15. October 2014 · Comments Off on Effects of Temperature on Preservation and Restoration of Cryonics Patients · Categories: Cryonics, Science

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

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

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

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