Tag: Vitrification

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.

The purple prose of suspended animation

Esquire magazine features an article on scientist Mark Roth and his research into “suspended animation.” As the website title “The Mad Scientist Bringing Back the Dead…. Really” indicates, this is not supposed to be a detailed account of Ikaria’s recent advances in induction of depressed metabolism but a sensationalist piece on mad scientists. Although the piece states that “Ikaria’s first suspended-animation product” has “completed Phase 1 trials in Australia and Canada” and is “being tested on humans, to make sure it’s safe” it remains to be seen if this technology involves major advances in rapid induction of depressed metabolism in humans or offers just another treatment option for various hypoxic-ischemic conditions as the press release (pdf) seems to indicate.

The article misses a number of opportunities to set the record straight on the proper use of terminology and prevailing definitions of death. The ability to resuscitate an organism from circulatory arrest, depressed metabolism, or suspended animation implicates that the organism was not dead to start with. This is not just a matter of semantics. The phenomenon of death is surrounded by many cultural and religious taboos and the difference between saying that we can  bring back the dead instead of  observing that recent advances in science and medicine requires us to redefine our definition of death  is not a trivial matter. Most religious people do not object to cardiopulmonary resuscitation or hypothermic circulatory arrest because they do not believe that a patient who is resuscitated in such medical procedures was (temporarily) dead. The word death should be reserved for a condition in which integrated biological function cannot be restored by either contemporary or future technological means.

Increasingly, the phrase “suspended animation” is thrown around to describe a number of distinct phenomena ranging from modest drops in metabolism to complete metabolic arrest. If the word  is taken literally, however, only complete metabolic arrest constitutes real suspended animation. Such a state cannot be achieved in humans by the use of hydrogen sulfide (or its injectable derivatives) and requires either the use of extreme cold such as practiced through vitrification in cryonics or the use of advanced nanotechnology in warm biostasis.

Popular reports on recent developments in “suspended animation” do not carefully distinguish between the results obtained with hydrogen sulfide and carbon monoxide in C. elegans and mice and its applications in humans. Until more detailed information is available on the use of these substances in large animals or humans it should not be assumed  that rapid pharmacological induction of depressed metabolism in humans is a clinical possibility.

Cryonics sets example for emergency medicine

One of the most neglected aspects of cryonics is that its procedures, and the research to support them, can have important practical applications in mainstream fields such as organ preservation and emergency medicine. Contrary to popular opinion, cryonics does not just involve an optimistic extrapolation of existing science but can set the standard for these disciplines. As a matter of fact, that is exactly what cryonics, and cryonics associated research, has been doing over the last 25 years.

The most striking example is the progress in vitrification as an alternative for conventional cryopreservation. Although the idea of eliminating ice formation at low subzero temperatures has been discussed since the beginning of cryobiology, vitrification as a serious research agenda was largely driven by the demand for ice-free preservation of the human brain. Over the last decades this research has culminated in the development of the least toxic vitrification agent to date, 21st Century Medicine’s M22.

The contributions of cryonics to mainstream science and medical practice are not confined to cryobiology. Researchers Jerry Leaf and Mike Darwin made impressive progress in the formulation of bloodless whole body organ preservation solutions to resuscitate dogs from ultraprofound hypothermic temperatures, an intervention that is increasingly being recognized as essential to stabilize trauma victims. In the mid 1990s, Mike Darwin and Steve Harris conceived and developed the idea of using liquid breathing with perfluorocarbons as a method to induce rapid hypothermia. They further validated a multi-modal medications protocol to resuscitate dogs from up to 17 minutes of normothermic cardiac arrest without neurological damage.

Although progress has slowed considerably in the non-cryobiology research areas over the last 10 years, it is encouraging to observe that some of the procedures that are routine in cryonics  stabilization protocol  are starting to catch on in mainstream emergency medicine practice as well. For example, contemporary cryonics stabilization protocol has been strongly shaped by the idea that the best strategy to limit brain injury after cardiac arrest is to combine a number of different interventions: cardiopulmonary support, induction of hypothermia, and administration of circulation-supporting and neuroprotective medications.

It is therefore very encouraging to learn that the Wake County EMS group in North Carolina has achieved impressive results in treating out-of-hospital cardiac arrest victims using a protocol that closely follows elements of current cryonics stabilization protocol. Systematic implementation of immediate induction of hypothermia, continuous compression CPR, and the use of an impedance threshold device (ResQPOD) produced an almost 400% improvement in survival and vast improvements in neurological outcome. A PowerPoint presentation about their experience and protocols are available at their website.

Such real world outcomes do not only inspire confidence in the procedures cryonics organizations can use to protect patients from brain damage after cardiac arrest, it should also serve as a wake-up call to relaunch an aggressive research agenda to push the limits of hypothermic and normothermic resuscitation. In absence of this, it will only be a matter of time before cryonics activists can no longer claim that “we did it first.”

HT Mike Darwin

Famous preserved body parts

The website TopTenz recently published a list of the Top 10 Most Famous Preserved Body Parts. The list includes Galileo’s finger and Albert Einstein’s brain. As has been discussed on this blog before, the preservation of human brains (no matter how frivolous the intention) raises a number of important questions about the nature of death and the possibility of  future resuscitation. The brain constitutes the physical basis of the person and, under ideal conditions such as prompt vitrification, preserving the brain is akin to preserving that person.

Not mentioned in this list is the strange fate of the brain of Benito Mussolini, the fascist leader of Italy. It is claimed that parts of Mussolini’s brain are contained in a box together with his remains in a tomb in his birthplace Predappio in Italy. In his travel diary “They Stole Mussolini’s Brain (Well, Almost),” industrial musician Boyd Rice published a hilarious account of his visit to Predappio and involvement in an (ultimately abandoned) attempt to steal Mussolini’s brain.

Further reading:

Albert Einstein’s brain and information-theoretic death

Also on TopTenz:

Top 10 Researchers who Experimented on Themselves

Cryoenzymology and cryoprotectant toxicity

The major limiting obstacle to reversible cryopreservation of complex organs is cryoprotectant toxicity. Elimination of ice formation through vitrification requires high concentrations of cryoprotective agents. These high concentrations of cryoprotectants can be toxic to tissues. Over the years, major advances by the cryobiology research company 21st Centrury Medicine have been made to reduce the toxicity of vitrification agents, culminating in the least toxic vitrification agent to date, M22.

In 2004, Fahy et al. published a landmark paper that proposed a model to predict general cryoprotectant toxicity. Although the authors speculate about the mechanisms of cryoprotectant toxicity in the discussion section of the paper, the emphasis of their investigations is to formulate less toxic vitrification solutions. Whereas general cryoprotectant toxicity is proposed to reflect cryoprotectant-induced perturbation of intracellular water, the mechanisms underlying specific cryoprotectant toxicity involve the effects of individual cryoprotective agents on macromolecules (for example, metabolic conversion of glycerol to a toxic compound).

A number of viability measures are available to investigate the toxicity of cryoprotective agents. One such measure is the potassium/sodium ratio. In complex organs such as the brain, other viability measures  are possible such as measuring electrical activity after vitrification and rewarming. These viability measures can be used to improve vitrification agents but they do not throw much light on the actual mechanisms of cryoprotectant toxicity. More “sophisticated” viability assays such as measurements of post-vitrification gene expression are available to help elucidating those mechanisms. Another technique that may hold promise for investigating cryoprotectant toxicity is cryoenzymology.

Cryoenzymology is the study of of enzymes at subzero temperatures in fluid solvents. The study of enzymes at low subzero temperatures overcomes two problems in studying enzyme reactions in steady state conditions: 1) the rapidity of the reactions and 2) the low concentrations of intermediates present. By starting enzyme-catalyzed transactions at low subzero temperatures the progressive transformation of intermediates into a subsequent one can be studied as the temperature is gradually increased.  This method can produce detailed structural and kinetic information of substrate-enzyme reactions which are not available at room temperature.

Because cryoenzymology requires a fluid aqueous environment at low subzero temperatures, organic cosolvents are used to prevent ice formation. Because the organic solvents used in cryoenzymology serve a similar function as cryoprotectants in vitrification, it is not surprising that we often find the use of the same solvents such as DMSO and ethylene glycol. An ideal solvent for cryoenzymology should inhibit ice formation without adverse effects on the structure or kinetics of the molecules that need to be studied. Researchers in cryoenzymology have also found that the presence of high concentration of organic solvents decreases the temperature at which proteins denaturate. Similarly, in cryobiology, there is a need  to expose biological tissues to low subzero temperatures without causing cryoprotectant-induced protein denaturation.

Although an ideal organic solvent for cryoenzymology is not necessarily an ideal cryoprotectant, observations of the interaction of organic solvents and proteins at subzero temperatures can throw light on  phenomena such as solvent-induced versus temperature-induced protein denaturation, chilling injury, cold shock, and solvent-water-protein interactions. The field of cryoenzymology also had to address a lot of challenges encountered in cryobiological research such as selection of proper buffers for use with organic solvents at cryogenic temperatures and the effect of solvent on solution viscosity.

Cryoenzymology is also of interest to other areas in biology such as the study of life under extreme conditions. The study of extremophiles is flourishing because of its relevance to astrobiology, the study of life (or the potential for it) in the universe.

Review papers on cryoenzymology:

Fink AL: Cryoenzymology: the use of sub-zero temperatures and fluid solutions in the study of enzyme mechanisms (1976)

Fink AL, Geeves MA: Cryoenzymology: the study of enzyme catalysis at subzero temperatures (1979)

Douzou P: Cryoenzymology (1983)

Travers F, Barman T: Cryoenzymology: how to practice kinetic and structural studies (1995)

Vitrification agents in cryonics

Today’s post on 21st Century Medicine’s vitrification agent M22 completes the series on vitrification agents in cryonics. To date, three different vitrification agents have been used for cryopreservation of humans: B2C (at Alcor from 2001-2005), VM-1 (at the Cryonics Institute since 2005) and M22  (at Alcor since 2005).

Perhaps the most encouraging development in cryonics is that Alcor’s current vitrification agent, M22, is not only the least toxic cryoprotectant in the history of cryonics, it is also the state of the art in mainstream cryobiology research for vitrification of complex organs.

It is doubtful if the state of the art in vitrification in cryobiological research would be where it is today without the incentives provided by cryonics to search for a cryoprotectant that enables reversible vitrification of the brain without ice formation and minimal toxicity.

The first vitrification agent in cryonics: B2C

Vitrification agents in cryonics: VM-1

Vitrification agents in cryonics: M22

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.

The first vitrification agent in cryonics: B2C

In 2001 the Alcor Life Extension Foundation licensed its first vitrification agent from the cryobiology research company 21st Century Medicine (21CM) to be used for its neuropatients. The composition of this agent, called B2C, has now been made public on Alcor’s website. The published composition is:

Dimethyl sulfoxide 24.765% w/v
Formamide 17.836%
Ethylene glycol 17.401%
Polyvinyl pyrrolidone K12 2%
Polyvinyl pyrrolidone K30 2%
X-1000 ice blocker 1%
Z-1000 ice blocker 1%

B2C was formulated as a “hyperstable” variant of 21CM’s VM3 to virtually eliminate the risk of ice formation. B2C differs from VM3 in two important ways. It has increased concentrations of DMSO, formamide, and ethylene glycol and contains two different molecular weights of the non-penetrating polymer polyvinyl pyrrolidone (PVP). Where VM3 includes 7% w/v of PVP K12, B2C includes 2% PVP K12 and 2% PVP K30.

Like VM3, B2C reflects the technical advances in 21CM-based vitrification solutions: neutralization of the toxicity of formamide by DMSO, reduced toxicity by substitution of ethylene glycol for propylene glycol, and addition of non-penetrating polymers and “ice-blockers.” Because the primary objective of B2C was to protect the brain against ice formation, not maintain viability, the concentrations of DMSO, formamide and ethylene glycol are higher than in the older 21CM solutions in addition to the advantages of the non-penetrating polymers and “ice-blockers” of the newer solutions.

In 2005, the state of the art at Alcor and 21CM converged when the vitrification agent M22 was introduced for cryopreservation of both neuro and whole body patients. As can be seen in the electron micrographs on the Alcor website, perfusion and vitrification with M22 produces better ultrastructural results than B2C. Perhaps the only advantage of B2C over M22 is that such a highly concentrated vitrification agent (9.93 Molar for the penetrating cryoprotectants) is more suitable for remote vitrification and transport on dry ice.

Because B2C was not available to Alcor’s whole body patients, from 2001 until 2005 Alcor offered a temporary combination option in which a patient’s brain would be perfused with B2C and the rest of the body with higher molarity glycerol. Currently all Alcor patients who have made whole body arrangements are perfused with M22. Although whole body patients could still benefit from separate treatment of the body and the brain, Alcor currently does not offer the option of whole body cryopreservation with preferential treatment of the brain.

Why is cryonics so unpopular?

In his 1998 essay “The Failure of the Cryonics Movement” (part 1, part 2), Saul Kent stresses that cryonics has remained so unpopular because nobody thinks it will work. One observable implication of this view is that we would expect to see broader acceptance of cryonics as its technical feasibility increases. Unfortunately, there is not much evidence that this is the case. During its existence a number of research and technical breakthroughs have been achieved in areas such as normothermic and hypothermic resuscitation, cryopreservation, and long term care, that should strengthen the case that cryonics will work. In particular, the change from conventional cryopreservation to vitrification should have appealed to critics who questioned whether the neurological basis of identity can survive freezing. But the transition to vitrification did not have any noticeable effects on membership growth at Alcor, or later at the Cryonics Institute. In 2007, researchers at 21st Century Medicine announced that they were able to observe long-term potentiation (LTP) in vitrified brain slices, further supporting the claim that current cryonics procedures should be able to preserve the physical basis of memory.

The view that acceptance of cryonics is being held back by the perception that it is not technically feasible is hard to reconcile with the observation that increased technical progress in cryonics does not translate into rapid membership growth. It is also hard to reconcile with the fact that millions of people hold on to views that cannot be falsified with any scientific method whatsoever. Perhaps there is a scientific tipping point beyond which people will sign up in droves for cryonics. For example, some cryonics activists argue that demonstration of reversible vitrification of a small animal will have such an effect. This may or may not be the case, but it still leaves the puzzle unresolved as to why cryonics organizations were not swamped with membership requests after publishing electron micrographs that demonstrated excellent ultrastructural preservation of brain tissue after vitrification.

There are many myths about cryonics, but in light of the fact that the costs of researching these issues pales in comparison with the expected rewards of finding a treatment to a terminal illness (some cryonics advocates even propose that cryonics will enable humans to become immortal), it is hard to understand why these myths persist and the total number of cryonics members and patients is currently less than 2000.

Although it can be argued that existing cryonics organizations do not do a very good job of explaining the technical feasibility of cryonics, this seems to be unlikely. If making cryonics arrangements is so appealing there would be no shortage of other people repacking the message and relaying it to others. It is also well known that there are a considerable number of people who find the technical feasibility of cryonics persuasive, have the financial resources, and even support it as a form of medical care, but have not made cryonics arrangements for themselves or their families. It is clear that something else is holding such people back from making cryonics arrangements.

Another explanation that has been offered is that people do not want to reflect on their own mortality. There seems to be some truth to this as far as casual observations of raising the issue of death is concerned. When people are young they generally do not think about death in personal terms in such a way as to induce them to make cryonics arrangements, and when they are old they may no longer be in a state to do so, or lack the financial resources. But we know that people do routinely reflect on their own mortality and make arrangements for their family in the form of life insurance and executing a living will.

One solution to the “death” issue is to present cryonics as a form of long term critical care medicine. Instead of presenting cryonics as the science of freezing “dead” people in the hope of future revival, cryonics can be presented as a branch of medicine that employs metabolic arrest to allow critically ill patients to reach a time when effective treatment is available to treat their disease. Presenting cryonics as a form of critical care medicine does not only stress the fact that human cryopreservation is a logical extension of conventional medicine, it should also minimize religious objections concerning “raising the dead,” “immortality,” and “playing God.” Just like mainstream religion has embraced modern medicine, so it can embrace cryonics as a novel but logical extension of it.

We know that terminally ill people are often willing to go to great lengths, and accept considerable uncertainty of outcome (even risks), to find a cure for their disease or to extend their life. In this sense, the lack of complete certainty of resuscitation of cryonics patients should not present a formidable obstacle to the acceptance of human cryopreservation. Perhaps the more fundamental difference between conventional medicine and cryonics is the duration of care. Although mainstream medicine already utilizes the benefits of cold temperatures to safely induce circulatory arrest in patients who need to undergo complex heart or brain surgery, these periods of unconscious depressed metabolism routinely take minutes, not hundreds of years. In this sense, contemporary cryonics is intrinsically linked to a far and unknown future.

Perhaps the biggest obstacle for people in making cryonics arrangements is that they realize that cryonics implies the potential loss of everything that gives meaning to their existing lives. They may be resuscitated in an unknown world without their family, friends, home, personal belongings, and savings. Sterling Blake mentions the writer Ray Bradbury who expressed interest in any chance to see the future. But “thinking about cryonics made him realize that he would be torn away from everything he loved. What would the future be worth without his wife, his children, his friends?” (Sterling Blake, “A Roll of the Ice: Cryonics as a Gamble” in Immortal Engines: Life Extension and Immortality in Science Fiction and Fantasy). And we know what happened with Arthur C. Clark, who strongly believed in the technical feasibility of cryonics.

Of course, this is more likely in a world where cryonics is not very popular, but it reveals a serious problem within the fabric of cryonics marketing. An important condition for most people to accept cryonics is that they will be restored to good health with everything they know and care for. But such a scenario is most likely to occur if a substantial number of people already have made cryonics arrangements and created an infrastructure to minimize loss and alienation.

There is no magic bullet to “selling” cryonics, but presenting cryonics as a form of medicine, encouraging community building, facilitating legal instruments to retain financial assets during long term care, and assisting families in making cryonics decisions may lessen the psychological barrier to choose cryonics. One sense in which the technical feasibility of cryonics and its acceptance are related is that advances in the science of cryopreservation and development of advanced cell repair technologies will reduce the time between start of long term care and resuscitation. If the duration of care presents a formidable obstacle to signing up for cryonics, supporting progress in the science of cryonics may lead to broader acceptance of the idea after all.

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.