Annotated bibliography of cryoprotectant toxicity

Introduction

Cryoprotectant toxicity should be distinguished from other mechanisms of cryopreservation injury such as chilling injury (injury produced by too low temperatures as such) and cold shock  (injury produced by rapid cooling). Cryoprotectant toxicity itself can again be divided into general cryoprotectant toxicity and specific cryoprotectant toxicity. General cryoprotectant toxicity involves concentration (water substitution) effects of cryoprotectants and specific cryoprotectant toxicity involves the effects of individual compounds on cellular viability. General cryoprotectant toxicity presents a formidable obstacle for cryopreservation methods that require very high concentrations of cryoprotectant agents (such as vitrification).

Another mechanism of injury that is rarely discussed in the cryobiology literature but that can complicate cryopreservation of complex organs is “non-specific” dehydration injury. In light of the fact that the current generation of vitrification agents are delivered in hypertonic carrier solutions and contain non-penatrating cryoprotective agents which do not cross the blood brain barrier, this form of damage may be especially important in cryopreservation of the brain.

Systemic reviews of cryoprotectant toxicity are rare but some mechanisms for (specific) cryoprotectant toxicity have been proposed including, but not limited to, protein denaturation, modification of biomolecules, membrane injury, destabilization of the cytoskeleton, oxidative damage, and ATP depletion. It is important to stress that some of the mechanisms may be downstream effects of other mechanisms. For example, ATP depletion can cause oxidative damage. And as Gregory Fahy has pointed out, cryoprotectant toxicity should be distinguished from injury associated with the method of introduction and washout of the cryoprotectant. In 2004, Fahy, Wowk et al., proposed a compositional variable to predict general cryoprotectant toxicity.

Cryoprotectant toxicity can also vary by species and organ type. Cryoprotectants that are moderately toxic in one species can be highly toxic in others. Similarly, cryoprotectants that are moderately toxic in one organ can be highly toxic in others (or even between different types of cells within organs). This raises the question of whether universal non-toxic cryoprotective agents are attainable (a requirement for reversible vitrification in complex organisms).

Cryoprotectant toxicty can be investigated by cryopreserving an organ (or cell) and measuring its viability after rewarming and washout of the cryoprotective agent. To eliminate the influence of other mechanisms of injury associated with cryopreservation (such as ice formation), a cell can just be loaded and unloaded with the cryoprotectant without cryopreservation. The effects of hypothermia on viability can be eliminated altogether by normothermic perfusion of the organ. This, of course,  introduces a challenge for hypoxia sensitive organs such as the heart and the brain because cryoprotective agents may not be good oxygen carriers.

Papers

Baxter SJ, Lathe GH (1971). Biochemical effects of kidney of exposure to high concentrations of dimethyl sulphoxide.
Biochemical Pharmacology. Jun; 20(6): 1079-91.

Baxter and Lathe investigated the effect of high concentrations of DMSO on kidney preparations. In a series of illuminating experiments, the investigators established that anaerobic glycolysis was reduced in slices and homogenates as a result of increased activation of the gluconeogenesis enzyme Fructose 1,6-diphosphatase (FDPase). DMSO-induced activation of FDPase can be inhibited by adding an amide or lysine to DMSO. The finding that a combination of DMSO and an amide allows for less toxic cryoprotectants formed the basis of subsequent investigations of GM Fahy for potent vitrification solutions.

Fahy GM (1983). Cryoprotectant Toxicity Neutralizers Reduce Freezing Damage.
Cryo-Letters 4: 309-314.

In this paper GM Fahy reports the ability of toxicity neutralizers urea, formamide, and acetamide (all amides) to reduce injury of cryopreserved renal cortical slices with DMSO. In later research papers Fahy will establish that DMSO neutralizes the toxicity of formamide, and not the other way around.

Fahy GM (1984). Cryoprotectant toxicity: biochemical or osmotic?
Cryo-Letters 5: 79-90.

If osmotic stress is an important cause of injury during introduction and removal of cryoprotectant agents, improved viability can be obtained by reducing the rate of cryoprotective agent introduction and removal. Fahy reviews the literature and presents data obtained in renal cortical slices that indicate that substantial hypertonic osmotic stress does not produce major changes in viability. Conversely, reducing exposure time to higher concentrations of the cryoprotectant can contribute to improved viability. These results suggest that biochemical toxicity, not osmotic stress, is the major factor in cryoprotectant-induced injury.

Fahy GM (1984). Cryoprotectant toxicity: specific or non-specific?
Cryo-Letters 5: 287-294

Fahy reviews the argument (Morris, Cryoletters 4, 339-340, 1983) that the lower toxity of cryoprotectant solutions that contain DMSO and amides can be entirely explained by the lower absolute concentration of DMSO. Fahy points out that the original Bexter and Lathe experiments demonstrated that solutions with the same absolute amount of DMSO (4.6 M) but with or without amides had different effects on glucose utilization. The author also presents data showing that “simple substitution (“dilution”) of one agent for another strikingly fails to reduce overall toxicity over a very critical range of DMSO concentration.” Also briefly discussed is the possibility of mutual toxicity neutralization between DMSO and amides, a topic that would be further explored by Fahy in future research.

Fahy GM, MacFarlane DR, Angell CA, Meryman HT (1984). Vitrification as an approach to cryopreservation.
Cryobiology.  Aug ; 21(4): 407-26.

In this paper on vitrification as an alternative to conventional cryoprotection, Fahy et al., list a number of methods for reducing cryoprotectant toxicity:

Primary (direct) methods:

  1. Maintain temperature as low as possible;
  2. Select an appropriate carrier solution;
  3. Keep exposure time at higher concentrations to a minimum;
  4. When possible, employ specific cryoprotectant toxicity neutralizers.

Secondary (indirect) methods:

  1. Avoid osmotic injury;
  2. Mutual dilution of cryoprotectants may be helpful in some instances;
  3. Use extracellular cryoprotectant to reduce exposure to intracellular cryoprotectant when possible.

The most important insights, some of which are still maintained in the current generation of vitrification solutions, concern toxicity neutralization, the choice of an appropriate carrier solution, and the use of extracellular cryoprotectants.

Fahy GM (1986). The relevance of cryoprotectant “toxicity” to cryobiology.
Cryobiology. Feb; 23(1) :1-13.

Fahy presents evidence that cryoprotectants themselves can present a source of injury. As a consequence, the advantages of higher concentrations of the cryoprotective agents does not necessarily produce higher viability after freezing, even when this allows for greater ice inhibition. He reviews data on “cryoprotectant-associated freezing injury” for DMSO, ethylene glycol, methanol, ethanol, and glycerol.  Because vitrification requires very high concentrations of cryoprotective agents, toxicity is the key limiting factor in reversible vitrification of organs.

Fahy GM, Lilley TH, Linsdell H, Douglas MS, Meryman HT (1990). Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms.
Cryobiology. Jun; 27(3): 247-68.

Fah,y et al., delineate 6 criteria that must all be met simultaneously in order for a putative mechanism of cryoprotectant toxicity to be implicated:

  1. The relationship between observed biochemical alteration and cellular viability must be clear or easily plausible;
  2. The maginitude of the cryoprotectant effect must be large enough to be significant;
  3. The effect must be irreversible over a reasonable time span after removal of the cryoprotectant;
  4. The time course of the observed effect must be consistent with the time course of observed injury;
  5. The cryoprotectant effect must be possible under conditions that could reasonably be encountered inside a living cell being prepared for freezing or being subjected to freezing and thawing itself;
  6. The cryoprotectant effect must be due to the cryoprotectant itself and not due to the technique of introduction and washout.

The authors investigate the proposed mechanisms for the biochemical effects of DMSO toxicity in the 1971 Baxter study and find that a) the effect of DMSO on FDPase activation is too small to affect the normal respiration of the cell and therefore fails to meet criterion 2 to be a significant mechanism of cryoprotectant toxicity; b) the presence of formamide does not affect the interaction between DMSO and lysine; and c) toxicity is not consistently reduced by blocking alteration of FDPase rather than substituting those compounds for DMSO.

The authors further present results that do not support the theory that generalized  protein denaturation is related to cryoprotectant toxicity.  The article ends with a referenced list of phenomena possibly related to mechanisms of cryoprotectant toxicity.

Fahy GM, da Mouta C, Tsonev L, Khirabadi BS, Mehl P,  Meryman HT (1995). Cellular injury associated with organ cryopreservation: Chemical toxicity and cooling injury.
Editors: John J. Lemasters, Constance Oliver. Cell Biology of Trauma, CRC Press

Fahy, et al., review different mechanisms of cryoprotectant toxicity with a particular focus on DMSO-medicated chemical injury. Mechanisms discussed include fructose-1,6-bisphosphatase activation, sulfhydryl oxidation, activation of extracellular proteinases and endothelial cell detachment and death. The article lists a number of interventions that do not change CPA-medicated injury such as inhibition calcium mediated injury or protein denaturation. The authors also report how the toxicity of formamide can be completely reversed by addition of DMSO.

Bakaltcheva IB,  Odeyale CO, Spargo BJ (1996). Effects of alkanols, alkanediols and glycerol on red blood cell shape and hemolysis.
Biochimica et Biophysica Acta. 1280: 73-80

In this elegant and thoughtful paper, the authors use the human red blood cell to study cryoprotectant toxicity. Morphological observations, quantification of hemolysis, measurements of the dielectric constant of the incubation medium (Ds) and the dielectric constant of the erythrocyte membrane in the presence of organic solutes (Dm), are used to investigate cryoprotectant toxicity in a series of alkanols, alkanediols, and glycerol. The authors propose that toxicity of a cryoprotectant is related to its ability to change the ratio of Ds/Dm. Changes in this ratio reflect changes in the difference between hydrophobicity of the solution and the membrane, with decreases in this ratio leading to increased exposure of membrane surface area and vesiculation, and increases in this ratio leading to decreased exposure of membrane surface area and cell fusion. The authors suggest that the design of less toxic cryoprotective agents should involve the maintenance of dielectric homeostasis of the medium and the membrane. Their findings also throw light on the observation that combinations of various cryoprotectant agents (such as DMSO and formamide) can reduce the overall toxicity of a solution.

Fahy GM, Wowk B, Wu J, Paynter S (2004). Improved vitrification solutions based on the predictability of vitrification solution toxicity.
Cryobiology. Feb; 48(1): 22-35.

This seminal paper on non-specific cryoprotectant toxicity represents a major contribution to the cryobiology literature in general, and enabled the authors to formulate less toxic vitrification solutions for the cryopreservation of whole organs. In the paper the authors propose a new compositional variable that reflects the strength of water-cryoprotectant hydrogen bonding called qv*. Contrary to the cryobiology wisdom to date, the authors found that weaker glass formers favor higher viability. As a consequence, vitrification agents with higher concentrations of cryoprotective agents are not necessarily more toxic. Although qv* is not helpful in predicting specific cryoprotectant toxicity, this paper, and the research that is reflected in it, suggests that non-specific cryoprotectant toxicity is mediated through the effects of penetrating cryoprotectant agents on the hydration of biomolecules.

Intermediate temperature storage in cryonics

The recent issue of Cryonics magazine features a comprehensive update on intermediate temperature storage (ITS). This article contains an important observation:

Acoustic events consistent with fracturing were found to be universal during cooling through the cryogenic temperature range.  They occurred whether patients were frozen or vitrified.  If cryoprotection is good, they typically begin below the glass transition temperature (‑123°C for M22 vitrification solution).  If cryoprotective perfusion does not go well, then fracturing events begin at temperatures as warm as -90°C.  Higher fracturing temperatures are believed to occur when tissue freezes instead of vitrifies because freezing increases the glass transition temperature of solution between ice crystals.  The temperature at which fractures begin is therefore believed to be a surrogate measure of goodness of cryoprotection, with lower temperatures being better.

This is an important observation because one of the arguments that has been made against intermediate temperature storage is that Alcor routinely records fracturing events above the nominal glass transition temperature (Tg) of the vitrification solution. But if we recognize that such events can be (partly) attributed to ice formation due to ischemia-induced perfusion impairment it should be obvious that the recording of fracturing events above Tg as such cannot be an argument against ITS. After all, we also do not argue against the use of vitrification solutions because ice formation will still occur in ischemic patients that are perfused with vitrification solutions. Because cryonics patients almost invariably suffer some degree of ischemia prior to cryoprotective perfusion and cryopreservation, our knowledge about fracturing events in “ideal” human cases remains incomplete.

But even if ITS would only be successful in reducing fracturing events, instead of completely eliminating them, this should not be an argument against ITS. To argue that a technology should not be used because it does not completely eliminate a problem would constitute a sharp departure from the philosophy that has informed Alcor since its formation. In many areas, the evolution of Alcor’s technologies has been one of incremental evidence-based progress towards better procedures and storage conditions, not one of radical change.

The worst argument against ITS is that mature repair technologies will be able to repair clean fractures. It is a poor argument because one could similarly argue that advanced cell repair technologies will also be able to reverse the biochemical effects of short periods of ischemia and moderate degrees of ice formation. What distinguishes Alcor from other cryonics organizations is that it aims to secure viability of the brain as far into its procedures as it practically can. In ideal cases, this currently means meeting the challenge of further reducing cryoprotectant toxicity during cryoprotectant perfusion and reducing/ eliminating fracturing.

Perhaps the biggest obstacle to offering ITS to the general Alcor membership is cost. An obvious solution would be to offer ITS in addition to conventional liquid nitrogen storage. An alternative would be to gradually phase out conventional liquid nitrogen storage by no longer offering it to new neuro members and to raise cryopreservation minimums accordingly. The (preliminary) cost estimates in the article indicate that this would bring the cost of ITS for neuros closer to that of conventional liquid nitrogen whole body cryopreservation. The article does not provide specific information on the “greater capital costs” of whole body ITS systems but the reported lower liquid nitrogen consumption per patient for whole body systems suggests that it might be possible to offer whole body ITS without putting it beyond the reach of most (new) members with adequate funding.

The double standard about cryonics

One of the most predictable features of public debates about cryonics is that those arguing in favor of cryonics are held to more rigorous standards than those seeking conventional medical treatment. Advocates of cryonics do not just have to prove that cryonics will work, they are also supposed to solve problems like overpopulation and the presumed boredom arising from expended lifespans. To some, people who make cryonics arrangements have an inflated perception of their own importance and should just forgo such selfish attempts to extend their lives. The default position seems to be that people should not exist and that life needs justification. Could you imagine such antinatalist rhetoric being employed when a person seeks conventional medical treatment to extend their life? We can’t, and such responses are quite indicative of the fact that people are not interested in serious evaluation of the cryonics argument.

The most striking case of cryonics being held to higher standards than conventional medicine concerns the requirement that “cryonics” needs to “work.” Even people who have made cryonics arrangements routinely say something like, “I estimate the probability of cryonics working as 5% but life insurance premiums are low and I have nothing to lose if it does not work.” To see how strange such a statement is, let’s look at these two terms, “cryonics” and “working.”

Cryonics is an experimental medical procedure to stabilize critically ill patients at low temperature to benefit from future advances in medicine. Such a definition can include a wide variety of cases, ranging from ice-free cryopreservation (vitrification) as an elective medical procedure in a hospital to the freezing of a person who is found days after circulatory arrest. Considering the enormous variability under which people can be cryopreserved, to claim that “cryonics” will not work without specifying under what conditions a cryopreservation is performed is akin to saying that “emergency medicine” or “chemotherapy” does not work — an absurd claim.

Usually when people argue that cryonics does not work they refer to the mistaken view that cryopreservation that is not initiated within hours, or even within minutes, after death does not make sense because the brain has “died” at that point. Such a view completely ignores the fundamental cryonics argument that lack of function of the brain does not imply that the neuroanatomical basis of identity is irreversibly destroyed.

But let us accept this position the sake of the argument. What such a critic is basically saying is that cryonics cannot work because cryonics patients are cryopreserved under conditions that do not allow it to work. To see how strange such a position is, imagine a country where law would prohibit CPR until 15 minutes of death. Would anyone be impressed if someone would argue that CPR does not work because patients suffer irreversible brain damage after 15 minutes of circulatory arrest? Of course not. We would instead insist that such obstacles should be removed so that these life-saving technologies can be employed as soon as needed. Clearly, whatever the merits of cryonics are, it is not reasonable to conflate the conditions under which cryonics is often conducted with the idea of cryonics as such.

Now let’s look at the second term. What does it mean for cryonics to “work?” Naturally, we would like a medical procedure to cure the disease and restore the patient to the condition than he was in prior to the disease. In real life this often happens, especially in the case of minor infections and minor insults. But there are also many cases where (heroic) medical interventions are aimed at keeping the patient alive without expecting a full recovery without side effects. This is often the case in acute cardio-respiratory arrest and stroke. Would we prefer a complete recovery for such patients? Of course. But would we say that interventions that aim to save a patient’s life did not work if we fail to meet such an ideal – say, a permanent loss of movement in one arm or reduced memory function? No, our first concern would be with the patient’s survival and his perception of the quality of his “new” life.

In the case of cryonics things are not much different. We hope that advanced cell repair technologies will be successful in completely restoring the patient to good health in a rejuvenated state. For some patients complete inference of the original structure of the brain might not be possible, but advanced neural archeology and neurogenomics may restore a significant degree of the original person. We do not heap scorn on such scenarios in today’s medicine and there is no reason to hold cryonics to higher standards, especially if one also advocates the very restrictions that are responsible for such less than perfect outcomes. In fact, there is no reason to be scathing about any credible attempts to save or prolong a life, even if the attempt will not necessarily succeed. Such a perspective is a given in conventional medicine or rescue operations.

One objection to this position is to argue that cryonics cannot work even under the most favorable conditions. Such an argument would basically entail that if a critically ill patient is stabilized without ischemic delays, without ice formation, and without fracturing, it should be categorically ruled out that technologies will ever be developed to repair the original disease of the patient and any form of injury that occurs during the cryopreservation process itself. I personally would consider such a position extremely dogmatic (would anyone argue such a position of long-term technological stasis if the cryonics context were dropped?) but it raises a fundamental question about the burden of proof. Should it rest with the person who aims to prolong life or should it rest with the person who aims to prohibit such attempts? Asking the question is answering it.

The case against cryonics

What is striking about cryonics is that those who have taken serious efforts to understand the arguments in favor of its technical feasibility generally endorse the idea. Those who have not made cryonics arrangements usually give non-technical arguments (anxiety about the future, loss of family and friends, etc), lack funding or life insurance, or are (self-identified) procrastinators. In contrast, those who reject cryonics are almost invariably uninformed. They do not understand what happens to cells when they freeze, they are not aware of vitrification (solidification without ice formation), they think that brain cells “disappear” five minutes after cardiac arrest, they demand proof of suspended animation as a condition for endorsing cryonics, etc.

This does not mean that no serious arguments could be presented. I can see two major technical arguments that could be made against cryonics:

1. Memory and identity are encoded in such a fragile and delicate manner that cerebral ischemia, ice formation or cryoprotectant toxicity irreversibly destroy it. Considering our limited understanding of the nature of consciousness, and the biochemical and molecular basis of memory, this cannot be ruled out. Cryonics advocates can respond to such a challenge by producing an argument that pairs our current understanding of the neuroanatomical basis of identity and memory to a cryobiological argument in order to argue that existing cryonics procedures are expected to preserve it. An excellent, knowledgeable, response of this kind is offered in Mike Darwin’s Does Personal Identity Survive Cryopreservation? Cryonics skeptics in turn could produce evidence that existing cryonics procedures fall short of this goal.

2. The cell repair technologies that are required for cryonics are not technically feasible. This argument should be presented with care and rigor because the general argument that cell repair technologies as such are not possible contradicts existing biology. A distinct difference from the first argument is that it is harder, if not impossible, to use existing empirical evidence to settle this issue. After all, making cryonics arrangements is a form of decision making under uncertainty and such decisions are not straightforwardly “correct” or “incorrect,” “right” or “wrong.” What can be done is to provide a detailed scientific exposition of the nature and scope of the the kind of repairs that are necessary for meaningful resuscitation and to argue that both biological and mechanical cell repair technologies are not conceivable – or are conceivable.

One thing that becomes immediately clear from this exercise is that there is no single answer to the question of whether cryonics can work because the answer to this question depends on the conditions and technologies that prevail during the cryopreservation of a patient. This introduces a set of more subtle distinctions concerning the question of what kind of cryonics should be assessed. It also produces an argument in favor of continuous improvement of cryonics technologies, and standby and stabilization services.

This short examination of technical arguments that could be made against cryonics gives advocates of the practice two talking points in discussion with skeptics or hostile critics:

(a) If a critic flat-out denies that cryonics is technically feasible, it is not unreasonable to ask him/her to be specific about what (s)he means by cryonics. This simple question often will reveal a poor understanding of existing cryonics technologies and procedures.

(b) A decision made on the basis of incomplete knowledge cannot be “right” or “wrong” and should be respected as one’s best efforts to deal with uncertainty.

Chemical preservation and cryonics research

In the 2009-4 issue of Alcor’s Cryonics magazine I review the technical and practical feasibility of chemical preservation. One of the most interesting aspects of chemopreservation is that it could play a useful role in the cryopreservation of ischemic patients.

There is accumulating evidence that vitrification agents cannot prevent ice formation in ischemic patients. This raises the question whether some cryonics patients could benefit from chemical fixation prior to transport and cryoprotective perfusion.

Such protocols raise a number of obvious concerns but the question is not so much whether these procedures are inferior to vitrification of non-ischemic patients, but whether fixatives can improve the situation of some ischemic patients compared to the prospect of substantial ice formation, or even straight freezing (cooling without cryoprotection). This is an empirical question which needs to be settled by experimental research.

Chemopreservation: The Good, The Bad and the Ugly

Cryonics in the media

The Detroit News features a story about cryonics that is a good illustration of the upward battle that cryonics faces in the media. First and foremost, this story reinforces the idea that cryonics concerns the practice of freezing dead people:

Preparation of the body is a five-day procedure. It begins with keeping the body as cool as possible before arriving at the facility, to slow decomposition. Upon arrival, the body is put in a sleeping bag, strapped to a backboard and put in a cooling box.

There is no mention of the practice of replacing the blood with a so called cryoprotective agent to inhibit ice formation in the text, but the practice is explained in a sidebar.

Then the inevitable “expert” (John K. Critser, president of the Society for Cryobiology) makes the predictable error of  offering his opinion on the feasibility of cryonics based on our current ability to achieve real suspended animation.  If a scientist cannot conceptually distinguish between a technology that is meant to halt decomposition to allow resuscitation efforts in the future and a technology that is able to cool down a complex organism and recover it with contemporary technology, cryonics has a serious “marketing” problem.

There has been much debate about how to persuade more people to consider cryonics. Renewed efforts should be made to end  misunderstanding about the following three basic points about cryonics:

1. Cryonics is not the freezing of dead people, but involves the attempt to halt decomposition of people that have been given up by contemporary medicine through the use of low temperatures. Legal death is not biological death.

2. The objective of cryonics is to protect critically ill patients against ice formation at cryogenic temperatures by replacing the blood with a cryoprotective agent. Vitrification solutions attempt to inhibit ice formation altogether.

3. Cryonics is not suspended animation and should not be evaluated as such. Expecting people to destroy their brains because suspended animation is not feasible yet is neither prudent nor caring. Our current burial and cremation practices reflect a simplistic view of death and a desire for instant gratification and closure.

The pursuit of cryonics as medicine

The biggest obstacle to the acceptance of cryonics is medical myopia; the idea that someone who has been pronounced dead by contemporary medical criteria will still be considered dead by future criteria. Advocates of human cryopreservation strongly argue against this. There are few things more discomforting than the idea that medical professionals of the future will look back in horror and wonder why we gave up on people who still possessed the neuroanatomical basis of their identities and memories.

But there is another kind of myopia in the public discussion of cryonics that warrants consideration. It is taken for granted by some critics of contemporary cryonics that cryonics has always been framed as a form of medicine. Nothing could be further from the truth. The history of cryonics is replete with debates between advocates of the medical model and those who believe that timely transport of the patient to a cryonics facility for low temperature storage should be adequate for future resuscitation by advanced nanotechnology. It is only because  cryonics advocates with medical and research backgrounds such as Mike Darwin and Jerry Leaf vigorously argued for adopting conventional medical techniques and protocols that today’s cryonics organizations can even be criticized  for falling short of these criteria.

There is a silver lining to a lot of the controversy that surrounds today’s cryonics . Critics now adopt the premise that cryonics is a form of medicine to make a case against practices they consider suboptimal.  It was not long ago that public critics of cryonics simply dismissed the whole idea as pseudo-science. This was never a sophisticated response but ongoing advances in cryobiology (such as vitrification of the central nervous system) and synthetic biology/nanotechnology have made this position even more of a showcase of ignorance. When people read the news about animals being cloned from straight frozen DNA they will be less receptive to tendentious claims that existing cryonics technologies are hopelessly inadequate to preserve the identity of a person.

The current development in which cryonics is being criticized from a clinical framework should have positive effects on how cryonics will be approached from a regulatory framework. It does not make sense to argue that cryonics is a pseudo-science and offering false hope but at the same time insist that cryonics organizations adopt high standards of medical care. The acceptance of the concept of “patient care” in cryonics would be incoherent without (implicitly) embracing the premise that cryonics patients have interests and deserve legal recognition of that fact. As more public information is disseminated about the quality of brain vitrification that is possible today, the need to recognize cryonics as an elective medical procedure will receive more attention from bioethicists and medical professionals.

There are those who believe that the acceptance of cryonics itself is being held back by amateurism. If this is the case there should be unexploited profit opportunities for cryonics providers that pursue the highest standards of medical care.

Reversible cryopreservation

On the forum of the Immortality Institute there is an interesting exchange about the feasibility and time line for reversible cryopreservation. Cryobiologist Brian Wowk weighs in with some interesting observations:

I think in the next 20 years more small animal organs, and perhaps some human organs, may be reversibly cryopreserved. The best scenario for cryonics would be improved, and possibly demonstrably reversible, cryopreservation of animal brains. It has been long observed that if reversible solid-state brain preservation could be demonstrated, then cryonics revival becomes a purely technical problem (albeit very complex one) of tissue regeneration. There would be no remaining doubt about whether the preservation itself was viably preserving human beings….Reversible solid-state cryopreservation of whole mammals is a very difficult problem with existing technology. This is why when asked about it people will often defer to nanotechnology. References to nanotechnology as a solution to a medical problem basically say, “We have no idea how to solve this problem with existing tools, but future abilities to completely analyze and repair tissue at the molecular level will be implicitly sufficient.” It’s a valid argument, but saying that a medical problem will be solved when someday technology exists to solve *every* medical problem is not very illuminating about time lines or nature of the problem.

Advocates of cryonics often push for demonstration of reversible small animal cryopreservation as  a means to persuade the medical establishment and the general public of the technical feasibility of cryonics. The limitation of this approach, however, is that this goal cannot be achieved until we are able to successfully vitrify all vital organs of the animal, including such difficult organs  as the lungs and the kidney. A more promising approach is to keep improving vitrification of the central nervous system. As argued in a recent piece for Alcor’s Cryonics Magazine, if organized electrical activity can be demonstrated after whole brain cryopreservation a strong case can be made for the acceptance of cryonics as a medical procedure and improved legal protection of cryonics patients.  It should be noted, however, that these research efforts constitute only one objective in cryonics. Another objective of cryonics research is to optimize procedures and protocols for existing patients, who invariably suffer some degree of circulatory arrest.

Basile J. Luyet on the instability of solidified solutions

Basile J. Luyet (1897-1974) can be considered the father of modern cryobiology. His book “Life and Death at Low Temperatures” is a classic in the field and his journal “Biodynamica” evolved into a publication solely dedicated to the study of low temperature biology. Luyet identified the possibility of solidification without crystallization at low temperatures (vitrification) of biological materials, an approach that was later worked out as a practical method for organ preservation by the cryobiologist Greg Fahy.  Vitrification solutions are also used in human cryopreservation to prevent ice formation in patients during cooldown and storage at liquid nitrogen temperature.

In the following Biodynamica study (1966) Luyet investigates the issue of structural instability and molecular mobility in solidified aqueous solutions. In these initial investigations he anticipated the phenomena of re-crystallization and de-vitrification upon rewarming, which later would present formidable challenges during the early years of applied vitrification research in large organs. Although Luyet briefly mentions the possibility of molecular mobility as such at temperatures down to absolute zero, his main focus is on ice formation that can occur during the rewarming of solutions. Cryonics Institute President Ben Best has done some theoretical explorations into the issue of molecular mobility at low temperature, a topic that raises important questions about the desirability of intermediate temperature storage (ITS) of cryonics patients.

B. Luyet – The Problem of Structural Instability and Molecular Mobility in Aqueous Solutions “Solidified” at Low Temperatures (1966) PDF