Viability as a Standard for Cryonics Research
by Ben Best
Research devoted to cryopreservation of organs is rare, but research devoted entirely to the objectives of cryonics is even more rare. Although there is much to be learned from cryobiologists, especially cryobiologists dedicated to organ preservation, the different objectives of cryonics may require different methods.
The objectives of organ cryopreservationists is to maximize viability, ie, to maximize living functionality of organs rewarmed from cryopreservation. By contrast, the objective of cryonics research is to preserve organs -- especially the brain -- in such a way as to optimize restoration by future science. (Admittedly, this involves second-guessing the capabilities of future science.)
If, for example, cryopreservation method A resulted in 60% viability & 80% structural preservation, whereas method B resulted in 50% viability & 90% structural preservation --the organ cryopreservationist would prefer method A, whereas the cryonicist might prefer method B. Normally, good structure leads to good function, but it is possible that (for example) methods to minimize damage upon rewarming could entail methods that are suboptimal for structural preservation on cooling.
Vitrification requires the use of cryoprotective agents ("antifreeze compounds") to prevent ice formation and/or freezing damage by increased viscosity, by hydrogen-bonding with water molecules, by dilution of electrolytes and/or by colligative interference. Ice formation is a two-step process involving first the formation of a nucleus (nucleation) and second the growth of an ice crystal from the nucleus.
Most people are unaware that the temperature at which pure water will freeze is much lower than the temperature at which pure water will melt. The reason tapwater freezes at just below 0ºC -- close to the temperature at which it melts (Tm = 0ºC) -- is because of impurities that serve as nucleating agents. Water that is perfectly pure is unlikely to freeze at -5oC because an ice crystal cannot grow at that temperature without achieving a critical crystal nucleus mass of 45,000 water molecules. With fewer than 45,000 water molecules the nucleus is dissolved because heat of fusion warms the nucleus too much and the dissolving effect of the surrounding medium is too great. Pure water freezes at about -40ºC when a nucleus need only contain 70 water molecules to be large enough to grow.
Both the melting temperature of water (Tm) and the freezing temperature of water which contains no nucleating agents (Th, -40ºC for homogenous -- ie, pure -- water) are lowered by the addition of cryoprotective agents. If enough cryoprotective agent is added the freezing temperature Th becomes so low that it reaches Tg, the temperature at which the water-cryoprotectant mixture forms a glass (vitrifies) rather than freezes into ice crystals. [CRYOBIOLOGY 21:407-426 (1984)]. Tg is typically about -130ºC for cryoprotectants used in cryonics.
The Devitrification Problem arises from the fact that the temperature range of maximum nucleation (maximum nucleus formation) is much lower than the temperature range of maximum ice crystal growth rate. Maximum nucleation temperature is in the -80ºC to -120ºC range, whereas maximum crystal growth rate temperature is in the -40ºC to -80ºC range. When cooling with cryoprotectant, the maximum growth rate range is not of concern because there are no nuclei to grow. The main concern on cooling is the maximum nucleation range, which increases near Tg. The ideal is to cool quickly enough to reach Tg without any nuclei forming, but if nuclei are formed the rapid cooling & low temperature will not allow the nuclei to grow very much and thus prevents them from being very harmful. Large ice crystals cause the most damage, whereas small ice crystals cause less damage -- and may cause no damage at all if they are small enough. The situation is very different, however, upon rewarming.
Not far above Tg the maximum nucleation temperature must first be passed-through. Then as temperature increases further, the range of maximum growth rate is reached. The formation & growth of ice crystals during rewarming of a vitrified solution is called devitrification. The fact that the ice crystal formation & growth is so much worse on rewarming than on cooling is called the Devitrification Problem.
One solution to the Devitrification Problem is to rewarm at extremely high rates.
Microwaves cannot be used for this purpose because microwaves produce uneven warming leading to head-denaturation of some parts of tissue, while other parts are still too cold and vulnerable to ice growth. But radio frequencies can heat more uniformly than microwave frequencies. David Pegg (editor of the journal CRYOBIOLOGY) has recently achieved warming rates of 600ºC per minute using radio frequencies without damaging tissues. A second way of preventing devitrification is the use of anti-nucleators, such as polyvinyl alcohol. A third way would be to use cryoprotectant mixtures which are so concentrated that no ice can ever form.
The Devitrification Problem arises because of the desire to maximize viability. All cryoprotectant agents are toxic, some more than others. Vitrification can be achieved by using a small amount of cryoprotectant and by cooling fast enough to avoid nucleation. If cryoprotectant toxicity was not a concern, however, and the most toxic cryoprotectants were used in high concentration, there would be no devitrification at any cooling rate.
Cryoprotectant toxicity is probably caused by denaturation of proteins. Recent discoveries provide clues as to why the substances giving the most powerful cryoprotection at low concentration are the most toxic. The cryoprotectants that vitrify most powerfully are those that hydrogen-bond most strongly to water, thereby interfering with the water-to-water hydrogen-bonding that is the basis of ice [CRYOBIOLOGY 48:22-35 & 157-178 (2004)]. But those same cryoprotectants may also hydrogen-bond most strongly to proteins, causing the most unfolding and the most protein (enzyme) denaturation. The least toxic cryoprotectants prevent ice formation by weak hydrogen-bonding, but more importantly by colligative interference with ice formation. For this reason, a major breakthrough for organ cryopreservation was achieved by substituting ethylene glycol for propylene glycol in VS55 (aka VS41A) solution (which is 3.1 Molar DMSO, 3.1 Molar formamide and 2.2 Molar propylene glycol in EuroCollins solution).
At least 90% of the water in a cell is what can be called "bulk water". Bulk water is the water that can freeze and/or move out of the cell in response to changes in osmotic pressure (a hypertonic solution outside the cell, which could be due to the formation of extracellular ice). By contrast, about 10% of cell water is "bound water" that is tightly hydrogen-bonded to the hydrophilic surfaces of macromolecules (proteins, nucleic acids or the polar end-groups of phospholipids) -- or water molecules that are tightly hydrogen-bonded to the molecules directly bound to the macromolecules. Bound water does not osmotically leave the cell due to extracellular freezing or other causes of extracellular hypertonicity. Bound water can only be removed by heading in a vacuum. Removal of bulk water does not cause dehydration damage, but removal of bound water does cause such damage -- because macromolecules chemically interact when they do not have the protective covering provided by bound water.
An alternate explanation to the protein denaturation hypothesis I have presented as an explanation for the greater toxicity of the cryoprotectants that hydrogen-bond most strongly is the dehydration damage hypothesis -- that the toxic cryoprotectants are causing dehydration damage by binding to water molecules. There is much evidence against this explanation. Dehydration damage is only caused by the removal of bound water. Bound water is 20 to 100 times more viscous than bulk water. Powerful cryoprotectants toxic at low doses would find plenty of bulk water to hydrogen-bond.
Access to bound water would be improbable due to the viscosity, and any bound-water molecule hydrogen-bonded by a cryoprotectant molecule would be instantly replaced by a bulk water molecule. Trehalose is a non-toxic cryoprotectant which displaces bound water and protects cell membranes by hydrogen-bonding to proteins and the polar ends of phospholipids more strongly than bound water [ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 245(1):134-143 (1986)]. Toxic cryoprotectants could prevent dehydration damageby the same means, while nonetheless causing protein denaturation which the more weakly hydrogen-bonding trehalose does not. Finally, evidence is presented on pages 582 & 583 of LIFE IN THE FROZEN STATE by Barry Fuller, et. al (2004) that good cryoprotectants are those that are preferentially excluded from proteins – thereby stabilizing them and opposing their unfolding or denaturation. Such cryoprotectants would be hydrophobic enough to cross cell membranes readily -- cryoprotectants cross cell membranes far more readily than would be predicted by their molecular weight [Figure 5, PHYSIOLOGICAL CHEMISTRY AND PHYSICS AND MEDICAL NMR 25:177-208 (1993)] -- and would be hydrophilic enough to readily mix with water. Thus, the less toxic cryoprotectants act more by colligative interference with ice formation than by hydrogen-bonding with water.
How important is cryoprotectant toxicity for cryonics? Can future technology easily replace denatured proteins? Enzymes should be easy to replace, and denatured membrane proteins may not cause so much structural damage as to prevent faithful reconstruction. If denatured proteins are replaceable, then viability should not be the most important standard for cryonics research. We may be able to achieve the best structural preservation with the least ice formation by the use of highly toxic, but powerful cryoprotectants like butane-2,3-diol (aka, 2,3-butylene glycol). Optimizing the preservation of brain structure, rather than possible commercial applications should be our focus (although our procedures could be of interest to neurohistologists).
Cooling reduces viability even before freezing temperatures are reached, in a form of damage known as "chilling injury". Substantial evidence exists that chilling injury in animal tissues and cells is due to phase transitions in cell membranes [CRYOBIOLOGY 42:88-102 (2001)]. Just as freezing temperatures are not required for grease in a frying pan to form a gel, lipids in cell membranes undergo a liquid-to-gel phase transition in a range between 0ºC and 20ºC. Higher phase transition temperatures are seen for membranes containing higher percentages of unsaturated fat. Maximum leakage of membranes is seen at the phase transition temperature due to packing defects in the phase boundaries between the liquid and gel portions of the membrane. Far less leakage is seen below (and above) the "thermotropic phase transition" temperature. Surfactants (detergents) can worsen this damage.
Much of the damage due to chilling injury is due to aggregation of membrane components -- such as proteins and "rafts" of unsaturated fat -- at, and just below, the thermotropic phase transition temperature. For this reason the main way of avoiding chilling injury in cryopreservation has been to cool rapidly so as to minimize time spent in the phase transition region. Chilling injury has been reduced in fish & pig embryos by removing lipids (yolk) [CRYOBIOLOGY 39:236-242 (1999)]. Antifreeze glycoprotein from polar fish reduces chilling injury in platelets by inhibiting aggregations in the membrane, but the technique cannot be used clinically because of the difficulty of removing the antifreeze proteins [CRYOBIOLOGY 43:114-123 (2001)]-- a consideration of less concern to cryonicists who are more interested in preservation of structure than in viability on rewarming. Along the same lines, membrane leakiness has been exploited by using DMSO to facilitate entry of trehalose into cells for protection of intracellular membrane surfaces. Toxicity of DMSO upon rewarming should not be of so much concern in cryonics applications. In fact, membrane damage by chilling injury may affect viability much more than general structure and therefore be of less concern in cryonics.
A lesser cause of chilling injury is protein denaturation. Just as proteins are denatured by heat, proteins can also be denatured by cooling. The exposure of hydrophobic regions of proteins becomes less energetically unfavorable at lower temperatures and thus proteins will unfold as temperatures are lowered. Again, rapid cooling may be the best means of reducing protein denaturation (although heat shock proteins could help reduce unfolding if a means was found to increase their expression). Again, if the injury is primarily by denaturing enzymes, this effect on viability may be of much less concern to cryonicists insofar as structural damage would be minimal.
Although chilling sensitivity has been reduced in plants by increasing the degree of fatty acid unsaturation [NATURE 356:710-713 (1992)], most of the chilling injury in plants has been attributed to free radical damage [JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 47:2410-2414 (1999)]. Evidence for free radical damage during chilling has also been seen in houseflies, however [CRYOBIOLOGY 447-458 (1996)]. Uncoupling of mitochondrial enzymes and ATP synthesis by chilling has been demonstrated, and coincident with the increased oxidative stress is a reduction of antioxidant enzyme activity due to chilling. Increased calcium influx can activate phospholipases that break-down membranes by releasing fatty acids. Exclusion of air (oxygen) has been shown to reduce chilling injury in plants and insects. Free radicals can destroy structure if they are permitted to operate for extended periods. It seems likely that chilling injury due to free radicals must also occur to some extent in mammalian tissues due to cold ischemia as well as similar mechanisms to those seen in plants.
At the Cryonics Institute Dr. Pichugin has been using intracellular K/Na (potassium-sodium) ratios to assess the effectiveness of our vitrification formula.
The intracellular ratios of potassium to sodium are generally taken to be a consequence of the ability of cells to pump sodium out-of and potassium into the cell. If the intracellular K/Na is high, that is taken as an indication that the sodium pump is operational, ATP is being generated to run the pump, the cell membranes are intact and the tissue cells are alive (viable).
The biggest advantage of K/Na assay is that it is inexpensive and easier to measure than structural damage seen in micrographs. The assays are quantitative and can be obtained quickly. It is certainly less costly than taking electron micrographs (EMs), although the latter is the ultimate standard for the absence of structural damage due to ice. Both K/Na ratios and EMs -- viability and structural integrity measures -- require rewarming (and possible devitrification). If it were economically feasible for us to assess histological structure at -130ººC -- cryohistology not requiring rewarming -- that would give us the best assessment of how well we are preserving structure for long-term storage.
In my opinion CI research should minimize the use of K/Na ratios to assess vitrification formulations until (1) structural damage has been eliminated completely and (2) K/Na ratios are high enough to be close to that of living tissue. It is also my opinion that we should first concentrate on eliminating structural damage through LMs (and eventually EMs) and that we should use the most powerful (and most toxic) cryoprotectants necessary to eliminate structural damage. We can then try to work our way toward less toxic formulations so long as structure is not compromised.
Although I am the President of CI, I am not the dictator. The standards for research are determined by Dr. Pichugin, Robert Ettinger and to a lesser extent by Directors, Advisors and Members -- in addition to me. I have purchased a camera for CI research, light microscope with digital with general approval of Mr. Ettinger and Dr. Pichugin.
***
The following is Dr. Pichugin's response to Ben Best's article.
I would like to give my opinion on the topic of Ben's article.
The K/Na assay (as well as electron microscopy (EM)) is a very sensitive indicator for the presence of damage due to ice and devitrification, but the K/Na assay is much cheaper and much simpler than EM. According to K/Na assay, if the rat brain slices were subjected to damage from ice or devitrification, slice survival was very low or zero.
The main causes why the K/Na ratio assay cannot be employed in some of cryonics research experiments are that:
For now we cannot use -25EC but only +4EC at funeral homes to perfuse CI patients. The time period for complete saturation of patient brains with VM to do vitrification will usually be longer than the optimal one. This will also decrease tissue survival. The use of the K/Na assay requires washout procedures of brain tissues from cryo-protective agents that result in additional damage.
As a result of all these factors and some other ones, the rat brain tissues were similar to the most actual cryonics brain tissues after the procedures (even after vitrification). They had very low survival according to the K/Na assay. The K/Na assay seems to be useless for the most real patient cases and so we need to use less sensitive, morphological tests such as light and electron microscopy. However, these morphological tests are much more expensive, more complicated, less available, less clear than the K/Na assay. If I began to work out a new CI cryonics suspension method using models of the most actual patient cases but not the rat live brain tissues and using histology plus electron microscopy only instead of the K/Na assay, we could spend much more money and time than we previously needed to get the results we have had now.
I used rat live brain tissues and the proper tissue evaluation method (the K/Na ratio assay) to compare the new various cryopreservation procedures with the glycerol CI method. I demonstrated that the glycerol method resulted in 0% brain tissue survival and an improvement of this method can give 15-20% survival in the best conditions. But these are not available for CI patients because they require very fast cooling rates. The best vitrification method that do not require fast cooling rates can give average 80% brain tissue survival.
This is a proof of the superiority of the new method over the old one. It is another matter to evaluate a state of the actual patient brain tissues after the actual application of the new CI method. In this case, the K/Na assay cannot work or can work badly in comparison with morphological methods because the tissues can have very low viability.
We already have a better cryopreservation method than the CI glycerol method. However, we need to adapt the new CI method to obtain better results for patients cryopreserved in funeral homes.
Yuri Pichugin Ph.D