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.

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)

Robert Prehoda in Cryonics Reports

Now online is an old interview with Robert W. Prehoda. Prehoda was a prolific science writer who published on topics such as aging, life extension, and technological forecasting. In 1969 Prehoda published the book “Suspended Animation: The Research Possibility That May Allow Man to Conquer the Limiting Chains of Time.” In this visionary book, Prehoda covered a variety of means to extend the maximum human life span including, but not limited to, chemical  anabiosis, human hibernation, suspended animation, and controlling the aging process.

Although Prehoda was involved in the James Bedford cryopreservation, he did  not advocate offering cryonics services before reversible cryopreservation could be demonstrated in a mammal. In this he does not differ from many other (cryobiological) researchers. A major problem with this perspective is that future technologies may be able to reverse the damage caused by today’s preservation methods. It offers no hope for people who are terminally ill today. And as recent history has demonstrated, engaging in cryonics now can also create a stronger infrastructure to support legitimate cryobiology research. The least toxic vitrification agent to date, M22, would not have existed today without an existing cryonics infrastructure.

Despite attempts from Mike Perry and Mike Darwin to locate Robert Prehoda, it is not known if he is still alive.

Interview with Robert W. Prehoda (1969)

Philip Ball on water in biology

Philip Ball, author of “Life’s Matrix: A Biography of Water”, and publisher of the excellent blog, Water in Biology,  reports on recent papers about the interaction of water and bio-molecules, including a recent study on trehalose:

H. Nagase of Hoshi University in Tokyo and his coworkers have continued their exploration of the molecular mechanisms of anhydrobiosis and how trehalose acts as a bioprotectant in this regard (H. Nagase et al., J. Phys. Chem. B. 112, 9105-9111; 2008 – paper here). They have studied the crystal structure of trehalose anhydrate, and find that it contains a one-dimensional channel threading between the trehalose molecules which may be filled with water in the dihydrate form of solid trehalose.

Such investigations, and research in related fields like cryoenzymology, are of great importance to elucidating the molecular mechanisms of cryoprotectant toxicity. Cryoprotectant toxicity is the foremost obstacle to reversible vitrification of the mammalian brain without loss of long term viability.

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

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.

Viability in brain cryopreservation

Because the current generation of vitrification agents permit cryopreservation of the brain without ice formation, the current objective of cryonics research is maintenance of viability of the brain during cryopreservation. The most popular viability assay that has been used in cryonics and cryonics-associated cryobiology research is the potassium/sodium ratio (K+/Na+ ratio). Because the ability of a cell to regulate its ionic composition reflects and affects many other biochemical processes, the K+/Na+ ratio is a good measurement of viability in general. For example, all the brain slice experiments to validate the Cryonics Institute’s vitrification agent VM-1, were assessed using the K+/Na+ ratio as a measure of viability.

In the case of the brain, demonstrating such “basic” viability after vitrification is a necessary, but perhaps not sufficient, condition for reversible vitrification of the brain without adverse (long term) effects. Recovery of function in the brain is a more subtle concept than in other organs. In 2007, 21st Century Medicine reported maintenance of long-term potentiation (LTP) in vitrified brain slices. Chana de Wolf proposed more specific experiments to demonstrate maintenance of memory after cryopreservation. And more specific molecular assays could assist in illuminating the effects of cryoprotective perfusion, cryoprotectant toxicity, and cryogenic cooling on the brain. Such viability and functional assays can be correlated and combined with structural assays to assist in developing cryoprotective solutions, and perfusion and cooling protocols that will permit successful resuscitation of whole brains after vitrification.

Further reading: Securing Viability of the Brain in Cryonics

Vitrification agents in cryonics: VM-1

A major public misperception is that cryonics involves the freezing of dead people. The objective of cryonics is not to preserve dead people with the hope of future revival but to place critically ill patients in a state of biostasis until a time when more advanced medical technologies might be available to treat and cure them. Currently, all major cryonics organizations induce metabolic arrest of the brain by attempting vitrification rather than freezing.

Unless a patient has suffered a long period of circulatory arrest, after which perfusion of the body or brain is no longer possible, metabolic arrest is induced by cooling down the patient to cryogenic temperatures. Vitrification can be defined as “the process of converting a material into a glass-like amorphous solid that is free from any crystalline structure.” Because vitrification of pure water would require extremely rapid cooling rates, vitrification in cryonics is achieved by substituting the water of patients with a highly concentrated cryoprotectant agent before cooling.

In 2001, Alcor introduced its first vitrification agent (B2C) for neuropatients and extended this technology to whole body patients in 2005 with the introduction of M22. The Cryonics Institute introduced its own vitrification agent, VM-1, to its membership in 2005. VM-1 was developed by Dr. Yuri Pichugin and stands for Vitrification Mixture-1, which indicates that it was the first vitrification agent to be introduced at CI. Before VM-1, CI generally used the cryoprotective agent glycerol. VM-1 consists of 35% ethylene glycol and 35% dimethyl sulfoxide (w/w). It is introduced in a carrier solution called m-RPS-2, consisting of potassium chloride, glucose, and TRIS (alternatively called THAM). A more detailed review of the research and components of the solution can be found on the Cryonics Institute website.

VM-1 has been formulated and validated specifically for cryonics patients. Although encouraging viability results have been obtained in brain slices, the agent first and foremost reflects the search for a vitrification agent that is an affordable, but also strong and stable, glass former. M22 is the culmination of many years of research (mostly on kidney slices) by Greg Fahy et al. to find vitrification agents that can successfully recover organs from cryogenic temperatures for organ transplantation. M22 is being licensed to Alcor by the cryobiology company 21st Century Medicine.

An interesting similarity between the two agents is that both contain the same core components: ethylene glycol and DMSO. 21st Century Medicine solutions additionally contain formamide, which has a low toxicity in the presence of DMSO, allowing formulation of solutions of lower overall toxicity. Solutions containing DMSO, an amide, and ethylene glycol are protected by 21st Century Medicine’s M22 patent.

The strong glass forming ability and stability of VM-1 is further evidenced by the following research findings. Pichugin did not observe ice formation or devitrification when 20 ml glass vials of 60% and 65% VM-1 were cooled and warmed with cooling and warming rates as low as 0.1 degrees Celsius per minute. 65% VM-1 solutions with “homogenized rat brain tissues containing natural nucleators” did not show visible ice crystals after 14 days at dry ice temperature (-78.5 degrees Celsius). The stability of large volumes (2 liters — unfiltered) of VM-1 was investigated and no ice crystals were observed after 21 days of storage at dry ice temperature. These results raise the intriguing question of whether patients can be perfused with high concentration VM-1 in the field and shipped to a cryonics facility on dry ice. In cryonics we do not ship vials of cryoprotectant solution, or fully equilibrated brain slices, but patients that have been exposed to variable degrees of warm and cold ischemia. Questions about the nature and extent of ice damage of poorly perfused areas during long holding and transport periods at dry ice temperature still remain.

Unlike M22, VM-1 is not a suitable agent for perfusion of whole body patients. In CI patients where whole body perfusion was attempted with one of the components, ethylene glycol, serious edema resulted. Similar results have been encountered in the past with DMSO as a mono-agent. Improved results may be obtained by modifying the carrier solution of VM-1 to include an oncotic agent and/or using glycerol for the rest of the body. Although high molar glycerol can be perfused in (non-ischemic) patients without serious edema, CI currently discourages members from choosing whole body perfusion in order to ensure optimal perfusion of the brain.

The current carrier solution of VM-1, m-RPS-2, is basically a stripped down and modified version of Fahy’s Renal Preservation Solution-2. As such, it does not contain components that reduce free radical damage (such as glutathione) or ATP precursors (such as adenine) to assist energy generation during hypothermia. Perhaps a more controversial choice is the lack of an oncotic agent to prevent and counter edema during perfusion. Pichugin questions the value of such agents for perfusion of the brain. In practice, CI has not encountered much edema during brain perfusion of its patients, many of which have been exposed to considerable periods of warm and cold ischemia. m-RPS-2’s lack of hypertonicity does not seem to make the carrier solution suitable to inhibit chilling injury.

There are still some open questions about VM-1. Although VM-1 is designed as a low cost agent to allow preservation of the brain without ice formation, no published electron micrographs are available that show the quality of ultrastructural preservation that can be obtained with VM-1. VM-1 has been validated solely on the basis K+/Na viability assays. As electron micrographs of brains perfused with vitrification agents B2C and M22 indicate, agents that can inhibit ice formation can still produce strikingly visible differences in terms of ultrastructural alterations. Although good hippocampal slice viability results imply good ultrastructural preservation, actual empirical evidence of this could make a stronger case.

During the final step of cryoprotective perfusion at CI, the current protocol is to introduce 70% VM-1 at -7 degrees Celsius to reduce the time required to achieve a minimum target concentration of 60% as measured by refractometry. It is not clear what the biochemical effects of exposing the patient to such concentrations of VM-1 are, although the “ideal” temperature for the final step is lower than what is currently used by Alcor for M22. In practice, it is doubtful that the patient’s brain is at such temperatures during a typical perfusion. Such a protocol would require more rigorous control of the perfusate and brain temperature using a subzero chiller.

Another advantage would be to introduce VM-1 in a more “linear” fashion using a “closed circuit” in which the concentration is gradually increased. Because such a protocol requires a more expensive, complicated, and challenging perfusion circuit, the costs and risks of such a protocol need to be weighed against the potential advantages. One straightforward compromise might be to do “open circuit” perfusion but to close the circuit after target concentration has been reached to allow for good equilibration of the cells before terminating perfusion.

With VM-1, CI seems to have introduced an extremely cost effective and stable vitrification solution. If CI will find the resources to do new experiments to improve its composition and protocol, obtaining actual images of brain slices perfused (and vitrified) with the solution seems to be an important priority. It also needs to be stressed that results obtained in brain slice experiments in different species are not necessarily a good indicator of what can be expected in actual human cryonics patients who generally have been exposed to long terminal periods, warm and cold ischemia, and longer perfusion times at higher temperatures. It is clear that is there is an urgent need for a research program that investigates the relationship between such variables and outcome in terms of ice formation, viability and ultrastructure. Investigations that have been done by Darwin and Pichugin under more realistic conditions will be discussed in the future.

Ben Best publishes on cryonics in Rejuvenation Research

A technical cryonics article to be published in the conference proceedings of a customarily peer-reviewed scientific journal, entitled “Scientific Justification of Cryonics Practice (pdf),” by Ben Best, President of the Cryonics Institute, will appear in the next issue (Volume 11, Issue 2) of Rejuvenation Research. (A previous article by Ralph Merkle, “The Technical Feasibility of Cryonics,” was published in the journal Medical Hypotheses in 1992, an editorial board-reviewed journal.)

As can be surmised, Mr. Best’s paper expounds upon the scientific basis for engaging in and supporting the practice of cryonics. He begins by providing the reader with an overview of cryonics as it is practiced today, including the mathematical basis for rapid reduction of body temperature in order to reduce chemical reaction rates to slow down the cascade of harmful events culminating in neuronal damage due to ischemia after cardiac arrest. He reports that six minutes of warm ischemia at 37 degrees C would take 100 sextillion (10^23) years to occur at liquid nitrogen temperature (-196 degrees Celsius).

The article continues with a discussion of vitrification and cryogenic storage, including a discussion of the history of cryoprotectants and vitrification solutions in cryonics. A concise treatment of resuscitation experiments lending credence to the possibility of “reversible death” is then provided, alongside a short discourse on investigations into limiting apoptosis (“programmed cell death”) and ischemic damage / reperfusion injury. He further stresses the fact that “conservative cryonics strives to minimize damage and minimize reliance on future molecular repair technologies.”

The article concludes with a section describing contemporary cryonics procedures, followed by a persuasive argument regarding the nature of science and the validity of using indirect evidence as a basis for the practice of cryonics. The author provides numerous examples of scientific endeavors that have benefited from “model-building based on extrapolations from indirect evidence,” as well as modern instances of cryopreservation of human stem cells and animal DNA in anticipation of future technology.

Mr. Best states, “It is not unscientific to risk modest or heroic medical treatments that are justified by indirect evidence for some probability of success, rather than absolute guarantee of success.” This is the most persuasive, and yet least appreciated, argument in favor of cryonics. We can only hope that continued publication of scientific and technical cryonics papers in peer-reviewed literature will engender wider acceptance of such humanitarian efforts.