temperaturedependence of radiation damagein biological samples at cryogenictemperatures. Proc Natl Acad Sci U S A.2010 Jan 19;107(3):1094-9.

14. Chinte U, Shah B, Chen YS, PinkertonAA, Schall CA, Hanson BL. Cryogenic(<20 K) helium cooling mitigates radiationdamage to protein crystals. Acta CrystallogrD Biol Crystallogr. 2007 Apr;63(Pt 4):486-92.

15. Walters C, Wheeler L, Stanwood PC.Longevity of cryogenically stored seeds.Cryobiology. 2004 Jun;48(3):229-44.

16. Wowk B. Thermodynamic aspectsof vitrification. Cryobiology. 2010Feb;60(1):11-22.

17. Leadbetter AJ; The Thermodynamicand Vibrational Properties of H$_2$O Iceand D$_2$O Ice. 1965 Sep;A287:403-425.

18. Paige DA, Siegler MA, Zhang JA,Hayne PO, Foote EJ, Bennett KA,Vasavada AR, Greenhagen BT, SchofieldJT, McCleese DJ, Foote MC, DeJong E,Bills BG, Hartford W, Murray BC, AllenCC, Snook K, Soderblom LA, Calcutt S,Taylor FW, Bowles NE, Bandfield JL,Elphic R, Ghent R, Glotch TD, WyattMB, Lucey PG. Diviner Lunar Radiometerobservations of cold traps in the Moonssouth polar region. Science. 2010 Oct22;330(6003):479-82.

19. Costanzo JP, Lee RE Jr, DeVries AL,Wang T, Layne JR Jr. Survival mechanismsof vertebrate ectotherms at subfreezingtemperatures: applications in cryomedicine.FASEB J. 1995 Mar;9(5):351-8.

20. Noday DA, Steif PS, Rabin Y.Viscosity of cryoprotective agents nearglass transition: a new device, technique,and data on DMSO, DP6, and VS55. ExpMech. 2009 Oct;49(5):663-672.

21. Merkle, RC. The Molecular Repair ofthe Brain. Cryonics. 1994 Jan;15(1):16-31.

22. Merkle, RC. The Molecular Repair ofthe Brain. Cryonics. 1994 Apr;15(2):18-30.

23. Fennimore AM, Yuzvinsky TD,Han WQ, Fuhrer MS, Cumings J,Zettl A. Rotational actuators based oncarbon nanotubes. Nature. 2003 Jul 24;424(6947):408-10.

Go here to see the original:
Cryonics | Evidence-Based Cryonics

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 preve
nt 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 withnanotec
hnology, 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

1. Chi AH, Clayton K, Burrow TJ, Lewis R,Luciano D, Alexis F, Dhers S, Elman NM.Intelligent drug-delivery devices based onmicro- and nano-technologies. Ther Deliv.2013 Jan;4(1):77-94.

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.

6. Wowk B. Systems for IntermediateTemperature Storage for FractureReduction and Avoidance. 2011 ThirdQuarter;32(3):7-12.

7. Okamoto M, Nakagata N, Toyoda Y.Cryopreservation and transport of mousespermatozoa at -79 degrees C. Exp Anim.2001 Jan;50(1):83-6.

8. Angell CA. Entropy and Fragility inSupercooling Liquids. Journal of Researchof the National Institute of Standardsand Technology. 1997 March-April;102(2):171-185.

9. Mizunoa F, Belieresa J.-P, KuwatabN, Pradelb A, Ribesb M, Angell CA.Highly decoupled ionic and protonicsolid electrolyte systems, in relation toother relaxing systems and their energylandscapes. 2006 Nov;352(42/49):5147-5155.

10. Mazur P. Freezing of living cells:mechanisms and implications. Am JPhysiol. 1984 Sep;247(3 Pt 1):C125-42.

11. Glenister PH, Whittingham DG,Lyon MF. Further studies on the effectof radiation during the storage of frozen8-cell mouse embryos at -196 degrees C. JReprod Fertil. 1984 Jan;70(1):229-34.

12. Weik M, Ravelli RB, Silman I,Sussman JL, Gros P, Kroon J. Specificprotein dynamics near the solvent glasstransition assayed by radiation-inducedstructural changes. Protein Sci. 2001Oct;10(10):1953-61.

13. Meents A, Gutmann S, Wagner A,Schulze-Briese C. Origin and temperaturedependence of radiation damagein biological samples at cryogenictemperatures. Proc Natl Acad Sci U S A.2010 Jan 19;107(3):1094-9.

14. Chinte U, Shah B, Chen YS, PinkertonAA, Schall CA, Hanson BL. Cryogenic(<20 K) helium cooling mitigates radiationdamage to protein crystals. Acta CrystallogrD Biol Crystallogr. 2007 Apr;63(Pt 4):486-92.

15. Walters C, Wheeler L, Stanwood PC.Longevity of cryogenically stored seeds.Cryobiology. 2004 Jun;48(3):229-44.

16. Wowk B. Thermodynamic aspectsof vitrification. Cryobiology. 2010Feb;60(1):11-22.

17. Leadbetter AJ; The Thermodynamicand Vibrational Properties of H$_2$O Iceand D$_2$O Ice. 1965 Sep;A287:403-425.

18. Paige DA, Siegler MA, Zhang JA,Hayne PO, Foote EJ, Bennett KA,Vasavada AR, Greenhagen BT, SchofieldJT, McCleese DJ, Foote MC, DeJong E,Bills BG, Hartford W, Murray BC, AllenCC, Snook K, Soderblom LA, Calcutt S,Taylor FW, Bowles NE, Bandfield JL,Elphic R, Ghent R, Glotch TD, WyattMB, Lucey PG. Diviner Lunar Radiometerobservations of cold traps in the Moonssouth pola
r region. Science. 2010 Oct22;330(6003):479-82.

19. Costanzo JP, Lee RE Jr, DeVries AL,Wang T, Layne JR Jr. Survival mechanismsof vertebrate ectotherms at subfreezingtemperatures: applications in cryomedicine.FASEB J. 1995 Mar;9(5):351-8.

20. Noday DA, Steif PS, Rabin Y.Viscosity of cryoprotective agents nearglass transition: a new device, technique,and data on DMSO, DP6, and VS55. ExpMech. 2009 Oct;49(5):663-672.

21. Merkle, RC. The Molecular Repair ofthe Brain. Cryonics. 1994 Jan;15(1):16-31.

22. Merkle, RC. The Molecular Repair ofthe Brain. Cryonics. 1994 Apr;15(2):18-30.

23. Fennimore AM, Yuzvinsky TD,Han WQ, Fuhrer MS, Cumings J,Zettl A. Rotational actuators based oncarbon nanotubes. Nature. 2003 Jul 24;424(6947):408-10.

Go here to see the original:
Cryonics | Evidence-Based Cryonics