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.