Cryonics Magazine, July 2013

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

An understanding of probable future repair requirements for cryonics patients could affect current cryostorage temperature practices. I believe that molecular nanotechnology at cryogenic temperatures will probably be required for repair and revival of all cryonics patients in cryo-storage now and in the foreseeable future. Current nanotechnology is far from being adequate for that task. I believe that warming cryonics patients to temperatures where diffusion-based devices could operate would result in dissolution of structure by hydrolysis and similar molecular motion before repair could be achieved. I believe that the technologie for scanning the brain/mind of a cryonics patient, and reconstructing a patient from the scan are much more remote in the future than cryogenic nanotechnology.

Cryonicists face a credibility problem. It is important to show that resuscitation technology is possible (or not impossible) if cryonicists are to convince ourselves or convince others that current cryonics practice is not a waste of money and effort. For some people it is adequate to know that the anatomical basis of the mind is being preserved well enough ― even if in a very fragmented form ― that some unspecified future technology could repair and restore memory and personal identity. Other people want more detailed elaboration.

Books have detailed what nanotechnology robots (nanorobots) will look-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 contains articles detailing repair of cryonics patients by nanorobots at cryogenic temperature, in particular, “A Cryopreservation Revival Scenario using Molecular Nanotechnology” by Ralph Merkle and Robert Freitas as well as “‘Realistic’ Scenario for Nanotechnological Repair of the Frozen Human Brain.” Despite the detailed descriptions, calculations, and quantitative analyses that have been given, any technology as remote from present capabilities as cryogenic nanotechnology is certain to be very different from whatever anyone may currently imagine. It is difficult to argue against claims that all such descriptions are nothing more than handwaving, blue-sky speculations.

Current medical applications of nanotechnology are mainly limited to the use of nanoparticles for drug delivery.1 Nanomachines are being built, but they are little more than toys ― including a rotor that can propel a molecule2 or microcantilever deflection of DNA by electrostatic force.3 In classical mechanics and kinetic theory of gases, on a molecular level, temperature is defined in terms of the average translational kinetic energy of molecules, which means that the lower the temperature the slower the motion of the molecules. According to the Arrhenius Equation, the rate of a chemical reaction declines exponentially with temperature decline. It would be wrong to conclude that nanomachines would barely be able to move at cryogenic temperatures, however. Nanomachines operate by mechanical movement of constituent atoms, a process that is temperature-independent. In fact, nanomachines would probably operate more effectively at cryogenic temperature because there would be far less jostling of atoms in the molecular structures upon which nanomachines would operate. Nanomachines would also be less vulnerable to reactions with oxygen at cryogenic temperature, although it would nonetheless be preferable for cryogenic nanorepair to occur in an oxygen-free environment.

Although under ideal circumstances ice formation can be prevented in cryonics patients, circumstances too often result in at least some freezing―such as inability to perfuse with vitrification solution, or poor perfusion with vitrification solution because of ischemia due to delayed treatment. Past cryonics patients were perfused with the (anti-freeze) cryoprotectant glycerol, whereas cryonics patients are currently perfused with cryoprotectant solutions that include ethylene glycol and dimethylsulfoxide (DMSO). Unlike water, which forms crystalline ice when solidifying upon cooling, cryoprotectants form an amorphous (non-crystalline, vitreous) solid (a “hardened liquid”) when solidifying upon cooling. The “hardened liquid” is a glass rather than an ice. The temperature at which the solidification (vitrification) occurs is called the glass transition temperature (Tg).

For M22, the cryoprotectant used by Alcor to vitrify cryonics patients, Tg is typically between −123°C and −124°C (depending on the cooling rate). Tg is about the same for the cryoprotectant (VM-1) used for cryonics patients at the Cryonics Institute. Although freezing can be reduced or eliminated by perfusing cryonics patients with vitrification solution before cooling to Tg, eliminating cracking is a more difficult problem. Cryonics patients are cooled to cryogenic temperatures by external cooling. Thermal conductivity is slow in a cryonics patient, which means that the outside gets much colder than the inside. When the outside of a sample cools more quickly than the inside of the sample, thermal stress results. A vitrified patient subjected to such thermal stress can crack or fracture. No efforts have been made to find additives to M22 that would have a similar effect as boron oxide has on allowing Pyrex glass to reduce thermal stress.

If a vitrified sample is small enough, and if cooling is slow enough, the sample can be cooled far below Tg ― down to liquid nitrogen temperature ― without cracking. A rabbit kidney (10 milliliter volume) can be cooled down to liquid nitrogen temperature in two days without cracking/fracturing.6 Cryonics patients are much too large to be cooled to liquid nitrogen temperature over a period of days without cracking. The amount of time required for cooling vitrified cryonics patients to liquid nitrogen temperature without cracking is unknown, and would probably be much too long.

In 1990 cryobiologist Dr. Gregory Fahy published results of cracking experiments that he performed on samples of the cryoprotectant propylene glycol.4 Tg for propylene glycol is −108°C, but in RPS-2 carrier solution the Tg is −107°C. In one experiment he demonstrated that cracking began at lower temperatures for smaller samples, specifically: −143°C for 46 mL, −116°C for 482 mL, and −111°C for 1412 mL. (The last volume is comparable to the volume of an adult human brain.) Dr. Fahy also demonstrated that cracking could be delayed by cooling at slower cooling rates. But when cracking did occur, the cracks formed at the lower temperatures were finer and more numerous.

Based on evidence that large cracks formed at higher temperatures by more rapid cooling results in a relief of thermal stress that prevents the fine and more numerous cracks formed when cracking begins at lower temperature, the Cryonics Institute (CI) altered its cooling protocol for cryonics patients. CI patients are cooled quickly from −118°C to −145°C, and then cooled slowly to −196°C.5 In order to minimize or eliminate cracking in cryonics patients, proposals have been made to store the patients at temperatures lower than Tg (−124°C), but higher than liquid nitrogen temperature (−196°C).6 Such a cryo-storage protocol is described as Intermediate Temperature Storage (ITS). Alcor currently cares for a number of ITS patients at −140°C, but a consensus has not yet been reached about what ITS temperature will be chosen when this service is made available to all Alcor members.

Although Alcor’s vitrification solution M22 can prevent ice formation with some samples and protocols, M22 cannot prevent ice nuclei from forming at cryogenic temperatures. Ice nuclei are local clusters of water molecules that rotate into an orientation that favors later growth of ice crystals when a solution is warmed. Ice nuclei are not damaging, but the fact that ice nuclei can form indicates molecular mobility which could be damaging. Specifically, between the temperatures of −100°C and −135°C, ice nuclei can form in M22, with the maximum ice nucleation rate occurring near Tg. At −140°C the ice nucleation rate for M22 is undetectable. But nuclei will be probably formed in cooling to −140°C.

Although cryostorage at −140°C is an attempt to minimize cracking and minimize nucleation, this ITS neither eliminates cracking nor ice nuclei formation. Cryonics patients slowly cooled from Tg to −140°C will surely experience some ice nucleation. Alcor places a listening device (“crackphone”) under the skull of its cryonics patients for the purpose of monitoring cracking events. My understanding is that for most Alcor patients the crackphone detects cracking at Tg or only slightly below Tg, although there was reportedly one M22-perfused patient for which the first fracturing event occurred at −134°C. The propylene glycol experiments would support the view of cracking occurring slightly below Tg, but vitrified biological samples resist cracking better than pure cryoprotectant solutions.

With ice formation, cracking could occur at temperatures higher than Tg. Although ITS may prevent the formation of cracking that could occur in cooling below −140°C, it does not prevent the cracks that occur in cooling from Tg to −140°C. I have wondered whether there are forms of damage which would occur in a cryonics patient stored at −140°C that would not occur during storage at −196°C. A solid cryogenic state of matter does not prevent molecular motion. Molecular motion in a biological sample held at cryogenic temperature could result in damage to that sample.

Ions generated by radiation are much more mobile than molecules. An ionic species (probably protons) in trimethylammonium dihydrogen phosphate glass is nine orders of magnitude more mobile than the glass molecules—and sodium ions in sodium disilicate glass are twelve orders of magnitude more mobile than the glass molecules.9

Cryobiologist Peter Mazur has stated that below −130°C “…viscosity is so high (>1013 Poise) that diffusion is insignificant over less than geological time spans.” He adds that “…there is no confirmed case of cell death ascribed to storage at −196°C for some 2-15 years and none even when cells are exposed to levels of ionizing radiation some 100 times background for up to 5 yr.”10 Frozen 8-cell mouse embryos subjected to the equivalent of 2,000 years of background gamma rays during 5 to 8 months in liquid nitrogen showed no evident detrimental effect on survival or development.11

In attempting to evaluate damaging effects of temperature and radiation, it could be valuable to analyze chemical alterations, rather than complete cell death or viability. Acetylcholinesterase enzyme subjected to X-ray irradiation shows conformational changes at −118°C, but no conformational changes when irradiated at −173°C.12 X-ray irradiation of insulin and elastase crystals resulted in four times as much damage to disulfide bridges at −173°C compared to −223°C.13 Another study showed a 25% crystal diffraction lifetime extension for D-xylose isomerase crystals X-ray irradiated at less than −253°C compared to those irradiated at −173°C.14

One study showed that lettuce seeds show measurable deterioration when stored at liquid nitrogen temperature for periods of 10 to 20 years. Rotational molecular mobility was quantified. A graphical plot was generated showing increasing times for when 50% of lettuce seeds would fail to germinate as a function of decreasing temperature. Those times were estimated to be about 500 years for −135°C and about 3,400 years for −196°C.15 Translational vibrational motion has been given as an explanation for seed quality deterioration at cryogenic temperatures.16 The mean square vibrational amplitude of a water molecule is not even zero at 0 Kelvins (−273°C), and has been determined to be 0.0082 square Angstroms. The mean square vibrational amplitude is 0.0171 square Angstroms at −173°C and 0.0339 square Angstroms at −73°C.17

Realistically, however, 3,400 years is much longer than cryonics patients are likely to be stored. Storage in liquid helium at −269°C or in a shadowed moon crater at −235°C18 would certainly be more trouble than it is worth. Northern wood frogs spend months in a semi-frozen state at −3°C to −6°C, and are able to revive with full recovery of heartbeat upon re-warming.19 An empirical study of a cryoprotectant very similar to M22 (VS55)
showed viscosity continuing to increase exponentially below Tg, just as viscosity increases exponentially with temperature decrease above Tg.20 The exponential decrease in viscosity (molecular mobility) that makes ice nucleation cease at −135°C indicates that there is probably little molecular mobility at −140°C, despite the possibility of damage from ionic species or vibrational motion. All things considered, however, my personal preference is for storage in liquid nitrogen, rather than some intermediate temperature above −196°C. I would also prefer for cryogenic nanorobot repair to be at liquid nitrogen temperature.

I am by no means a nanotechnology expert, but I can give a brief description of my own views of how cryogenic nanotechnology repair of a cryonics patient would proceed. I must thank Ralph Merkle for his assistance in allowing me to consult with him to formulate and clarify many of my views. I believe that repair of cryonics patients at cryogenic temperature would be a combination of nano-mining and nanoarcheology. Nanorobots (nanometer-sized robots) would first clear blood vessels of water, cryoprotectant, plasma, blood cells, etc. The blood vessels would become mining shafts that would provide access to all body tissues. Nanometer-sized conveyor belts or trucks on rails could remove blood vessel contents. Where freezing or ischemia had destroyed blood vessels, artificial shafts would be created. Unlike the nano-mining that simply removes all blood vessel contents, the creation of artificial shafts would have the character of an archeological dig. Care would be taken in removing material to avoid damaging precious artifacts that might indicate original structure ― which could
be discovered at any unexpected moment.

Section 13.4 of K. Eric Drexler’s book Nanosystems provides diagrams and details of a nanorobot manipulator arm. Such a “diamondoid” component would contain about four million atoms, and could be fitted with a variety of tools at the end of the arm. A variety of tips with varying degrees of chemical reactivity could allow for reversible, temporary chemical bonds that could be used for grabbing and moving molecules. These could range from radicals or carbenes that would form strong covalent bonds, to boron that can form relatively weak and reversible bonds to nitrogen and oxygen, to simple O-H groups that can form even weaker hydrogen bonds. Tools for digging need not be so refined. The manipulator arm is depicted as being 100 nanometers long and 50 nanometers wide, although nanorobots would need to be larger to include capability for locomotion, computation, and power. A complete nanorobot could be as large as a few thousand nanometers in size. A capillary is between 5,000 to 10,000 nanometers in diameter, so there should be plenty of room for many such nanorobots to operate. Ralph Merkle estimates that 3,200 trillion nanorobots weighing a total of 53 grams could repair a cryonics patient in about 3 years.21,22 Like many of the calculations associated with nanotechnology, I take these figures with a pound of salt. It is certainly true, however, that it could take years to repair a patient, and that there should not be a rush to finish the job.

Merkle & Freitas have suggested that nanorobots be powered by electrostatic motors. Stators and rotors would be electric rather than magnetic. Tiny moving charged plates are easier to fabricate than tiny coils and tiny iron cores, but more fundamentally, magnetic properties do not scale well with reduced size (i.e., molecular-scale magnetic motors don’t work), whereas electrostatic properties do scale well with reduced size. Electrostatic actuators are already being used in microelectromechanical systems (MEMS).23 High density batteries could provide power for days, and recharging stations could be located throughout the patient. Alternatively, nanotube cables could bring power to the patient from the outside. Such cables could also be a means of transmitting and receiving computational data. Nanotube cables could also be used to reunite fracture faces
created by cracking. Scanning and image processing capabilities would need to evaluate what needs to be fixed.

As much as possible I would favor replacement rather than repair, which would greatly simplify the process. It would be much easier to replace a kidney than to repair the diseased kidney of an elderly patient who died of kidney disease. Curing disease and rejuvenation would thus become part of the repair of a cryonics patient. Of course, neuro patients would require an entirely new body. The brain would be the major exception to replacement strategy because the brain could not be replaced without loss of memory and personal identity.

Even within the brain, however, it could be feasible to replace many components without loss of memory and personal identity. It could be feasible to replace many organelles such as mitochondria, lysosomes, etc., and many macromolecules such as proteins, carbohydrates, and lipids. DNA could be repaired, and possibly even modified to cure genetic disease, but epigenetic expression in neurons may be critical for reconstruction of synaptic structure. Synaptic connections would not only be restored, but the quantity and quality of neurotransmitter contents should be restored. It is not simply a matter that some neurotransmitters are inhibitory and others are stimulatory. There are more than 40 different neurotransmitters used in the brain, and there must be a good reason why such variety is necessitated.

Part of the repair process could involve removal of ice nuclei, nearly all of which would be extracellular. Re-created blood vessel contents would include fresh cryoprotectant, water, plasma, and blood cells without the original ice nuclei. Although some repair scenarios favor different types of repair above cryogenic temperature, I doubt that this is necessary or desirable. Alternative repair scenarios involve splitting the brain in half, and halving the halves repeatedly at cryogenic temperature—with digitization at each step—until the brain has been totally digitized.21,22 Or digitization could be done by repetitive nano-microtomes at cryogenic temperature. The digital data could be used for full reconstruction. Some people might object that if one individual could be created from digital data, many such individuals could be created—raising questions of which are duplicates and
which is the original. There is detailed discussion of the duplicates problem/ paradox in the philosophy section of my website BENBEST.COM.

Although other repair scenarios could prove to be feasible, I believe that cryogenic nanotechnology will be required for all cryonics patients in the foreseeable future until the problem of cryoprotectant toxicity can be solved. With effective nontoxic cryoprotectants, sufficient cryoprotectant could be used to prevent ice nuclei formation at all temperatures, prevent devitrification (freezing) upon rewarming, and eliminate all toxic damage. In such a case, there could be true reversible cryopreservation (suspended animation).

What is needed to create the nanotechnology required for repair of cryonics patients? Small machines will need to build parts for smaller machines, which would in turn build even smaller machines. Many details of machine
operation must be perfected at each stage. Current modern technological civilization began with cave people pounding on rocks. Ralph Merkle has said that compared to future technology, current technology is pounding on rocks.

References

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

2. Kudernac T, Ruangsupapichat N, Parschau M, Maciá B, Katsonis N, Harutyunyan SR, Ernst KH, Feringa BL. Electrically driven directional motion of a four-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 DNA hybridization efficiency by pH-driven nanomechanical bending. Langmuir. 2012 Apr 17;28(15):6494-501.

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

5. Best B. The Cryonics Institute’s 95th Patient. Long Life. 2009 Sept-Oct; 41(9- 10):17-21.

6. Wowk B. Systems for Intermediate Temperature Storage for Fracture Reduction and Avoidance. 2011 Third Quarter;32(3):7-12.

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

8. Angell CA. Entropy and Fragility in Supercooling Liquids. Journal of Research of the National Institute of Standards and Technology. 1997 March-April; 102(2):171-185.

9. Mizunoa F, Belieresa J.-P, Kuwatab N, Pradelb A, Ribesb M, Angell CA. Highly decoupled ionic and protonic solid electrolyte systems, in relation to other relaxing systems and their energy landscapes. 2006 Nov;352(42/49):5147- 5155.

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

11. Glenister PH, Whittingham DG, Lyon MF. Further studies on the effect of radiation during the storage of frozen 8-cell mouse embryos at -196 degrees C. J Reprod Fertil. 1984 Jan;70(1):229-34.

12. Weik M, Ravelli RB, Silman I, Sussman JL, Gros P, Kroon J. Specific protein dynamics near the solvent glass transition assayed by radiation-induced structural changes. Protein Sci. 2001 Oct;10(10):1953-61.

13. Meents A, Gutmann S, Wagner A, Schulze-Briese C. Origin and temperature dependence of radiation damage in biological samples at cryogenic temperatures. Proc Natl Acad Sci U S A. 2010 Jan 19;107(3):1094-9.

14. Chinte U, Shah B, Chen YS, Pinkerton AA, Schall CA, Hanson BL. Cryogenic (<20 K) helium cooling mitigates radiation damage to protein crystals. Acta Crystallogr D 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 aspects of vitrification. Cryobiology. 2010 Feb;60(1):11-22.

17. Leadbetter AJ; The Thermodynamic and Vibrational Properties of H$_2$O Ice and 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, Schofield JT, McCleese DJ, Foote MC, DeJong E, Bills BG, Hartford W, Murray BC, Allen CC, Snook K, Soderblom LA, Calcutt S, Taylor FW, Bowles NE, Bandfield JL, Elphic R, Ghent R, Glotch TD, Wyatt MB, Lucey PG. Diviner Lunar Radiometer observations of cold traps in the Moon’s south polar region. Science. 2010 Oct 22;330(6003):479-82.

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

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

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

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

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

From June 3 to 6, 2012 I attended the annual Society for Cryobiology meeting, which in 2012 was held in Rosario,Argentina.

Attending with Argentine biogerontologist and Cryonics Institute Member Rudy Goya may have reduced the interaction I had with the cryobiologists. There were fewer sessions than usual, and thus more free time. The welcome reception was not held until the evening of the first day of the sessions.

The first session dealt with an aspect of Argentine cultural heritage, the Llullaillaco children — three Inca children who had been mummified by dehydration high on a volcano and preserved for over 500 years. Two of children were selected by the Incas because they were “perfect” (beautiful and pure) at 6 or 7 years of age. It was believe to be an honor to go directly to heaven, not really death or sacrifice. The children were given an intoxicant and buried alive atop the Llullaillaco volcano. Much of the session focused on the conditions that caused the children to be so well-preserved, and the conditions the curators should use to preserve the children for the future — involving careful regulation of temperature, atmosphere, humidity, and an environment inhospitable to most microbes.

If reanimated cryonicists receive anything like the care these children are receiving, there should be no concerns about being welcome in the future. In a sense, the Incas had it right when thinking they were sending the children to heaven. Of course the Inca children were deprived of life and are unable to experience or enjoy their treatment by modern curators — and cryonicists should not encourage hastening death based on reliance on unproven future technologies.

At this conference there were special “How to do it?” sessions overlapping part of the lunch hour that focused on practical techniques unrelated to the experimental results and theoretical considerations covered in the regular sessions. Sunday’s topic was proteomic analysis, which covered removal, isolation, and identification of proteins from cells. The presenter (from the Institute of Molecular Cell Biology in Rosario) claimed that instrumentation allowing high throughput and resolution had given proteomics a maturity comparable to genomics.

The afternoon sessions were concerned with cell and tissue preservation. Elza Cabrita reported on improved cryopreservation of fish sperm through a combination of cryoprotectants and antioxidants. Locksley McGann reported on experiments sequentially exposing human articular cartilage to four CPAs (DMSO, glycerol, propylene glycol, and ethylene glycol) at lowering temperature (0ºC, −10ºC, −15ºC). Vitrified samples were cooled to liquid nitrogen temperature, and demonstrated 75% cell recovery when rewarmed.

Adam Higgins reported on an improved procedure for washing glycerol from red blood cells. Currently about 99% of banked blood is stored at refrigerator temperature (2-4ºC), with a shelf life of 42 days. Only 1% of blood (mostly rare blood types) is cryopreserved with glycerol and stored at −80ºC, with a shelf life of ten years. A major deterrent preventing more blood from being banked at −80ºC is the 30-60 minute glycerol washout procedure. Adam’s group developed a procedure that can wash the glycerol out in 30 seconds, but 5 seconds longer or shorter results in too much hemolysis. A three minute washout procedure is less time sensitive (one minute longer or shorter is tolerable), but the method needs to be scaled-up from the 0.5 milliliter test volumes being used.

On Monday, Peter Mazur reported that in vitrifying mouse oocytes, it is the warming rate and not the cooling rate that is most critical for success. He spoke of microwave warming and the problem of thermal runaway (uneven warming). Ice blockers would not cross cell membranes, and thus would not be of use against intracellular ice formation. Pier Morin reported on miRNA microarray assessment of miRNA expression of the freeze-tolerant insect goldenrod gall fly at control (+5ºC) and freezing (−15ºC) temperatures. mIR-210 was down-regulated and mIR-1 was up-regulated at freezing temperature (the latter is involved in cell cycle regulation).

Ali Eroglu reported on epigenetic perturbation resulting from human oocyte cryopreservation techniques. Both the slow freezing and vitrification methods he used resulted in down-regulated expression of H19 and Ube3a genes. Igf2r was down-regulated by vitrification, but not by slow freezing.

Monday’s “How to do it?” session described a combination of nanotechnology and stem cells for tissue engineering. Specifically, electrospinning can be used to create a nanometer scale web of biodegradable fibers that can be populated with mesenchymal stem cells by electrospraying. The main challenge is vascularization of the tissue. Vascular Endothelial Growth Factor (VEGF) increases cell adhesion, but not necessarily vascularization.

Barry Fuller reported on successful hypothermic perfusion of liver. A kidney hypothermic perfusion machine has been in operation for ten years, but liver has been more challenging, because of its large size and the fact that two vessels supply the organ (hepatic artery and portal vein). The liver hypothermia perfusion machine uses two pumps.

PhD student Na Guan described her study of gene expression changes associated with chilling injury of rat liver slices. Cryoprotectant solutions supplied by 21CM (Greg Fahy) were used to ensure no ice formation interfered with the process. ATP levels indicated that the cryoprotectant solutions used were causing no damage, although the composition of those solutions was not disclosed. 1108 genes were observed, of which 251 were up-regulated and 77 were down-regulated by chilling at −15ºC. Focusing on the top ten up- and down-regulated genes: inflammatory and DNA repair genes were considerably up-regulated, and genes associated with biosynthesis of cholesterol and polyunsaturated fatty acids were down-regulated. The latter seems paradoxical in light of the up-regulation of cell surface-linked signaling pathways, which indicate cell membrane injury.

During the question period, both Andreas Sputtek and Arthur Rowe were sharply critical of the undisclosed composition of the 21CM cryoprotectant solutions being used. Sputtek said that because science is about disclosure of methods and materials, Guan’s work was not science. Guan said she had begged 21CM for disclosure, but said she was told that anyone wanting to replicate the experiments could buy the solutions from 21CM. Tiantian Zhang said that gene analysis only done 30 minutes after chilling injury does not give the whole picture. She said that in her own work doing gene analysis of fish oocytes or embryoes after chilling injury, gene expression changes dramatically with time — that it is a mistake to only analyze the expression 30 minutes after exposure as Guan had done. After the presentation, Arthur Rowe spoke with Guan telling her how much trouble he has had over the years with her collaborator (Dr. Fahy) in connection with the non-disclosure issue. I spoke with Guan myself after her presentation. She told me that the greatest chilling injury occurs at −90ºC. She also said that she would be getting her PhD in July and did not know who would be continuing her work. When I spoke to Dr. Fahy about the presentation, he told me that the composition of the vitrification solution had been disclosed and that Guan was mistaken in believing that she could not disclose the composition.

Tuesday morning had been scheduled to begin with a lecture by Ken Storey. Storey typically has no interest in what other cryobiologists have to say, is fairly ignorant of areas of cryobiology outside of hibernation and effects of low temperature on animals in nature, and only comes for his own presentation before leaving. His ignorance is on display when journalists get him to do cryonics-bashing, which he does with relish, but the general public only sees the comments of a respected cryobiologist, not the ignorant misunderstandings of cryobiology. I would not have expected Storey to come all the way to Rosario, Argentina only for his own presentation, but this is what he attempted to do — and he missed one of his flight connections. Ironically, this year Storey was honored by being made a Fellow in the Society for Cryobiology. Storey does, admittedly, have a fabulous knowledge of molecular biology, and is an outstanding scientist in connection with his own work.

To compensate for Storey’s absence the conference organizers arranged a makeshift follow-up session on the Llullaillaco children. This wasn’t entirely a waste, because many issues had not been addressed in the first round. I was going to question using a 2% oxygen and 98% nitrogen atmosphere for the children rather than pure nitrogen, but Barry Fuller raised this objection before I was called upon. I did, nonetheless, suggest that the goal should be to perfect the preservation environment rather than try to recreate the conditions of the mountain. Even this had not been done because the relative humidity had been raised to 70% on the bad advice of an expert rather than held to the 40% present on the volcano. The children were reportedly gaining 300 grams per year, probably from the humidity. There is a lower humidity limit below which no microorganisms can grow, but 0% relative humidity in the −20ºC preservation chambers would run the risk of freeze-drying.

For the second session on Tuesday, John Crowe had been scheduled to lead a symposium composed of 3 other speakers besides himself, but all of the other 3 speakers cancelled-out. John, nonetheless, did an excellent job of speaking for the whole session on the basis of his own work. John is an expert in dehydration and freeze-drying of organisms as well as on tardigrades and trehalose. Drying DNA with trehalose prevents fragmentation, and drying proteins with trehalose prevents denaturation. John discovered that drying liposomes with trehalose prevents membrane fusion — although he lost most of the patent rights on commercially valuable processes by publishing too soon. Dehydration of samples containing sucrose drives the glass transition temperature (Tg) from 20ºC to 60ºC, but dehydration of samples containing trehalose raises the Tg from 20ºC to 120ºC. More recently, however, it has been found that LEA proteins can be as protective as trehalose, but in a way that is distinctive and complementary to trehalose — stopping liposome fusion, preventing protein aggregation, and changing sample Tg. Yeast cells are protected against dehydration damage not only by trehalose, but by the trehalose transporter protein which exports the trehalose to the exterior membrane surface and imports the trehalose to the internal membranes of organelles such as mitochondria. But although the genome of tardigrades has been sequenced, the tardigrade trehalose transporter has not yet been identified.

Barbara Reed is probably the world’s foremost expert on plant cryopreservation, and she has spoken a lot about the benefits of antioxidants for cryoprotection. But the presentation Barbara gave on Wednesday gave me the strongest indication that oxidative stress could be a significant mechanism of cryoprotectant toxicity. Not only because a variety of cryopreserved plants show improved viability with Vitamin E, Vitamin C (if iron is removed), lipoic acid, glutathione, and melatonin — but because oxidative damage was shown to increase significantly associated with cryoprotectant loading.

Roland Fleck works with the UK Stem Cell Bank. The Bank conducted studies indicating that a 2-step freezing protocol results in better viability than vitrification. But examining the results of 8 technicians showed that in the hands of the most experienced technician vitrification was as effective as the 2-step freezing protocol. Protocols should not be so highly dependent upon technician expertise. After his presentation, Roland told me he was concerned that he was only able to assay viability by the use of trypan blue, which only indicates membrane integrity and does not provide a very fine measure of cell function. He said that the requirement to use the trypan blue viability assay was imposed by bureaucrats or scientists who do not have much knowledge of cryobiology.

Igor Katkov said that he believes any sperm cell can be vitrified simply by choosing the right cooling and warming rate. He said he was advised by his patent attorney to drop seven slides from his PowerPoint presentation.

At the business meeting the Society membership was reported to be down to 186. The journal CRYOBIOLOGY continues to be profitable. CRYOBIOLOGY has a 33% rejection rate, a 1.83 impact factor, and 33 Members on the Editorial Board. The Society has $300,000, which the IRS thinks is too much for a charitable organization, but the IRS is allowing the society to retain tax-exempt status. Increasing travel awards is the preferred use of money, but there is a problem that on the one hand travel awards are a taxable benefit, and on the other hand it is illegal to pay the taxes on travel awards. The 2013 conference (the 50th annual conference) is to be held in Washington, DC, where the first conferences were held. The 2014 conference might be Istanbul, Turkey and the 2015 conference could be in Isreal, but definite decisions have not been made.

Last year’s new Society for Cryobiology Fellows Barbara Reed and John Crowe each gave presentations reviewing their careers. Barbara Reed began as a plant biologist in 1985, but was brought into the field of cryobiology by a need to preserve germ tissue. John Crowe said that after the Sputnik shock of 1957 the US government sought to encourage more young people to go into science, including him. As a teenager, John was sent to a number of different science laboratories on his summer vacations. John considers himself more of a “dryobiologist” than a cryobiologist. He entertained us with photos taken in the many exotic countries he and and his wife have visited since his retirement.

The two new Fellows for 2013 are Ken Storey and Mehmet Toner.

This conference was attended by not more than about 80 people, at least half of whom were South America. There were maybe 30 or so hard-core Society for Cryobiology Members. This was my 9th annual meeting in a row, but for the most part I made little effort to relate to the cryobiologists, although one of my intentions in attending these meetings has been to soften the hostility of cryobiologists to cryonicists. I sat near the front of the meetings with Rudy who told me that he learned a great deal about the cryobiology behind cryonics practices by attending this conference. Very many of the cryobiologists were reporting on using vitrification at this conference, and including articular cartilage and plant tissue as well as single cells. I was fairly active in my questioning and comments — about which a few of the cryobiologists complimented me.

I lost my sense of urgency about talking to Peter Mazur. Peter recently told a journalist that although it is not possible to prove that the chance of cryonics patients being reanimated are zero, “you can, I think demonstrated that the probability of its being done is so extremely low that effectively it is zero”  [CANADIAN MEDICAL ASSOCIATION JOURNAL; Monette, M; The Church of Cryopreservation; 184(7):749 (2012)] I am curious about the demonstration Peter has in mind, but I am also committed to learning from cryobiologists rather than arguing with them about cryonics. Peter walked away a few years ago when I asked him when solution effects rather than mechanical damage cause injury to cells due to slow cooling, so that may be a touchy subject with Peter as well.

I did, however, pepper John Crowe with questions — finding him to be friendly and informative. John confirmed what Peter Mazur had told me about cells being able to tolerate the loss of all osmotic water (freezable water, which constitutes at least 80% of cell water) without injury — a matter of great relevance in the vitrification of cryonics patients (assuming inter-cellular effects are not of great significance).

I sought-out Ali Eroglu, with whom I have had little interaction in the past, calling his attention to an article in the most recent issue of CRYOBIOLOGY about transfection of mammalian ovary cells with trehalose [CRYOBIOLOGY; Chakraborty,N; 64(2):91-96 (2012)]. Ali has microinjected oocytes with trehalose (along with low concentrations of DMSO to protect the mitochondria) [BIOLOGY OF REPRODUCTION; Eroglu,A; 80(1):70-78 (2009)]. Ali had not seen the CRYOBIOLOGY article, but he told me that ovarian tissue is easier to work with than oocytes.

At the final banquet I sat next to one of the conference organizers. He told me that John G Baust had been supposed to conduct a symposium, but had cancelled the whole thing a month before the conference without giving any explanation. He agreed with the comments I had made about the Llullaillaco children, and told me that a committee of cryobiologists was going to supplement the questionable advice that the Argentine government has been getting from a single advisor in New York. He told me that National Geographic had discovered the children and attempted to remove them from Argentina on a midnight flight, but the Argentine government got wind of the plan and intervened. Nonetheless, the children were simply kept in −20ºC freezers for several years while planning and building better preservation chambers.

The return bus trip to BA on Thursday took the entire afternoon — much longer than I would have expected. I sat next to Adam Higgins on the bus, and spoke with him much of the time, mostly about his life and work, as well as about our experiences in Argentina. Adam knew Spanish fairly well because he has spent four months of language immersion living in Equador (and visiting the Galapagos Islands). If he gets a patent for deglycerolizing blood, the University would get half the royalties and he would split his half with his collaborators. The advantages of his method would be the ten year rather than 42-day shelf life for banked blood, and the greatly reduced washout time. The latter is a significant savings in labor costs, but would have to be weighed against greater electrical costs for a −80ºC freezer as opposed to refrigeration. Even if he is successful in perfecting his methods, he thinks that the blood banking industry is too conservative to be captivated by superior storage methods. Adam has attended most of the annual conferences since I began attending in 2004, and told me that he would like to become a Governor of the Society. Not once did Adam ask me what work I do, and he evidently does not know because he was surprised when I told him I am not a Member of the Society for Cryobiology. Whether or not I am formally accepted as a Member, my attendance at these conferences is implanting me into the consciousness of the cryobiologists as being a member of their community.