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.

Neural cryobiology and the legal recognition of cryonics

It has been said that if you want to persuade someone, you need to find common ground. But one of the defining characteristics of cryonics is that proponents and opponents cannot even seem to agree on the criteria that should be employed in discussing cryonics. The cryonics skeptic will argue that the idea of cryonics is dead on arrival because cryonics patients are dead. The response of the cryonics advocate is that death is not a state but a process and there is good reason to believe that a person who is considered dead today may not be considered dead by a future physician. In essence, the cryonics advocate is arguing that his skeptical opponent would agree with him if he would just embrace his conception of death….

Cryonicists have named their favorite conception of death “information-theoretic death.” In a nutshell, a person is said to be dead in the information-theoretic sense of the word if no future technologies are capable of inferring the original state of the brain that encodes the person’s memories and identity. There are a lot of good things to be said about substituting this more rigorous criterion of death for our current definitions of death. However, in this brief paper I will argue that our best response does not necessarily need to depend on skeptics embracing such alternative definitions of death and that we may be able to argue that opponents of cryonics should support legal protection for cryonics patients or risk contradicting conventional definitions of death.

In contemporary medicine, death can be pronounced using two distinct criteria; cardiorespiratory arrest or brain death. A lot of ink has been spilled over the co-existence of those criteria and its bioethical implications but I think that most people would agree that the practice of medicine requires this kind of flexibility. What is interesting for us is that clinical brain death (or brain stem death) is defined as “the stage at which all functions of the brain have permanently and irreversibly ceased.” There are a number of ways how such a diagnosis can be made, but in this context I want to focus on the absence of organized electrical activity in the brain.

We first should note the use of the word “irreversible.” After all, if a patient is cooled down to a low core temperature to permit complicated neurosurgical procedures most of us would not say that this person is “temporarily brain dead.” As a matter of fact, one could argue that cryonics is just an experimental extension of clinical hypothermic circulatory arrest in which there is a temporal separation of stabilization and treatment. Now, we could argue that what may be irreversible by today’s standards may not be irreversible by future standards but then, again, we are trying to persuade the other person to accept our view of future medicine. It would be much better, and I hope much easier, to argue that contemporary cryopreservation techniques can preserve organized electrical activity in the brain. The advantage of this approach is obvious. Instead of arguing in favor of our own criterion of death we can argue that, according to mainstream criteria for determination of death, cryonics patients are not dead. This is an interesting case in which a scientist (i.e., a cryobiologist) may be able to make a major contribution to the legal recognition and protection of cryonics patients.

So where are we standing right now? How good are our preservation techniques? If we aim for reversible whole brain cryopreservation a cryoprotective agent should have two properties: (1) elimination of ice formation, and (2) negligible toxicity. In the early days of cryonics, we were not able to satisfy both criteria at once. Using just a little bit of glycerol would not be toxic but it would still allow massive ice formation. Using a lot of a strong glass former such as DMSO would eliminate ice formation but at the price of severe toxicity. Mostly due to the groundbreaking work of cryobiologists Gregory Fahy and Brian Wowk, in the year 2000 the Alcor Life Extension Foundation introduced a vitrification agent called B2C that eliminated ice formation and had a more favorable toxicity profile. In the year 2005, the separation between the state of the art in experimental cryobiology and cryonics practice was further narrowed when Alcor introduced M22 as their new vitrification agent. M22 is the least toxic vitrification agent in the academic cryobiology literature that permits vitrification of complex mammalian organs at a realistic cooling rate.

M22 and other solutions derived from the same cryobiological principles have been validated in the brain as well. Former Cryonics Institute researcher Yuri Pichugin and collaborators used a related vitrification solution for the preservation of rat hippocampal brain slices without loss of viability after vitrification and rewarming. At a cryonics conference in 2007, 21st Century Medicine announced that the use of M22-based solutions permitted the maintenance of organized electrical activity in rabbit brain slices. So, at this stage we can argue that our existing vitrification solutions have a reasonable chance of maintaining organized electrical activity in brain slices. The next challenge is to demonstrate this property in whole brains.

Whole brain cryopreservation is not just the cryopreservation of a great number of individual brain slices. Brain slices can be cryopreserved by (step-wise) immersion in the vitrification solution. Vitrification of whole brains (even small brains such as rodent brains) requires the introduction of the vitrification solution through the circulatory system. This aspect of whole brain vitrification presents a number of technical challenges. Electron micrographs of vitrified tissue from whole brains, however, indicate that these challenges can be overcome. The current research objective is to perfect perfusion techniques and optimize vitrification solutions to maintain organized electrical activity in whole brains. We know that this objective is possible in principle because the famous surgeon Robert White demonstrated retention of electrical activity in whole isolated brains after cooling them to ~2-3°C. Isolated brain perfusion is a complicated surgical procedure, but the current writer and cryobiologist Brian Wowk have recognized that validation of whole brain activity is also feasible in situ.

Reversible cryopreservation of the whole brain without losing organized electrical activity is not a trivial research objective but it should be easier to achieve than reversible cryopreservation of the whole body and, perhaps, some other organs. If and when we accomplish this, we will no longer be dependent on “rationalist” arguments that appeal to logic and optimism about the future. We can argue that our patients should not be considered dead by the most rigorous criterion for determination of death in current medical practice. We can then even mount some smart legal challenges to seek better protection for cryonics patients. If we can make this step forward we should also aim at improved protection of existing cryonics patients, which will allow them, among other things, to own assets and bank accounts. This is how science can be employed in legal strategies for asset preservation.

This article is a slightly revised version of a paper that accompanied a recent presentation on neural cryobiology and the legal recognition of  cryonics at the 5th Asset Preservation Meeting in Benicia, California.

Mind uploading, falsifiability and cryonics

On the cryonics discussion list Cryonet cryobiologist Brian Wowk weighed in on the topic of mind uploading in a post that merits quoting in its entirety:

I read with interest Bob Ettinger’s recent remarks about Mark Gubrud’s piece in The New Atlantis.

http://futurisms.thenewatlantis.com/2010/06/why-transhumanism-wont-work.html

Although I have not been around as long Bob, I have nevertheless observed arguments about uploading, identity duplication, and related subjects for decades.  In all that time there are two things I’ve never seen: (a) A truly new argument, and (b) Someone change their mind.  What is seen are people who passionately believe they are correct, and who believe that they have just the argument to finally convince the other side that they are right.  They never do.

I have come to believe that the question of whether a computationally equivalent duplicate of a human mind (assuming equivalence in this context is even definable) constitutes a continuation of the original person may be objectively unanswerable.  It’s almost a matter of taste, like alternative interpretations of quantum mechanics that assume different underlying realities that give exactly the same measurable results.

Eventually the distant day will come when the computational processes of a human brain are duplicated in an electronic computer, or even in another identical organic substrate.  When that day comes, we can be certain of this: If the person who was “duplicated” believed before duplication that duplication constitutes survival of the self, then, by definition, the duplicated entity will insist vociferously that indeed they did survive.  This has ethical implications.  Conversely,
an entity derived from a person who did not believe in this form of survival might be quite unhappy to be told that they were the product of a destructive scan of somebody.  This too has ethical implications.

Philosophical truth aside, evolution selects against humans who spend time worrying about whether sleep, anesthesia, or biostasis endangers personal identity.  Similarly, it is easy to predict which side of the uploading and duplication debates will win in the long term.  There is no entity more invulnerable or fecund than one that believes it consists of information.

Recent discussions of the topic of mind uploading on the Cryonics Institute members mailing-list contradict Wowk’s claim about people changing their mind about mind uploading. Robert Ettinger posted an itemized list with objections against the idea of mind uploading as a strategy for personal survival and I weighed in on the (current) lack of experimental evidence to settle the matter. The effect was that some people changed their mind or became more agnostic about mind uploading.

Wowk may be correct that the question whether a “computationally equivalent duplicate of a human mind…constitutes a continuation of the original person may be objectively unanswerable.” The discussion about mind uploading and persistence of the original person has distinct similarities with discussions about solipsism, consciousness, and the existence of the external world. It is not inconceivable that in a world where mind uploading has become routine the debates will still continue because the hard problem of persistence of the person is not falsifiable in a meaningful manner.

There are mind uploaders and there are Mind Uploaders. The Mind Uploaders are a small but vocal minority who display little patience for the argument that the technical feasibility of mind uploading requires empirical verification and cannot be completely settled by logical deduction or thought experiments. As cryonics activist and ex-Alcor Board member David Pizer says, “Having existed with Uploading Lovers for many years now, I believe they are as firmly entrenched in their beliefs as  traditional religious persons believe that their souls are going to Heaven after death here on Earth.”

Cryonics is often associated with ideas like mind uploading and transhumanism. One negative consequence of this (un)intentional association is that some people who are considering cryonics feel that they have to embrace a much larger set of controversial ideas than what they are actually being asked to consider. As a result, there is a real risk that people reject cryonics for reasons that have little to do with the proposal of cryonics itself. Advocates of cryonics do not do themselves a favor by promoting the idea of human cryopreservation as part of a larger set of futurist ideas instead of just promoting cryonics as an experimental medical procedure to extend life. There is too much at stake to alienate people by piling more controversial ideas on top of what is already considered to be a radical idea. Such a low-key attitude will also produce a more consistent message because it extends the element of uncertainty that is inherent in cryonics to other areas of life as well.


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.

5 dangerous ideas about cryonics

The cryonics organizations Alcor and the Cryonics Institute have taken great care to correct some of the persistent myths about cryonics. With so much widespread misinformation being circulated in the media it seems trivial to pay attention to some of the misconceptions that some people who are sympathetic to cryonics hold. But the price of ignoring these opinions is that progress in the science of cryobiology and practice of human cryopreservation is adversely affected. What follows is a list of 5 “dangerous” ideas (or misconceptions) about cryonics and their consequences.

1. First in, last out.

A popular expression in cryonics is that the first person who was cryopreserved will require the most extensive repair technologies and therefore will be the last person to be resuscitated. The underlying assumption in this view is quite reasonable: when advances in cryopreservation technologies are made, demands on advanced future repair technologies will be lessened. The problem with this view, however, is that it assumes that advances in cryobiology and neuroprotection are the only factor influencing the quality of care in cryonics. Unfortunately, advances in the science of cryopreservation will not automatically translate into better patient care.  Other factors, such as the delay between time of “death” and start of procedures, and the protocols, equipment and personnel of the responding cryonics organizations, matter as well. For example, if a cryonics standby team is not able to get to a patient before 24 hours after cardiac arrest, pumps him full of air during remote blood washout, and ships him back to the cryonics organization at subzero temperatures, that patient will not benefit from advances in human cryopreservation such as rapid induction of hypothermia, neuroprotection and vitrification.

A professional cryonics organization with “old” technologies may on average do better than an incompetent cryonics organization with “new” technologies. The important lesson to be drawn here is that the concept of “patient care” is a meaningful concept  in cryonics and consumers of cryonics services need to evaluate their cryonics providers on their ability to provide good care.

2. Only the future will tell us how good our cryonics procedures are.

It is true that only the future will tell us whether cryonics patients will be resuscitated or not; but that does not mean that we cannot say anything meaningful about the quality of care in individual cryonics cases. The most obvious point is that we can compare actual patient care to the published protocols and objectives of the cryonics organization. More specific observations can be made during a cryonics case using medical equipment. In a well-run cryonics case a number of physiological and chemical measurements are made to determine the response of a patient to various interventions. As a general rule, the objective of cryonics stabilization procedures is to keep the brain of the patient viable by contemporary medical criteria. The danger of thinking of cryonics as one single experimental procedure that can only be evaluated in the future is that it ignores the fact that actual cryonics procedures consist of various separate procedures that can be monitored and evaluated using existing medical tools. The least that a cryonics consumer should expect from his cryonics organization is that it discloses its cryonics procedures to the general public and produces detailed case reports.

3. Cryonics patients are no longer being frozen.

Because not all cryonics patients will be “ideal” cases, this view is vulnerable to the same objections as the “first in, last out” rule, but there are some other issues that are important to mention in this context. The most important fact to be stressed is that ice formation is not a binary all or nothing thing but a continuum ranging from straight freezing (cryopreservation without cryoprotection) to complete elimination of ice formation. Although there have been many cases where patients have been frozen without the use of a cryoprotective agent, its opposite, complete vitrification, should be considered  a theoretical ideal. The degree of ice formation is determined by the nature and concentration of the cryoprotective agent. For example, low concentrations of the cryoprotectant glycerol will result in more ice formation than higher concentrations of glycerol.

What has changed in the recent years is that both major cryonics organizations are now offering cryopreservation using vitrification agents. Although these vitrification agents are formulated to eliminate ice formation, it is generally believed that such a result is not achievable in all tissues and organs in the human body at the moment.  Another important point to be made is that not all solutions that can eliminate ice formation are equal because they can differ greatly in toxicity.  The technical challenge in cryonics is not so much to eliminate ice formation but to develop vitrification solutions with no or limited toxicity. Although it is correct that contemporary vitrification solutions  can solidify without ice formation, delays in response time, poor patient care, and high toxicity can offset most of these advances.

4. The probability that cryonics will work is X.

Both critics and supporters have made specific probability estimates about how likely cryonics is to work. In its worst form such probability assessments convey nothing more than putting a number on overall feelings of pessimism or optimism. More serious attempts have been made to calculate a specific probability that cryonics will work. Such attempts usually go as follows: A number of independent conditions (or events)  for cryonics to work are distinguished, these conditions are “assigned” a probability, and the total (or joint) probability is calculated by multiplying them. Although such calculations give the semblance of objectivity, they are  equally vulnerable to the fundamental objection that assigning one single number to the probability that cryonics will work is just a lot of hand waving.  How many independent events are there and how do we know that they are independent? What is the basis for assigning  specific probabilities to these conditions? What are the effects of minor changes in the numbers?

Probability calculations are not completely useless.  They can help us in identifying important conditions that need to be satisfied for resuscitation. They can also help identify weak links  that can be improved. But probability estimates can be dangerous as well when we take them too seriously and discourage people from making cryonics arrangements. The point here is not that we should refrain from being skeptical but that if we make quantitative estimates we should be able to back up our statements with rigorous arguments or just confine ourselves to more qualitative statements. Another objection to  making cryonics probability estimates was made by the cryonics activist and mathematician Thomas Donaldson. He makes the common sense point that many of these conditions are not independent of what we do. We can make a contribution to increasing the probability that cryonics will work.

Last but not least, what does it mean when we talk about “cryonics working?” It is conceivable that cryonics will work for one person but not for another, reflecting improved technologies and protocols. Perhaps asking the question if cryonics patients can be “revived” is the wrong question. As the cryobiologist Brian Wowk has pointed out,  the real question is how much original personality would survive the many possible damage/repair scenarios, not revival per se.  Survival in medicine is not a simple black-and-white issue, as evidenced by people who recover from stroke or cardiac arrest but with personality and memory alterations.  And it is worth  mentioning once more that how much of our personality survives is depended on what we do to improve the quality and long-term survival of our cryonics organizations.

5. I will sign up for cryonics when I need it.

It should be obvious without much reflection why this is a dangerous idea. At the time a person really needs cryonics, he may no longer be able to communicate those desires, lack funding to make arrangements, or encounter hostile relatives. A more subtle variant concerns the person who expects that aging will be solved before cryonics will be necessary. This person may or may not be right, but such optimism may not make him more immune to accidents than other people. This mindset is often observed among young “transhumanists” and practicing life extensionists. A related, but rarer, variant is to postpone making cryonics arrangements until the cryonics organization makes a number of changes including, but not limited to, hiring medical professionals, stop wasting money, becoming more transparent, giving members the right to vote, etc. Such issues are important, and need to be addressed, but a safer response would be to join the organization and influence its policies, or, if this will be necessary, combine with others to start a competing cryonics organization without such flaws.

There are not many people who think that it is sensible to make cryonics arrangements, but there are even fewer people who have actually made such arrangements.

As we have seen, some of these dangerous ideas share the same or related assumptions and produce identical effects: decreased scrutiny of cryonics organizations and a decreased chance of personal survival. An important common theme is that cryonics cannot be treated as one single monolithic technology and that the fate of our survival depends as much on the state of the art in human cryopreservation technologies as on the competence of cryonics providers. Caveat emptor!

Suspended animation is not cryonics

On the Immortality Institute cryonics forum, Alcor Board member and researcher Brian Wowk has posted some insightful comments on the difference between suspended animation and cryonics. Although  impressive technical advances in cryonics to date, such as vitrification, have failed to translate into increased membership growth for cryonics organizations, many cryonics observers believe that demonstration of reversible vitrification of a small mammal will be a turning point in cryonics.

But as Brian points out, the key idea of cryonics is that patients should continue to be cared for, even if contemporary technologies cannot reverse cryopreservation. As has been reiterated on this blog before, even when suspended animation is perfected, there still will be a need for cryonics to care for patients that cannot be treated by contemporary medical technologies. Dismissing cryonics until there is proof of successful suspended animation ignores the fundamental, and humane, premise of cryonics to use  low temperature  biostasis  so that critically ill people may benefit from medical technologies that have not yet arrived.

Suspended animation is not cryonics. The paradigm shift of cryonics is something different. It is a paradigm shift that could happen before suspended animation is perfected, or perhaps not even after suspended animation is perfected. The key idea of cryonics– the paradigm shift of cryonics –is the idea that patients should continue to be cared for even if they are beyond recovery by contemporary means. It’s the idea that almost everything that medicine calls “death” in a particular era is destined to become a treatable pathology in a later era. That is an idea that transcends suspended animation, and that is so far from normal social mores that it may never be accepted by the mainstream whether there is suspended animation or not. It is a paradigm shift that requires overturning the idea of closure, which is a deeply uncomfortable proposition for most people regardless of demonstrated technology.

When people say that they hope they never need cryonics, I’m not sure in what sense they mean this. Do they mean that in the same sense that we all hope we never have to go to a hospital, even though the probability of eventually being hospitalized for some reason converges to near certainty? Or do they actually believe that they may never need cryonics? Such a belief is equivalent to the belief that one will never suffer a medical crisis that is untreatable by available medicine. I suppose an alternative possibility is the belief that one’s first and last major medical crisis will be vaporization. That doesn’t seem very likely. We live in a time when for the foreseeable future, Singularity or not, virtually everybody is going to need some form of cryonics at some time.

Brian Wowk quotes cryonics advocate Thomas Donaldson:

If you’re involved in cryonics, you’ve got to make your peace with the unknown, because it will always be there. You’ve simply got to make your peace with it.

Vitrification agents in cryonics: M22

M22 represents the culmination of decades of work in applied cryobiology by researchers Gregory Fahy , Brian Wowk, and others to develop a vitrification agent that can recover complex organs (such as the kidney) from cryogenic temperatures without ice formation and minimal toxicity. In 2005, M22 was licensed by the patent holder 21st Century Medicine (21CM) to the Alcor Life Extension Foundation to replace their previous vitrification agent B2C. As a result, the least toxic vitrification agent for complex organs that has been documented in peer review journals is currently being used for cryonics patients at Alcor.

M22 incorporates a number of important discoveries in cryobiology:

1. High concentrations of a cryoprotective agent (or a mixture of different cryoprotective agents) can prevent ice formation during cooldown and warming.

2. The toxicity of some cryoprotectants can be neutralized by combining them with other cryoprotective agents.

3. The general toxicity of a vitrification agent can be predicted by using a measure called qv*, allowing for the rational formulation of less toxic vitrification agents.

4. Within limits, non-penetrating agents can reduce the exposure of cells to toxic amounts of cryoprotectants without reducing vitrification ability.

5. Synthetic “ice blockers” can be included in a vitrification mixture to reduce the concentration of toxic cryoprotective agents necessary to achieve vitrification.

6. Substituting methoxyl (-OCH3) for hydroxyl groups (-OH) in conventional cryoprotective agents can decrease viscosity, increase permeability, and reduce the critical cooling rate necessary to avoid ice formation.

7 Chilling injury can be eliminated by introducing the vitrification agent with a hypertonic concentration of non-penetrating solutes.

8. In cryonics, with a minor proprietary modification, M22 can be used for whole body perfusion without causing severe edema that has been a problem for some other solutions.

Vitrification is the solidification of a liquid without crystallization. When a solution is cooled down to the glass transition point (-123.3°C for M22) the extreme elevation in viscosity will produce a glass in which all translational molecular motions are arrested. Although water vitrifies at cooling rates exceeding a million of degrees Celsius per second, such cooling rates are relaxed when other solutes are substituted for water. In cryobiology solutions with high concentrations of cryoprotective agents can be used to vitrify complex organs such as the kidney or the brain.

Vitrification has a number of clear advantages over conventional cryopreservation. The most important advantage is the elimination of ice formation. Although the adverse effects of ice formation can be mitigated by the use of cryoprotective agents (glycerol, DMSO) and optimization of cooling rates, massive ice formation does not permit recovery of complex organs with full viability. Another advantage is that vitrification eliminates the need to strike a balance between the risk of intracellular freezing induced by fast cooling on the one hand, and cell dehydration and solution concentration induced by slow cooling on the other hand.

The challenge in formulating successful cryoprotective agents is to design vitrification solutions that are non-toxic but allow for vitrification at realistic cooling and warming rates. For more than a decade the least toxic vitrification agent was Greg Fahy’s VS41A, which is an 55% weight/volume equimolar mixture of DMSO and formamide plus propylene glycol. The “1A” in VS41A reflects the solution’s ability to vitrify at normal atmosphere pressure (as opposed to an older, more dilute solution, VS4, which requires 1000 atmospheres of pressures to vitrify). The equimolar concentrations of DMSO and formamide reflect Baxter and Lathe’s research who concluded that amides can neutralize the toxicity of DMSO, a finding that Greg Fahy later revised in favor of the theory that it is actually DMSO that neutralizes the toxicity of formamide. The ability of DMSO to neutralize the toxicity of formamide (up to certain concentrations) allows for the formulation of vitrification agents with reduced toxicity. This finding has been so fundamental that an equimolar concentration of DMSO and formamide remains the core of M22.

Another major step was made when the researchers at 21CM found that high concentration of (penetrating) cryoprotectant agents do not necessarily increase toxicity. Contrary to conventional cryobiology expectations, Fahy et al. found that weaker glass formers favor higher viability. They proposed a new compositional variable called qv* to predict the general toxicity of vitrification solutions. Using qv* they made the “counter-intuitive” decision to substitute a higher concentration of the weaker glass former ethylene glycol for propylene glycol to create a solution called Veg, which produced a substantial improvement in terms of viability as measured by K+/Na+ ratios.

Because cells contain higher concentrations of protein, the intracellular space is more favorable to vitrification than the extracellular space. As a consequence, the concentration of penetrating (toxic) cryoprotectants can be reduced in favor of non-penetrating polymers like polyvinylpyrrolidone (PVP). Variations of Veg in which the concentration of DMSO and formamide was reduced in favor of PVP increased viability without decreasing its ability to suppress ice formation. The concentration of penetrating cryoprotectants can be further reduced by inclusion of non-penetrating “ice-blocking” polymers. These ice-blockers also reduce the critical cooling and warming rates necessary to avoid ice formation, which is an important requirement for solutions that are used to vitrify complex organs such as the human brain.

Because concentrated vitrification solutions depress the homogeneous nucleation temperature (Th) below the glass transition temperature (Tg), a major obstacle to successful vitrification is the presence of heterogenous nucleators. Some organisms have antifreeze proteins (AFPs) and anti-freeze glycoproteins (AFGPs) that mitigate heterogenous nucleation by binding to nucleators. Because adding such anti-nucleating proteins to vitrification solutions would be prohibitively expensive and less effective, Greg Fahy proposed the creation of synthetic ice-nucleation inhibiting polymers. In 2000 Wowk et al. published work that showed the effectiveness of a co-polymer of polyvinyl alcohol (PVA) and vinyl acetate in inhibiting heterogenous ice-nucleation. This co-polymer is now being sold by 21CM under the name “X-1000”. X-1000 is particularly effective in glycerol solutions, presumably because glycerol itself is a poor anti-nucleation agent. Increasing the concentration of X-1000 in vitrification solutions decreases ice formation and relaxes minimum cooling rates. Although X-1000 is presumed to be non-toxic, the maximum concentration in vitrification solutions does not exceed 1% w/v because no further benefits were observed beyond this concentration. In 2002, 21CM announced the discovery of another synthetic “ice-blocker” called Z-1000. Z-1000 is the polymer polyglycerol (PGL), which specifically inhibits ice nucleating activity caused by the bacterium Pseudomonas syringae. Mixtures of PVA and PGL are more effective in inhibiting ice formation than either agent alone, suggesting the PVA and PGL complement each other by inhibiting different sources (bacterial and non-bacterial) of ice nucleation.

A variant of Veg that includes the low molecular weight polymer polyvinylpyrrolidone K12, X-1000, and Z-1000 named VM3 improved viability in renal cortical slices and decreased the critical cooling and warming rates necessary to avoid ice formation and de-vitrification (ice formation during rewarming) while maintaining the same molar concentration as VS41A. The transition from Veg to VM3 reflects the two breakthroughs mentioned above: reduction of cryoprotectant toxicity by inclusion of non-penetrating polymers and ice blocking agents. VM3 also was the least toxic agent in vitrification of rat hippocampal brain slices, which is of particular importance for cryonics. The first vitrification agent ever to be introduced to cryonics was a hyperstable variant of VM3 called B2C. B2C was used until late 2005, when it was replaced by M22.

M22 takes advantage of two other discoveries: the ability to design better glass formers by methoxylation of conventional polyols, and inhibition of chilling injury by delivering the vitrification agent as a hypertonic solution. Because hydroxyl groups can bind either to water or hydroxyl groups on other cryoprotective agents, substituting methoxyl groups for hydroxyl groups should decrease interaction between cryoprotectants and increase interaction between the cryoprotectant and water. As a result, methoxylated compounds have stronger ice inhibiting ability, thus reducing the critical cooling rate for vitrification or reduce the concentration of (toxic) cryoprotective agents in a solution. Methoxylated cryoprotectants also decrease viscosity and increase cell permeability, allowing for shorter perfusion times, and thus reduced cryoprotectant exposure at higher temperatures. For example, the methoxylated glycerol derivative 3-methoxy-1,2-propanediol has a higher glass transition point and vitrifies at ~ 5% lower concentration than the corresponding conventional cryoprotective agent. Complete exploitation of these advantages is limited by the fact that they are more toxic than their non-methoxylated compound, as predicted by qv*. As can be seen in the table, the major difference between VM3 and M22 is the reduction of PVP K12 in favor of the penetrating cryoprotectants 3-methoxy-1,2-propanediol and n-methyl-formamide, and increased concentration of the ice-blocker Z-1000. The final molar concentration of 9.345 M demonstrates that more concentrated vitrification agents do not necessarily have to be more toxic.

VS41A

Veg

VM3

M22

Dimethyl sulfoxide

3.10 M

3.10 M

2.855 M

2.855 M

Formamide

3.10 M

3.10 M

2.855 M

2.855 M

Propylene glycol

2.21 M

Ethylene glycol

2.71 M

2.713 M

2.713 M

N-methylformamide

0.508 M

3-methoxy-1,2-propanediol

0.377 M

Polyvinyl pyrrolidone K12*

7% w/v

2.8% w/v

X-1000 ice blocker*

1% w/v

1% w/v

Z-1000 ice blocker*

1% w/v

2% w/v

Total Molarity

8.41 M

8.91 M

8.41 M

9.345 M

* Non-penetrating polymers are in w/v

M22, so called because it was intended to introduced at -22 degrees Celsius, constitutes a major landmark in vitrification of complex organs. In 2005 Fahy, Wowk et al. announced routine recovery of rabbit kidney slices from temperatures around -45 degrees Celsius. Although consistent recovery of vitrified organs is not yet feasible, continued progress in solution composition and perfusion techniques inspire optimism that this may be possible in the future. In 2007, Greg Fahy of 21CM reported recovery of electrical activity in vitrified brain slices and induction of long-term potentiation (LTP), which indicates that the structures for processing memory are maintained after vitrification, storage and rewarming of brain tissue. Visual evidence that M22 can preserve the ultrastructure of the brain better than B2C was published on the Alcor website in 2005.

M22 also needs to be used in a suitable carrier solution to support cell metabolism at low temperatures and decrease oxidative injury and edema. The carrier solution for M22 is called LM5 to reflect the 50% reduction of glucose (as compared to the older carrier solution RPS-2) in favor of equimolar concentrations of mannitol and lactose, to address compatibility problems with the ice blockers. The combination of the isotonic LM5 plus the non-penetrating polymers in M22 creates a hypertonic solution, which has been shown to eliminate chilling injury, which is the injury that is caused by exposure to low temperatures as such. For cryonics, the composition of M22 is further enhanced by including a proprietary components that allows perfusion of whole body patients without edema.

The research breakthroughs discussed above allow for a global reconstruction of the composition of M22 using the table. Maintained is the equimolar combination of DMSO and formamide from Fahy’s older vitrification solutions to reconcile strong glass formation ability and minimal toxicity. The discovery of the  compositional variable qv* allows for substitution of higher concentrations of the weaker glass former ethylene glycol for propylene glycol. Substitution of a non-penetrating polymer, PVP K12, and the ice-blockers X-1000 and Z-100 allow for further reduction of DMSO and formamide, reduction of critical cooling rates, and increased stability against ice formation. In M22, PVP K12 is reduced to optimize hypertonicity of the non-penetrating agents for suppression of chilling injury. Added are the methoxylated cryoprotectant 3-methoxy-1,2-propanediol and the highly permeable amide n-methyl-formamide, producing the least toxic but most concentrated vitrification solution to date.

The most striking differences between Alcor’s old perfusate and the newer vitrification agents licensed from 21CM are complexity and cost. Until 2002, Alcor patients were perfused with high molar glycerol in an MHP-2 based carrier solution. M22 itself consists of 8 (!) different components, putting the total number of components of M22 in carrier solution above 15. Such perfusates makes great demands on preparation skills and quality controls. Components such as the ice blockers and 3-methoxy-1,2-propanediol have put the cost of Alcor’s whole body perfusate alone close to the cost of complete cryopreservation arrangements at the Cryonics Institute (CI). This raises obvious questions about costs and benefits. As evidenced by CI’s VM-1, potent protection against ice formation can be achieved with a vitrification agent that solely consists of DMSO and ethylene glycol. It is plausible to assume that vitrification lessens demand on future repair technologies, but it speculative to assume that minor differences in toxicity between different vitrification agents will translate in earlier resuscitation and less expensive repair protocols. However, more toxic vitrification solutions, such as CI’s VM-1, may cause acute injury to endothelial cells. As Brian Wowk notes, “good cryoprotection depends on good perfusion, which depends on preservation of vascular integrity during perfusion. The ability to perfuse M22 into whole bodies with tolerable edema is likely to be intimately related to its low toxicity to vascular endothelium.” And of course, there are also PR advantages to the fact that a cryonics organization uses a vitrification agent that is also the state of the art in conventional cryopreservation of organs.

M22 produces substantial brain shrinking during perfusion of (non-ischemic) patients. As a matter of fact, cerebral dehydration may be a major contributing factor to vitrification of the brain and even allow for reduced concentrations of M22 for brain preservation. This does not mean that the (expensive) non-penetrating polymers could be replaced for any high molecular weight polymer because the ice blockers and non-penetrating cryoprotective agents also protect the extracellular space against ice formation and are effective in ischemic patients with a compromised blood brain barrier (BBB). The limited ability of some components of M22 to cross the BBB and, and differences in permeability of the various components of M22, does raise questions about the exact composition of M22 beyond the BBB and within brain cells after completion of cryoprotective perfusion.

Patients outside of the US may not fully benefit from cryopreservation with M22 because of the of long cold ischemic times during transport. This raises the question if cryonics patients can be perfused outside of the US and shipped in dry ice. Experiments with VM-1 in bulk solution indicate that this solution is very stable against ice formation, even during long storage periods. M22 in bulk solution seems to form ice crystals overnight if stored in dry ice. This does not necessarily mean that M22 cannot be used in combination with dry ice for overseas patients because human tissue perfused with M22 (or any cryoprotective agent) is not the same as M22 in pure solution. But regardless of M22’s compatibility with dry ice shipping, cryonics organizations may benefit from formulating a highly concentrated inexpensive vitrification solution that is extremely robust against formation of ice, which can be used for simple perfusion of non-US patients in combination with dry ice shipping. The decreased cold ischemic times of such a solution may outweigh the increased toxicity of such solutions.