Annotated bibliography of cryoprotectant toxicity


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.


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.

October 2010 Cryonics Symposium in Germany

On the first weekend of October, 2010 I was an invited speaker at “Applied Cryobiology – Scientific Symposium on Cryonics” held in Goslar, Germany: The meeting was the first effort by the German Society for Applied Biostasis (DGAB) to create a milieu for scientific discussion of cryonics-related issues as well as to elevate the scientific status of cryonics and bring more scientists into the field. DGAB hopes to have another such symposium in two years.

Goslar, Germany is a World Heritage Site and tourist center based on the fact that it was the beginning of German industry nearly a thousand years ago as a rich source for mining many minerals. Goslar became a free imperial city and was a favorite residence for many emperors. Goslar is also the city where the conference organizer lives.

With only about 10,000 tourists per year, and a location that is not close to a major city, Goslar can only be reached after several hours by car or train by those coming from outside of Germany. I chose to rent a car, partly because it was so much less expensive than train, and partly because of my curiosity about the Autobaun.

The German Autobahn is probably the only major highway system in the developed world that has portions without a maximum speed limit. I have no enthusiasm for speeding, but was curious to see what it is like to drive on the Autobahn. I reasoned that such a motorway would not be permitted to exist if it were littered with corpses and smashed vehicles. I found that much of the Autobahn had speed limits, there were many construction zones that restricted speed, and traffic jams were sometimes so bad that any forward motion was slow and intermittent. But there were a few times when I was traveling over 90 miles per hour in the flow of traffic, and being passed on my left by cars going so fast that I could have been standing still, relatively speaking. Nonetheless, it did not seem too dangerous.

The symposium was originally to be held mainly in German, but there were twice as many attending (about 50) as had been anticipated — and so many were from outside Germany that the organizers decided to have all sessions in English. Although many of the participants had impressive scientific backgrounds, they were overwhelmingly people with a personal interest in cryonics. The organizers struggled to get speakers with scientific credentials, but many of those who would have been otherwise interested and qualified did not want to risk their careers by participation. Peter Gouras, MD, PhD was the most credentialed scientist presenting. There was a medical examiner whose presentation concluded that cryonics can’t work in Germany, a perfusionist-turned-journalist, an embalmer who failed to attend, a nanotechnology PhD, and me. The other presentations were not about cryonics science.

I was scheduled to speak about challenges in cryonics technology, but became concerned that there was no general introduction to cryonics technology in the program. I requested that I give an introductory presentation as the first speaker, and give another presentation on technical challenges later in the program. Instead, the organizers gave me double the time for my presentation as first speaker (following the Mayor of Goslar). I believe that I did a good job combining introductory material with technical challenges in cryonics. My presentation and the question period that followed were recorded on video, which I am hoping will be put on YouTube.

Holger Zorn discussed his experience as a perfusionist who had worked in the field of hypothemia. He said that cannulation for cooling perfusion could be done in two minutes. When cooling for cardiac surgery they used diluted blood (low hematocrit). Holger discussed cases of forensic perfusion in which reperfusion was performed weeks after death on corpses to elucidate puncturing by knife or gunshot. He said he had worked with hypothermic perfusions down to 18 degrees Celsius, and had never seen a case of shivering. This conflicts with studies reporting shivering between 34 and 35.5 degrees Celsius in therapeutic hypothermia, requiring drugs for suppression:

There has been recent criticism of the use of drugs to suppress shivering in cryonics cases.

Dr. Peter Gouras, who is on the Cryonics Institute Scientific Advisory Board (and whose wife is German) has been involved in cryonics for many decades. He is an ophthalmology professor at Columbia University. He was introduced as the “father of retinal pigment epithelial transplantation.” He discussed his work studying macular degeneration in rhesus monkeys on calorie restriction, concluding that calorie restriction has less benefit for primates than for rodents. He expressed the view that enthusiasm for cryonics is genetic, and that any attempt at persuasion is fruitless. Somewhat contradicting this claim is his claim that reviving a whole mammal from cryopreservation would have a huge impact on the acceptance of cryonics.

The Nanotechnology and Cryonics presentation by Klaus Mathwig was somewhat standard nanotechnology fare for me. What I found most interesting was the question of how nanomachines would know how the correct structure would look after increasing levels of damage. It was suggested that there might be a need to scan the brain structure beforehand. So if your last scan was a month or two before your deanimation, you might be reconstructed as you were at that time. But with a good scan, what need is there for the original material? I thought the purpose of nanobots was to partly to discover the original structure.

Christoph Meissner is a medical examiner who works at the Department of Forensic Medicine at a university hospital. He had done an impressive amount of research on the subject of brain deterioration following stoppage of the heart. Many of the studies he cited were decades old because such studies would not currently be approved by ethics committees. In his experience, the corpse of a murder victim is not found in less than four hours. Under the best of circumstances he believes that a death certificate cannot be issued in Germany in less than one or two hours. He believes that it would not be possible to revive a cryonics patient who had experienced that amount of warm ischemia. During the question period he was asked why he would come to a cryonics conference if he had such a negative view of cryonics prospects. He answered that he is a scientist and that he was trying to make a reasonable assessment of cryonics. I believe that he is sincere and had no “ax to grind” about cryonics one way or the other. The fact that he was specific about probable delays in Germany being the source of his negative prognosis implied that he has not decided that cryonics is hopeless ifcryopreservation is prompt. Ironically, one of the studies he cited showed that rat brain neurons in cortical slices recover function upon reoxygenation as well after five hours post-mortem as they do after immediate post-mortem reoxygenation.

David Styles announce the beginning of Eucrio, an organization intended to give Suspended Animation, Inc -like standby/stabilization services to all the countries in the European Union, plus Norway. Cryonics patients would be vitrified in Europe with CI’s VM-1 vitrification solution, and then shipped on dry ice to Michigan or Arizona for cryostorage. Given the welter of European languages, laws, and insurance policies this is an ambitious undertaking. David has a lot of energy, intelligence, and determination, so if anyone can make this project work, he is one of the few. David spent much time discussing the equipment Eucrio has or is obtaining. Eucrio currently has seed capital for the first year of operation, and it is expected that Eucrio members paying 35 euros per month will keep the organization going even when there are no patients. Fees for service are calculated with a goal of breaking even, based on the assumption that one-third of insurance policies don’t pay (which has not been CI experience).

Sebastian Sethe is a lawyer who spoke on Ethical Problems in Cryonics. Sebastian asked many questions for which he gave no answers. When challenged on this matter, he said that ethicists are more interested in questions than answers, whereas scientists are the opposite. I sometimes think that ethicists are sadists who enjoy torturing people with questions. As a case in point, Sebastian asked whether if the CI facility caught fire, if Ben Best should be saved or the 100 cryonics patients in storage. Part of his question was entailed in Sebastian’s assertion that “It is reasonable to assume that cryonics is not going to work.” After the lecture I tried to pin Sebastian down on his assertion, asking him why his assertion should be more true than “It is reasonable to assume that cryonics is going to work.” He answered that the true opposite of his assertion is “It is unreasonable to assume that cryonics is not going to work.” I at least got him to say that cryonics has more than a zero chance of working, although I had a hard time nailing down what he thinks the most limiting considerations are — technical, organizational, societal, financial, etc. He suggested that the cryonics organizations are financially threadbare and vulnerable.

I considered discussing the preventative measures against fire that are in place at the Cryonics Institute, but did not do so.

Torsten Nam spoke on Cryonics and Transhumanism. He described transhumanists as people who want to use technology to improve their physical and mental abilities, and to overcome their (biological) limitations. He said that 8% of transhumanists are cryonicists, which by his calculations means that a transhumanist is 200,000 times more likely to be a cryonicist than someone in the general population. He called FM-2030 the father of modern transhumanism, while acknowledging Robert Ettinger’s transhumanist classic MAN INTO SUPERMAN. Among major milestones he listed Francis Fukuyama calling transhumanism the world’s most dangerous idea and a 2007 European Union report on human enhancement. In the early days it had been common to compare transhumanism to fascism (Nietzsche’s Superman), but now the subject is entering the academic mainstream. Some transhumanists want to dissociate themselves from cryonics in order to be more acceptable.

On Sunday the Robert Ettinger Medal for outstanding merits in the field of cryonics was awarded to its first recipient: Robert Ettinger.

Medal Front

Medal Back

I accepted the medal on behalf of Mr. Ettinger, which meant that I had to make a speech. I said that Robert Ettinger is above all a man ofideas, who nonetheless also felt obliged to exert his influence in the physical world by, among other things, helping found the Cryonics Institute because he was not satisfied with what the other cryonics organizations were offering. I also said that Mr. Ettinger deserves a lot of credit for the creation of CI’s fiberglass cryostats, something he is rarely credited for.

In the Round Table discussion I provocatively asked David Styles how Eucrio would provide good stabilization service in Germany, where they would have to wait 1-2 hours after cardiac arrest to get a death certificate before proceeding with cooling and Cardio-Pulmonary Support (CPS).The situation is worse in Italy where 24 hours must pass before getting a death certificate, and in France where cooling is not permitted. France and Italy both require embalming before a body can be shipped out of the country. There was a lengthy discussion/argument wherein David defended his ability to expedite obtaining death certificates and to adapt legal requirements to cryonics purposes. In my own talk I had cited studies showing that neurons are more durable than generally believed, and can survive hours of warm ischemia. Good vitrification in Europe and shipment in dry-ice would definitely be an advantage over the alternative of spending days in water-ice during shipment.

I mentioned the importance of vital signs alarm systems for cases of sudden death where no standby is possible — and the greater availability of such systems in Europe versus the United States, notably the  Vivago Care watch. Dr. Klaus Sames became very impatient and stressed that a scientific symposium should discuss more scientific issues. Dr. Peter Gouras then began beating the drum for raising money for cryonics research — and his preference for small animal whole body experiments. I re-emphasized that Aschwin and Chana de Wolf are doing the most focused cryonics research in their experiments that have found ways to improve perfusion in cryonics patients that have suffered ischemic damage (virtually every cryonics patient). I believe that it would be a great boon to cryonics science if there was money for Aschwin and Chana to do full-time research, rather than just on weekends.

Dr. Sames again felt that this subject is not purely scientific, which led to some discussion of methods of cryonics research. Dr. Sames questioned that the results of small animal experiments are applicable to large animals (humans). Dr. Gouras argued that mouse experiments are the basis of most modern medicine. I described the whole body vitrification experiments at 21st Century Medicine, and the electrophysiology studies on vitrified hippocampal slices. I noted that my information is three years old and that the next public update on 21st Centrury Medicine research is not likely to happen until the May 2011 Suspended Animation Conference in Florida.

Dr. Gouras repeated his claim that experiments on small mammals provides more rapid feedback than organ cryopreservation. No one seemed very inspired by my contention that the greatest breakthrough for cryonics would be elimination of cryoprotectant toxicity. We only have vague theories of what cryoprotectant toxicity is — there should be focused research on this topic, understanding the mechanisms of cryoprotectant toxicity would be a significant step toward understanding how to eliminate it. Whole body vitrification efforts are easily distracted by perfusion problems, and trying to analyze every organ at once makes the problem hopelessly complicated. Analyzing cryoprotectant toxicity on single organs, perhaps even with biochemical tools (because it is ultimately an issue in molecular biology), has the best chance of addressing the toxicity problem, in my opinion. But “cryomouse prize” and whole body vitrification approaches win the popularity contests by a large margin over a cryoprotectant toxicity “X-prize”. I believe that given adequate funding, Aschwin and Chana de Wolf could contribute significantly to finding less toxic cryoprotectants, and I would like to be involved in the project.

At the symposium I met many people whom I had not known before, many I had known, but not met, and quite a few others that I enjoyed meeting again. I will only mention one, however: Roland Missionnier.

In the late 1960s the Cryonics Society of France was the largest cryonics organization outside of the United States. Roland was the President and Anatole Dolinoff was Vice-President. Roland showed me a list of officers and directors of the organization, pointing-out who had been fighting with whom, and the fact that virtually all were dead without having been cryopreserved. Dolinoff believed that cryonics was illegal in France because of a decree issued by the French Minister of Health in 1968. On page 13 of the October 1989 issue of CRYONICS magazine, Saul Kent said that he would investigate challenging the French law if it had an substance, but if he did so, I never heard the result of his efforts. Roland has been trying to re-start a cryonics organization in France, but he is also planning to move to Florida where he can live close to Suspended Animation, Inc. Roland said that with some money and a lawyer, almost anyone could move to the United States.

Cryonicist Charles Tandy, PhD, wants to publish the symposium proceedings through his Ria University Press.

Those of you who read Finnish can read the summary by Ville Salmensuu or you can stick the link in Google translate:|en|

The 2009 SENS Conference

Once a year I try to attend at least one biogerontology conference. Although I attend biogerontology conferences out of personal interest, and at my own expense, they are the most fruitful grounds for promoting cryonics I have found, and this is especially true of SENS conferences.

I have missed none of the four SENS conferences that have been held at Cambridge University. “SENS” is Dr. Aubrey de Grey’s “Strategies for Engineered Negligible Senescence.”

SENS conferences attract scientists who are eager for science to achieve rejuvenation, and who have a strong belief that science has the capacity to do so. Not surprisingly, such people are often receptive to the idea that future science may be capable of reanimating humans who have been well cryopreserved.

Recently I have heard regret expressed about the aging of the cryonics community and the absence of a next generation of cryonics activists to replace the current ones. My experiences at the 2009 SENS conference dispelled much of my concern about this.

I took about a hundred CI brochures, but these were quickly taken by the 290 SENS conference attendees. I was continually approached by young scientists and researchers who were eager to meet me and who said they would make cryonics arrangements when they got out of graduate school and could afford to do so. Insofar as many of the attendees were Europeans, I was often asked whether the shipping delays to the United States would make cryonics not worth doing, and whether there were any plans by the Cryonics Institute to create a storage facility in Europe. (I was told about a group wanting to establish a storage facility in Switzerland, but I did not get any details. Apparently it is not a project with serious hope of success in the near future.)

I was astounded when a British student approached me and said that he would be devoting all of his graduate school work to the problem of cryoprotectant toxicity. He had already gotten Dr. Fahy to send him a copy of “Cryoprotectant toxicity neutralization,” a new paper to be published in an upcoming issue of CRYOBIOLOGY. The student is in the process of collecting other cryobiology publications that address the subject. I directed him to a relevant webpage in the cryonics section of my website.

A number of people from KrioRus were at the conference, notably Igor Artyuhov, who is their technical guru. The group also does life extension research. Igor showed me their poster showing extended lifespan of mice administered heat-shock protein through nose-drops. I was interviewed by a journalist who writes for the Russian edition of SCIENTIFIC AMERICAN.

I had met Nick Mayer, a Terasem employee, at the previous SENS conference, and Nick introduced himself to me again at this meeting. Nick manages “cyberbiological systems”, specifically a website that is being used like an on-line personal diary. As Nick described it to me, the website would be useful to store personal information that could be used to help in the reconstruction of someone who has been reanimated from cryopreservation. But when I looked at his website, it appears to be a project for reconstructing people from their diaries alone — without any saved biological material.

To my surprise, one of the presenters, Dr. Gunther Kletetschka, had a poster and an oral presentation dealing with eliminating the cracking problem in cryonics.

Cracking of vitrified tissue at cryogenic temperatures is a consequence of the fact that external cooling causes superficial tissue to contract more than deep tissue (thermal conductivity is low). Dr. Kletetschka’s approach is based on the idea that if a cryonics patient were perfused with a solution containing gadolinium (nanoparticles would be best), an entire vitrified brain could be cooled uniformly by the magnetocaloric effect.

From a practical point of view, his sample size was apparently very small, and he did his testing on ice rather than vitrified tissue. I had many other criticisms of his approach, which I attempted to discuss with him in a constructive, supportive manner. He was interested in what I had to say, and was very receptive. Insofar as he is so enthusiastic about doing cryonics-related research, and insofar as he lives in Maryland (not so far from Michigan) I suggested that he attend the CI Annual General Meeting on Sunday, September 27. He expressed an interest in doing so.

A European student told me that his mother is a stroke victim, but that he has not been able to induce her to consider cryonics. Having experienced the debilitating effects of stroke she is worried that faulty reanimation procedures would bring her back into an even more debilitated condition. I suggested asking her to assess the probability of that happening and how bad the downside would matter if the probability is small. I think that in the context of all of the other repairs that would be essential to cryonics working that it is unlikely that all such defects would not be fixed.

A middle-aged European woman wanted to speak with me about how to convince her husband that cryonics is a good idea. The couple are both religious, but she thinks “heaven can wait” because she enjoys life here on earth and she would like to share earthly life for a very long time with her husband. I gave her many arguments against the claim that cryonics is against religion, including the one concerning refusing a lifesaving medical treatment being equivalent to suicide (a sin).

I was reminded of the Depressed Metabolism posting about the “hostile wife phenomenon” in cryonics:  It occurs to me that when a male spouse is interested in cryonics, but his wife is not, that he can go ahead and make the arrangements. A financially dependent woman (as this woman is), less often has that option. I have also often seen cases of women interested in cryonics, but who dropped the interest when it became clear that their spouse would not join them in cryostasis. They would rather not live if they cannot be with their husbands. It reminds me of studies of working couples that show that a wife is much more likely to quit her job to follow her husband in a career change that involves moving — whereas the opposite happens much less frequently.

I won’t say much about the SENS conference itself, but I had lots of meetings and discussions that taught me a lot about biogerontology issues. I was particularly interested in discussing my recent article “Nuclear DNA Damage as a Direct Cause of Aging” which had been published in Rejuvenation Research, because it is a direct challenge to one of the tenets of SENS (that nuclear DNA damage only matters for cancer).

Not only was I able to have two private sessions in which I discussed the matter with Aubrey de Grey, but I was able to eat breakfast several times with Vera Gorbunova and her husband Andrei Seluanov, two DNA repair experts who were attending the conference. Vera and Andrei have written the only other review (other than my own) supporting the thesis that nuclear DNA repair capability declines with age.

I had cited that review in my own review. Vera had sat across from me at my first breakfast by chance. She had read my review and told me that she agreed with it. Most of the times that I went for a meal I was very pleased by at least one person randomly sitting near me, and had an interesting and productive discussion with them on a matter of interest. I discussed my cryonics alarm system problems with a woman who is getting a PhD in biomedical engineering.

I was very surprised and pleased to meet Kristen Fortney at the conference. Kristen is a University of Toronto student who attended some of our cryonics meetings in Toronto. She was a physics student and was planning to do graduate work in quantum physics. At the conference she told me she had changed to a PhD program focused on computational work with the human genome, focused on anti-aging strategies. She wrote a blog of the conference as it progressed on the Ouroboros academic blog for aging research.

The red blood cell as a model for cryoprotectant toxicity

Various approaches are available to investigate cryoprotectant toxicity, ranging from theoretical work in organic chemistry to  cryopreservation of complete animals. Because resuscitation of complex organisms after cryopreservation is not feasible at the moment, such investigations need to be confined to viability assays of individual cells and tissues or ultrastructural  studies.

One simple model that allows for “high throughput” investigations of cryoprotectant toxicity are red blood cells (erythrocytes). Although the toxic effects of various cryoprotective agents may differ between red blood cells, other cells, and organized tissues, positive results in a red blood cell model can be considered the first experimental hurdle that needs to be cleared before the agent is considered for testing in other models.  Because red blood cells are widely available for research, this model eliminates the need for animal experiments for initial studies. It also allows researchers to investigate human cells. Other advantages include the reduced complexity of the model  (packed red blood cells can be obtained as an off-the-shelf product) and lower costs.

Red bloods cells can be subjected to a number of different tests after exposing them to  a cryoprotective agent. The most basic test is gross observation of the red blood cells in a cryoprotectant solution. When high concentrations of a cryoprotectant are used (such as in vitrification), a stepwise approach is necessary to avoid  osmotic  injury. If a cryoprotectant solution is extremely toxic rapid hemolysis will follow, which often can be observed by a noticeable change of the color of the solution,  red cell debris sinking to the bottom of the test tube, or negligible difference between the pellet (if there is one at all) and the supernatant after centrifugation. But if the researcher is interested in agents that are not extremely toxic, or wants to compare variants of  similar agents with each other, quantitative methods and detailed observations are required using respectively spectrophotometry and light microscopy.

In 1996, Bakaltcheva et al. used the red blood cell model for an elegant and thoughtful study  of cryoprotective toxicity. The authors did not only use spectrophotometry to measure hemolysis  but also used microscopy to study the morphology of the red blood cell after exposure to various agents at different temperatures. The results of these different measurements were in turn correlated with each other in order to determine if there are general properties  affecting cryoprotectant toxicity. The authors propose that reduced toxicity can be achieved by keeping the dialectric constant of the medium and membrane close to that of an aqueous solution without solutes.  These findings can also explain why cryoprotective mixtures  of various agents (such as DMSO and formamide) can reduce toxicity.  A general rule of thumb for formulating vitrification agents with reduced toxicity seems to be to maintain most properties of water but eliminating the posibility of ice formation. It should not be a surprise that such an approach has guided the choice of solvents in areas such as cryoenzymology.

Greg Fahy on the cryopharmacology of vitrification solutions

In an abstract in Cryobiology 55 (2007), 21st Century Medicine researcher Greg Fahy reports on the biological (pharmacological or “cryopharmacological”) effects of vitrification solutions. He identifies four different mechanisms of toxicity:

1. “Specific toxicity,” or the effects of vitrification agents on well-defined biological pathways or sites.

2. Adverse effects on the hydration of biomolecules as a result of water-cryoprotectant interactions.

3. Protein denaturation by methylated cryoprotectants (such as N-methylformamide), or the vitrification solutions that include them.

4. Chemical reactions between DMSO and cellular sulfhydryl groups.

These investigations into the chemical and physical mechanisms of cryoprotectant toxicity may contribute to improved vitrification solutions that inhibit ice formation and maintain viability of complex organs.

Life in non-aqueous solutions

Can life exist without water? This is one of the questions that fascinates astrobiologists. The behavior of biomolecules in non-aqueous solutions is also of interest to cryobiologists and cryoenzymologists. Ice formation below zero degrees Celsius can be prevented by high concentrations of cryoprotective agents. But what are the effects of such vitrification agents on proteins?

In 1989 Alexander M. Klibanov published a paper called “Enzymatic Catalysis in Anhydrous Organic Solvents” that reports that enzymes are not only able to function in anhydrous organic solvents, but that some display remarkable properties in such environments like enhanced storage stability, solvent-induced changes of enzyme stereoselectivity, molecular memory, and reactions that are normally inhibited in aqueous solutions.

Upon reading the paper it is clear that when the author speaks of anhydrous solvents it is not implied that enzymes do not require water at all:

“…the key question should be not whether, but how much, water is required for enzymatic activity. Clearly, the enzyme molecule cannot ‘see’ more than a monolayer or so of water around it. Therefore, if this layer of ‘essential’ water is  somehow localized and kept on the surface of the enzyme, then all the bulk water should be replaceable with organic solvents with no adverse effects on the enzyme.”

To assure enzymatic activity in organic solvents two rules must be followed. First, hydrophobic solvents are preferred.  The authors propose that hydrophilic solvents ‘strip’ the essential water from the enzymes, and thereby reduce or eliminate the activity of enzymes. Second, the enzymes to be used in organic solvents need to be lyophilized (freeze dried) from aqueous solutions  with the pH optimal for their activity. This last requirement reflects the phenomenon of “pH memory” in which the enzymes retain the ionization state they had at that pH in the aqueous solution during freeze-drying and in organic solvents.

As surprising as some of these findings may be, the requirement of bound water for enzymes to  function is still consistent with the orthodox view that life requires water. At best, such findings can explain the existence or preservation of life in low water environments.

For cryobiologists, such findings raise interesting questions. In 2004, Fahy, Wowk et al. speculate that one of the mechanisms of cryoprotectant toxicity may involve “reduced hydration of biomolecules.” Understanding how solvents, and the combination of solvents, affect the intracellular milieu and the hydration and stability of biomolecules, should contribute to the design of less toxic vitrification solutions. Such vitrification solutions can be optimized for the human brain to allow for real suspended animation and improved prospects of resuscitation of cryonics patients.

Cryoprotectant toxicity: biochemical or osmotic?

The current generation of vitrification agents in cryonics permit elimination of ice formation using realistic cooling rates. But attempts to vitrify the brain require high concentrations of cryoprotective agents to inhibit ice formation. Such high concentrations of cryoprotectants can produce injury to tissues that is distinct from damage caused by ice formation.

Vitrification of complex tissues requires perfusion to substitute the cryoprotective agent for water. Because the cryoprotectant concentration necessary to vitrify (CNV) is higher than than the concentration of solutes in the cells, exposing cells to such high concentrations at once will result in cell injury as a result of osmotic stress. This osmotic effect of cryoprotectants requires that the introduction of the vitrification agent be gradual to allow the cryoprotective agent to be exchanged with cell water without injury.

How important is osmotic shock as a form of injury?

In 1984, Greg Fahy published a paper (Fahy GM, Cryoprotectant Toxicity: Biochemical or Osmotic? Cryo-Letters 5:79-90) to distinguish cryoprotectant-induced osmotic injury from biochemical injury. Fahy reviews the literature and presents his own 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.

A number of caveats for cryonics are in order. Osmotic stress as a result of rapid introduction of the cryoprotectant depends on the specific cryoprotective agent(s) and tissue. For example, glycerol, the prevailing cryoprotectant in cryonics until the more recent vitrification agents were introduced, has relatively high viscosity and poor permeability at low temperatures compared to other cryoprotective agents such as DMSO and ethylene glycol. W.M. Bourne et al. found that the highest concentrations of different cryoprotectants that did not cause a loss of human cornea endothelial cells were higher with the ramp method (gradual increase) for glycerol and higher for DMSO, 1,2-propanediol and 2,3-butanediol using a step method. These results indicate that more toxic cryoprotective agents with good penetration may benefit from a stepped approach to reduce cryoprotectant exposure times.

What the optimal introduction rate for specific cryoprotective agents (or mixtures of cryoprotective agents) is in the brain we do not know. The brain is also unique in the sense that an intact blood brain barrier (BBB) limits introduction of vitrification agents to the brain. This is especially important in case the vitrification solution includes non-penetrating agents such as polyvinylpyrrolidone (PVP) and ice blocking polymers. In many cryonics patients, the BBB may be compromised as a result of warm and cold ischemia, which introduces another variable that may affect the optimal introduction rate of the vitrification agent.

Osmotic shock as a result of too rapid diffusion of water from the cells should be distinguished from dehydration injury as such. Vitrification agents like M22 are assumed to confer some of their ice inhibiting effects by dehydration of the brain. Whether such (extreme) dehydration affects (long term) viability in the brain is another area that warrants investigation. Research that would investigate the effects of different introduction and removal protocols for various vitrification agents on the brain would be a step towards finding the right balance between the need for gradual introduction of the vitrification agent on the one hand and minimizing cryoprotectant toxicity on the other.