Brian Wowk began the presentations by describing a search at 21CM for new CryoProtectant Agents (CPAs), ie, chemicals that cause water to vitrify (cool to a glassy solid) rather than form ice crystals with lowering sub-zero temperatures. The search strategy is based on the idea of adding methoxyl groups to conventional CPAs. The conventional CPAs Brian named were glycerol (widely used for freezing blood & sperm -- and the CPA most commonly used for cryonics purposes), ethylene glycol (the most commonly used additive in automobile antifreeze) and propylene glycol (used to minimize ice crystals in ice cream). The chemical structure of these three compounds are:

21CM researchers replaced hydroxyl (-OH) groups with methoxyl (-OCH₃) groups to produce compounds that were less viscous (reduced self-interaction and increased interaction with water) and had more permeance (more penetrating through cell membranes due to greater oil miscibility). Substituting a methoxyl for a hydroxyl creates an ether (molecule with an oxygen between two carbons). One less hydroxyl group reduces hydrogen-bonding between cryoprotectants, but the ether oxygen can still hydrogen-bond with water. Brian summarized the results in the following table, which compares the original cryoprotectants with the methoxyl derivatives:

The "--" entries in the table represent "data not available", although Brian commented that 1-methoxy-2-propanol is much more permeant than propylene glycol. Also, precise data is not available for the 1,3-dimethoxy-2-propanol derivative of glycerol, but it is seen to be less viscous.

Brian showed a graph which compared the output of a Differential Scanning Calorimeter (DSF) for 45% glycerol and 45% 3-methoxy-1,2-propanediol (3-MPD). (A DSF measures heat flow in & out of small samples of cryoprotectant in water solution at various temperatures). Cooling at 20ºC per minute, a sharp drop in heat flow was seen at -60ºC for the glycerol due to ice formation, which was not seen with 3-MPD. Brian estimated approximately 30% ice formation in the glycerol solution as compared with less than 1% ice in the 3-MPD. The only dip in the DSF curve for 3-MPD was seen at about -110ºC, associated with the glass transition temperature (temperature of formation of a vitreous solid).

Brian said that as a rule of thumb, a methoxylated-derivative will vitrify at a concentration approximately 5% less than the corresponding conventional CPA. As another rule of thumb, he said that the glass transition temperature tends to be approximately 12ºC higher per methoxyl group than the conventional CPA. Brian suggested that higher vitrification temperatures might allow for long-term storage at higher temperatures at less cost, while reducing the cracking often seen at lower temperatures. The highest glass temperature that the 21CM group has yet found is -85ºC, but Brian still has hopes of being able to achieve vitrification above dry ice temperature (-79ºC).

Brian said that the hydrophobicity (lipophilicity) of methoxylated compounds is associated with their high toxicity. This may be due to dissolution of cell membranes. But Brian said that this toxicity can be alleviated by mixing the methoxylated compounds with other agents. Measuring viability by the ability of cells to pump ions (the K+/Na+ ratio), Brian compared a 50-50 mixture of ethylene glycol and 3-MPD with Greg Fahy's VS41A compound (the least toxic vitrification solution known for the last decade) for kidney slices. Both the 50-50 mixture and the VS41A showed about a 60% viability reduction as compared to control (ie, no CPA). By replacing the propylene glycol in VS41A with 3-MPD, they achieved a viability that was actually slightly greater than that seen for VS41A.

[A peer-reviewed paper describing the effects of methoxylation by Dr. Brian Wowk, et.al. appeared in CRYOBIOLOGY 39(3):215-222 (1999)] (The work was also patented -- Patent 5,952,168).

Dr. Greg Fahy then took the stage to discuss his work to improve upon his VS41A solution. His VS4 solution is an equimolar mixture of DMSO and formamide [14% (w/v) for each of DMSO & formamide -- known as "D(1)F" -- where DMSO neutralizes the toxicity of formamide] with about 11% (w/v) propylene glycol and about 10% (w/v) colloid added to make the total cryoprotectant concentration 49% weight/volume. VS4 is not very toxic, but it will only vitrify at 1000 atmospheres of pressure. VS41A is a mixture of the same cryoprotectants as are in VS4, but with about relative proportions -- but with about 17% (w/v) propylene glycol to make the total cryoprotectant concentration about 55% weight/volume. Unlike VS4, VS41A will vitrify at normal (atmospheric) pressure (the "1A" suffix refers to "1 Atmosphere"), but is pushing the edges of toxicity. Nonetheless, VS41A has been the least toxic vitrification solution known to man for an entire decade.

Greg said that the greater permeance of the methoxylated compounds contributes to their reduced toxicity because with increased permeance there is a reduction in exposure time required to achieve vitrification. But the methoxylated compounds were even less toxic when exposed for the same amount of time.

Greg said that the problem of designing a practical vitrification protocol involves not only choosing a vitrification solution, but a vehicle. He reviewed the vitrification solutions currently used by cryobiologists and divided them into 3 categories:

Greg concluded that these solutions were almost all witch's brew mixtures which were not based on rational design. He showed the results of an experiment he performed to assess the viability of 13 CPAs. He concluded that there are two kinds of cryoprotectant solutions: ones that contain an amide plus DMSO (like VS4 and VS41A) and ones that do not. There were no grounds for understanding why one particular solution results in more viability than another. He then attempted to correlate viability with the tendency of a CPA to denature protein, but his results made no sense. He also tried to correlate viability with CPA concentration and he saw little variation in viability over wide ranges of concentration. But when he used a new method of measuring CPA concentration (which he calls "qv*") he was able to produce straight-line graphs for amide and non-amide solutions (the amide solutions being slightly less toxic). At the time of the presentation Greg had a patent pending on qv* (he subsequently received the patent -- Patent 6,395,467).

Using qv* Greg was able to distinguish specific toxicity from non-specific toxicity and thereby able to minimize the specific toxicities. Based on the qv* concept Greg generated a vitrification solution which he called VX. The VX solution showed a viability of 85% of control, which is significantly better than the 60% viability for VS41A (which for 10 years had been the least toxic vitrification solution known). The viability of VX was comparable to that of VS4, but VS4 does not vitrify (except at 1000 atmospheres pressure), whereas VX is on the threshold of vitrifiability. Modifying VX to get a full vitrification solution, he found another compound, "VXD", which results in the same viability (K+/Na+ ratio) as VX. Further modifying VXD he found more promising mixtures. He said he now has about 15 solutions to examine, all of which appear to be superior to his previous "world champ", VS41A.

The standard means of assessing CPA vitrifiability is to measure small quantities of the CPA. But the results of small quantity assays do not always scale-up. For example, a 56% ethylene glycol solution vitrifies well in a test-tube, but for larger volumes -- such as 250 mL in an Erlenmeyer flask -- ice crystals are seen.

As has been revealed by Patent 6,395,467, q* refers to

                                    moles of water
  q*  =  ----------------------------------------------------
               moles of polar groups on penetrating cryoprotectant

and qv* refers to q* at a concentration needed to vitrify 5-10 ml of solution at a cooling rate of about 10ºC per minute (C_v). When penetrating cryoprotectants are plotted at their concentrations needed to vitrify a good correlation is seen between qv* and viability.

The qv* concept resulted in a conceptual breakthrough that has greatly improved cryoprotectant formulations, allowing neuro cryonics patients vitrified with less than 0.2% ice formation. The qv* concept has led 21CM to formulate cryoprotectant cocktails with the penetrating cryoprotectant (eg, ethylene glycol) selected being the one that has the weakest hydrogen-bonding when the cryoprotectant is at a concentration just adequate to vitrify (C_v). Using weaker cryoprotectants means that a greater volume of cryoprotectant is needed and that more water is displaced. But the weaker hydrogen-bonding allows the water that remains to still perform vital cell hydrating functions that were apparently not occurring with the stronger cryoprotectants -- leading to toxicity.

To assist in vitrification, Greg turned to additives which could inhibit ice crystal formation patterned after substances found in nature. The compounds that block ice crystal growth do so by means quite distinct from the physical process of vitrification -- and typically at higher temperatures. Ice-blocking proteins attach themselves to "nucleators" -- incipient ice nuclei or other similarly-shaped molecules that can act as nuclei for ice crystals to grow-around. Polar fish have proteins that inhibit ice crystal growth, allowing them to live below the freezing point.

Greg had tested VS4 with 5% anti-freeze protein in the hope of achieving a vitrification comparable to VS41A, but without much success. But by using another vehicle solution (which he called "vehicle 1") he reduced the ice formation in a VS4 solution by a factor of a thousand, using only 1% of fish anti-freeze protein. Increasing the cooling rate from 1ºC/minute to 3ºC/minute virtually eliminated ice formation. Using another vehicle ("vehicle 2") he was able to eliminate ice formation with VS4 plus 1% beetle anti-freeze protein with a cooling rate of only 1ºC/minute (a rate achievable in cooling human kidneys). Moreover, Brian had reported that less than 1% beetle anti-freeze protein in VS4 also prevents ice crystal formation on rewarming at 1ºC/minute (as compared with 200ºC/minute required without the protein). Dr. Fahy has seen ice crystal formation on rewarming of vitrified solutions as the major barrier to organ vitrification.

Brian then returned to the podium to describe his work to find non-protein synthetic ice blockers which could be used to perform the functions of anti-freeze proteins. He explained that synthetic ice blockers would be more robust than proteins, and less likely to be pH-sensitive or produce allergic reactions. Moreover, the beetle protein is prohibitively expensive at $1,000 per milligram.

Ice has two axes of crystal growth, a vertical "c-axis" ("z-axis", in Cartesian coordinates) and two horizontal "a-axes" ("x-axis" and "y-axis" in Cartesian coordinates). Ice crystal growth along the a-axes occurs more at higher temperatures, whereas c-axis growth occurs more at lower temperatures. Most natural anti-freeze proteins inhibit a-axis growth without inhibiting c-axis growth. Brian succeeded in developing the "21CM-X" series of ice-blockers. Unlike the natural ice blockers, the 21CM-X compounds show the greatest activity against the c-axis growth that occurs at lower temperatures (a valuable property for assisting in vitrification and devitrification). The ice blockers are of low toxicity and are a thousand times less expensive than beetle anti-freeze protein. [CRYOBIOLOGY 40:228-236 (2000)]

Although 50% DMSO in a test tube is required to prevent ice formation with a cooling rate of 7ºC/minute, adding 1% "X1" ice-blocker reduced the required DMSO concentration to 47%. Although 3% doesn't sound like much reduction, it can make a great difference in reducing toxicity.

Solutions which will vitrify without freezing often form ice crystals "like crazy" (Brian's words) on rewarming (devitrification). But ice blockers seem to be as effective in preventing ice formation on devitrification as on cooling. These effects have been seen with every CPA tested, not just DMSO. Moreover, the effects of X1 are not lost upon scaling-up to 250 mL volumes.

[A peer-reviewed paper describing the effects of ice-blockers by Dr. Brian Wowk, et.al. appeared in CRYOBIOLOGY 40:228-236 (2000)]

IMPROVED CRYOPRESERVATION OF CELLS, TISSUES & ORGANS

Dr. Fahy began his presentation by showing a photograph of a kidney he had vitrified in 1983. Although it has been possible to vitrify a kidney for 15 years, it has not been possible to vitrify kidneys without loss of viability -- or to prevent ice formation upon rewarming (devitrification).

Greg reviewed the reasons why it would be desirable to cryopreserve organs. 95% of kidneys are poorly matched to their recipients, and the matching for livers & hearts is even worse. The ability to bank organs for even a few weeks would allow time for matching of the organs with the recipients. And transplant surgeons would not be forced to work at inconvenient times and under inconvenient conditions in order to take advantage of organ availability.

Discussing the potential market, he suggested that 21CM could conceivably earn $10,000 per organ of about 20,000 vital organ transplants per year ($200 million). Adding nonvital organ transplants (limbs, esophagus, stomach, breasts, etc.) might bring-in another $300 million. Artificial organs and tissues (with no limit on supply) could bring in another billion dollars. And xenographs (transplant of animal organs to humans) could bring in more millions.

21CM is concentrating efforts initially on kidneys because kidneys have been the most thoroughly studied and are the closest to being cryopreserved. It is easy to transplant kidneys. Kidneys are currently the most frequently transplanted organ. And a failure to transplant does not result in patient-death, since the patient can be kept alive with dialysis machines.

In 1994, just before Greg and his team left the Red Cross, he showed that he could perfuse kidneys at -46ºC and have the kidneys survive 50% of the time. Since cooling damage is maximized at -46ºC, Greg guesses that further cooling down to glass transition temperature should result in no further injury. ("Cooling damage" is a mysterious injurious effect which is apparently in addition to CPA toxicity and nucleation of ice crystals.) But rewarming rates of 50ºC to 100ºC/minute are required to prevent ice formation with VS41A. Members of the new VX family of CPAs can be cooled at 5ºC per minute and rewarmed at 10ºC per minute (even without ice blocker) without loss of viability. Greg showed a 50% improvement in viability over VS41A on rewarming using the VX series.

Greg compared the vehicle solutions EC, RPS2 and MHP2. EC is more easily perfused, but is not as compatible with viability as RPS2. RPS2 is good for slices, but EC is better for whole kidneys. MHP2 is as nontoxic on kidney slices as RPS2, can be used to perfuse whole kidneys like EC, is compatible with vitrification solutions and X1 -- and 21CM has a patent on it.

Greg spoke of the contracts 21CM has negotiated/is negotiating for cooperative research with a university cardiac preservation laboratory and a liver transplant laboratory. 21CM will retain the patents on their proprietary compounds in this research. The kidney preservation work will be done by 21CM at the Cryobiology Division.

In a video, Greg used a cryomicroscope to demonstrate recrystallization on rewarming. The cryomicroscope uses liquid nitrogen vapor regulated by program-controlled pumps that can create desired temperatures under the microscope. Images go to a video monitor, which can be recorded (and quantified). Upon rewarming, ice crystals normally restructure themselves into larger ice crystals -- which can be extremely damaging to tissues. But ice blockers prevent this crystal restructuring, thereby preventing the tissue damage.

Returning to the subject of profitability, Greg discussed sperm, corneas, skin equivalents and routine cell preservation. Only 10% of human donors have sperm that can be cryopreserved. Human sperm is already a $20 million market, but with improved CPAs the potential market could be much larger. Human sperm is normally preserved in one molar glycerol, but Greg showed a video of sperm frozen in 2.5 molar VX solution with considerable improvement.

Currently, there are about 50,000 cornea transplants annually, but if corneas could be banked, the number would be at least 5 times greater. When most of the cornea is exposed to VS41A for long enough to cause vitrification, there is a cell loss of 21%, which is unacceptable. But Greg believes that the new compounds are so much more permeant and so much less toxic that chances are very good for achieving vitrification in the near future.

ADVANCES IN RESUSCITATION AND NONINVASIVE INDUCTION OF PROFOUND HYPOTHERMIA

Mike Darwin began his presentation by saying that his personal goal is to achieve resuscitation after 30 minutes of normothermic ischemia (lack of circulation at room temperature). The theoretical absolute limit is 60 minutes. He showed a video of a dog that had been subject to 17 minutes of normothermic ischemia -- and thereafter resuscitated to apparently perfect health.

In stressing the importance of his work, Mike said that sudden cardiac arrest (arrhythmic death) accounts for 54.8% of all cardiovascular deaths and 26% of total deaths in the United States. Approximately 30% of the victims are under 50 years of age. He described CPR as being worse than useless if it delays the time to Advanced Cardiac Life Support (ACLS).

Mike reviewed the drugs currently used in ACLS protocols and noted that they are all directed at the cardiovascular system (epinephrine, lidocaine, atropine, etc.). No drugs are included for ischemia-reperfusion injury. Yet the current clinical outcome after 4-6 minutes of normothermic cardiac arrest is about 20% survival with less than 1% of the total being free of any neurological deficit. The deficits are primarily the result of neurological damage caused when circulation is restarted (reperfusion injury).

Through the use of blood dilution, rapid cooling, multi-modal drug treatments, a heart-lung machine and metabolic support (pH, blood gas, glucose, etc.) Mike has repeatedly recovered dogs from 15-17 minutes of ischemia (an unofficial world record) in experiments costing $10-$15,000 each. Mike uses dogs because their neurological injuries would be readily noted and because a dog's response to brain ischemia is very similar to that of a human. By contrast, monkeys that live in trees have evolved to recover much more readily from brain ischemia -- up to 15 minutes without intervention.

Mike puts his dogs on heart bypass artificial circulation to control both metabolism and temperature. His protocol includes 22 drugs, which reduce blood viscosity, control pH, reduce free radicals, inhibit excitotoxicity and antagonize calcium (among other actions). To deal with decreased vascular capacitance, he must drain about half the blood volume initially.

In discussing the limitations of his protocol, Mike mentioned liver toxicity due to the drugs. He justified the necessity of pretreatment with heparin by saying that clotting is probably not a very important factor in ischemic damage, and that heparin-bonded catheters are very expensive. Use of barbiturate as an anaesthetic may afford some protection, but probably not much. Finally, the experiments could benefit from systematic and quantitative means to compare physiological & neurological status before & after application of trauma & protocol. Prospects for FDA approval are poor, because the FDA rarely approves multi-drug cocktails. The FDA would also be unlikely to approve an automated multi-modal drug delivery system.

The second half of Mike's presentation dealt with means of lowering body temperature. Putting someone in a stirred ice-water bath is one of the most effective contemporary methods, and the use of heart-lung machine bypass is another. A bypass can be done in 15 minutes.

Problems with external cooling include the fact that cooling rates cannot exceed 0.2ºC/minute and that external cooling causes blood vessels to constrict (cutting off blood flow). Cardiopulmonary bypass is invasive, requires anticoagulation, is costly & technically demanding, and is not portable. Peritoneal/pleural lavage is not much better than external cooling.

About previous Mike had the idea of using total liquid ventilation with perfluorocarbons in the lungs as a means of rapidly cooling. When he applied for a patent, he discovered that someone else had already patented the idea. Mike experimented with liquid ventilation and found that partial liquid ventilation (PLV) has advantages over total liquid ventilation. He was able to get a 1.5º-2ºC temperature drop within 180 seconds of initial application. Use of PLV for rewarming is more problematic, because nitrogen bubbles form in the blood ("the bends") with temperature differences over 5ºC.

Total liquid ventilation actually reduces heat exchange because of problems associated with moving viscous fluid in&out of the lung. With PLV, cold perfluorocarbon (2ºC) can be blown-in & suctioned-out at a fairly rapid rate. Carbon dioxide exchange is greatly improved by this method and good oxygenation is maintained, as well. A consistent cooling rate of 0.36ºC/minute (half of the maximal rate achievable with cardiopulmonary bypass).

Mike said the the areas of the body directly affected by the perfluorocarbon (small airways, blood volume, lung parenchyma) accounts for 17% of total body heat capacity, whereas 72% of total heat capacity is in the well-perfused central tissues (brain, liver, etc.) and 11% is in the poorly perfused tissue (intestinal contents, bone, etc.). For this reason there is temperature oscillation during PLV and there is rebound.

Mike envisions a portable device which could deliver PLV in the field without a great deal of difficulty. And he thinks clinical trials could begin in as soon as 3 years.

(The work was patented as part of Dr. Wowk's patent on alkoxylated compounds -- Patent 5,952,168).

HOW SOON CAN WE ACHIEVE SUSPENDED ANIMATION?

The panel began with Brian Wowk reviewing the current state of the art in cryonics, which he described as 7.0-7.5 molar glycerol perfusion with external cooling of 0.1ºC/minute. Brian showed electron micrographs of the brain of a dog which had been subjected to the 7.5 molar glycerol-0.1ºC/minute protocol. Extensive damage was evident.

Cooling a 7.5 molar glycerol solution in a 250 mL flask at 0.1ºC/minute results in a chalky white solution composed of millions of ice crystals. Adding 1% ice-blocker (X1) to 7.5 molar glycerol results in a solution that is about 90% vitreous for a 0.1ºC cooling rate -- and 2% ice blocker results in complete vitrification.

Complete vitrification is achievable for 7.3 molar glycerol with 1% X1 ice-blocker by increasing the cooling rate to 0.5ºC/minute. Such a cooling rate can be achieve in cryonics by the use of fluorocarbon perfusion. Using fluorocarbon, cooling rates of 9ºC/minute can be achieved at 20ºC and 1ºC/minute is still achievable at temperatures as low as -110ºC. Fluorocarbons do not mix with either water or oil, and they remain liquid to very low temperatures.

X1 ice blocker does not diffuse into cells, which could limit its effectiveness. But Brian mentioned that this is a matter of some dispute, since most freezing occurs in the extracellular space -- and existing intracellular proteins may be adequate to prevent damage inside cells. Increasing X1 concentration above 2% results in viscosity problems which would be much less severe in other 21CM-X ice-blockers.

Since 7 molar glycerol is toxic to cells, it makes more sense to perfuse with solutions such as VX rather than glycerol. Greg Fahy described experiments he recently performed on rabbit brains. In the first experiment he used perfluorocarbons and the minimal amount of required cryoprotectant. Electron micrographs showed intact brain structure: axons, synapses, pericapillary tissue, etc. Some areas of damage were seen, however. In the second rabbit, a higher concentration of CPA was used (9.5 molar) with no hint of freezing on cooling or warming. Electron micrographs showed excellent preservation of neural structure with no evidence of damage -- even in the CA1 area of the brain, which is the area most sensitive to ischemic insult.

The panel discussed a variety of problems. The blood brain barrier seems like an obstacle to cryoprotectant, but this could possibly be solved by implanting a catheter directly into the cerebrospinal fluid. Certain organs like bones & eyes might be particularly difficult to perfuse, meaning that it could take decades after organ cryopreservation (kidney, heart, brain, liver) has been achieved for complete suspended animation of a whole person to be achievable. Removal of perfluorocarbon might also be a difficult problem (although Hugh Hixon suggested that carbon tetrafluoride -- which evaporates readily -- might work for washout).

FURTHER EXPLANATIONS AND SPECULATIONS

The presentations impressed upon me how much witchcraft and how little science has gone into the study of cryoprotectant agents (CPAs). This might be understandable in light of the fact that most cryobiologists are, in fact, biologists. I suspect that a great deal could be accomplished by a thorough study of the physics of the chemistry of CPAs.

But considering that the other two conventional cryoprotectants Brian discussed (propylene glycol and ethylene glycol) are both considerably less viscous and more permeant than glycerol, I was mystified as to why glycerol has been the CPA of choice for cryonicists. I asked Brian and he admitted that he did not know for certain, but speculated that because edema was so common among early cryonics patients, and because glycerol is so dehydrating -- that glycerol was found in practice to result in fewer problems with edema. But this would have been irrelevant after Jerry Leaf & Mike Darwin began using HES (HydroxyEthyl Starch) as a colloid to prevent edema in ischemia-compromised cryonics patients. The toxicity of ethylene glycol at 38ºC (due to metabolism to oxalic acid) does not occur at 0ºC.

Sodium chloride (table salt) forms a crystal structure with a high melting point (800ºC) that is easily dissolved in water. It is the polar nature of water that allows it to separate the sodium and chlorine ions. I suspect that the capacity to vitrify is related to the ability of substances to intrude themselves between water molecules and to hydrogen bond to them. For organ preservation purposes, a CPA must combine low viscosity, polarity, lipophilicity and low molecular weight.

The ability of a solvent to separate charges is measured by a physical property known as dialectric constant. Consulting the HANDBOOK OF CHEMISTRY AND PHYSICS, I observe that CPAs typically have a high dielectric constant:

I included ethanol to show the contrast with 2-methoxyethanol, which Brian had presented as an ethylene glycol derivative. The addition of methoxy groups seems to reduce dialectric constant -- which makes sense from the point of view of their lipophilicity. Since Brian said that the addition of methoxy groups typically results in 5% less CPA to achieve vitrification, it would appear that the dialectric constant effect is "mitigated" by other factors. Since CPA cocktails seem to be more effective than single-agent CPAs.

Understanding the physics of the chemistry of CPA interactions (CPA-to-CPA and CPA-to-solvent) will be a great step beyond witchcraft. I would be curious as to the result of a substitution of a methoxy for a methyl group in DMF or N-methylformamide -- since this would most likely be increasing hydrogen bonding.

It may be that the high dialectric constant of formamide works in concert with other solvating/vitrifying properties of other CPAs. I leafed-through the HANDBOOK OF CHEMISTRY AND PHYSICS to see what other high dielectric-constant, low molecular-weight solvating compounds looked promising. I found the following:

N-methylpropamide may be too large & bulky, and hydrogen cyanide may be too reactive to seem appealing. But if most freezing occurs in the extracellular space, some bulkiness might not be too much of a problem. Reactivity is a function of temperature, so if hydrogen cyanide is of any use, it might be the last constituent added at the lowest temperature (which would leave the problem of how to remove it on rewarming).

Intermolecular forces between neutral molecules are typically less than 15% as strong as covalent or ionic bonds. My speculations have placed a great deal of emphasis on dipole-dipole forces, but two other intermolecular forces between neutral molecules are significant: London dispersion force and hydrogen bonding. And water can interact with not only other neutral molecules (such as cryoprotectants), but with non-neutral ions (ion-dipole forces) -- although it seems unlikely that much cryoprotective benefit can be gained by depressing the freezing point with added salt (in large part a colligative effect, although salts vary greatly in their freezing point depression).

London dispersion force is an induced dipole between large molecules that are close together -- especially large hydrocarbons. (Large molecules do not have such a tight grip on outer electrons, making spontaneous, temporary dipoles easier to induce.) But even for a solution of HCl, which has a strong dipole, London dispersion force accounts for about 80% of the attraction between molecules, with dipole-dipole interaction accounting for the other 20% (chlorine is too large to hydrogen-bond).

Hydrogen bonding is the strongest intermolecular force, but it is only seen between hydrogen and the smallest electronegative atoms -- namely oxygen, nitrogen and fluorine. In biological systems, oxygen & nitrogen hydrogen bonding would normally be most relevant. Hydrogen bonding energies are typically in the range of 4 KJ/mol to 25 KJ/mol.

Most likely, hydrogen bonding between water molecules and cryoprotectant molecules is the key to the mechanism of cryoprotectant action. The oxygens in formamide & DMSO and the nitrogen in formamide may play this role. Interestingly, formamide reduces the toxicity of DMSO by some unknown mechanism (the mechanism of the toxicity is also unknown). [For further discussion of cryoprotective agents, see my essays Vitrification in Cryonics and Perfusion & Diffusion in Cryonics Protocol.]

Replacing hydrogen atoms with fluorine atoms in hydrocarbon produces molecules known as fluorocarbons. A hydrocarbon (alkane) in which every hydrogen atom has been replaced with a fluorine atom is called a perfluorocarbon. The bond-strength and stability of these molecules is so great that they interact very little with other molecule -- being neither miscible with water nor oil.

A study of the average bond dissociation energies for carbon-halogen and carbon-hydrogen sheds some light upon the properties of chlorofluorocarbons and perfluorocarbons:

Chlorofluorocarbons (CFCs) became popular in refrigeration and as aerosol propellants because they are nontoxic, nonflammable, odorless and noncorrosive. The most widely used were Freon-11 (CCl₃F) and Freon-12 (CCl₂F₂) until evidence emerged that these substances deplete atmospheric ozone. These Freons have boiling points of -24ºC (Freon-11) and -30ºC (Freon-12). The Freons cool a refrigerator upon evaporation and then are mechanically recycled back to liquid by the pressure of a compressor.

Elemental oxygen normally exists in the two forms O₂ and O₃(ozone), with only a tiny portion in the form of ozone. But that tiny portion plays a very significant role in the earth's stratosphere (11-48 kilometers above the surface) where the absorption of ultraviolet light induces the reaction: O₃ -> O₂ + O^. which is normally the equivalent of: 2 O₃(ozone) -> 3 O₂

Because CFCs are so inert, they remain in the atmosphere and some eventually reaches the stratosphere. Ultraviolet light causes the release of chlorine radical (Cl^.) which depletes the ozone by producing a chlorine oxide radical (ClO^.) and oxygen: Cl^. + O₃ -> ClO^. + O₂.  The chlorine oxide radical then combines with oxygen to regenerate the chlorine radical: ClO^. + O^. -> Cl^. + O₂.  By such a chain reaction, a single chlorine radical from a single CFC molecule will deplete about 4000 ozone molecules from the stratosphere. The result has been increased crop damage, skin cancer and general injury to the world's plants&animals.

The strength of the Carbon-Fluorine bond has much to do with why Teflon (Dupont's name for what is effectively a perfluorocarbon polymer made from tetrafluoroethylene) is so inert and does not interact readily with other substances (hence, non-stick, corrosion-resistant cookware).

There was some question at the 21CM Seminar concerning the possible impact of government regulation of ozone-depleting substances upon perfluorocarbon availability. But unlike CFCs, perfluorocarbons do not deplete ozone because the carbon-fluorine bond is so strong that fluorine radicals are not released by ultraviolet light. In fact, hydrofluorocarbons (HFCs) have been promoted as an alternative to CFCs, and most new cars&trucks now contain air-conditioners that operate on HFCs (HFC systems cannot be made to operate on CFCs).

Although there is some concern about the fact that the stability (and long lifetime) of perfluorocarbons could increase global warming, the US Environmental Protection Agency has approved several perfluorocarbons as acceptable substitutes for CFCs for electronics cleaning solvents (see www.stanford.edu/dept/EHS/comply/alt.html). I am listing these in a table, along with the melting and boiling points listed for them in THE HANDBOOK OF CHEMISTRY AND PHYSICS

It is interesting to note that certain fluorinated hydrocarbons have more than twice the oxygen carrying-capacity as hemoglobin. For example, Fluosal DA, a 20% emulsion of perfluorodecalin and perfluorotripropylamine in water has been used as a blood substitute in mammals. A water emulsion of 1-bromoperfluorooctane (LiquiVent, perflubron) began clinical trials in the US in 1996. It could be that these substances could be used to augment the effects of straight perfluorocarbons to maintain oxygenation, while lowering temperature.

I have great hope that the researchers at 21CM will expand along the lines of applying chemistry & physics to the problems of cryobiology. In particular, there are many questions yet to be answered about the molecular interactions that result in CPA toxicity, cooling damage, ice formation, vitrification, etc. -- questions that must ultimately be answered in physical chemical terms.

For more recent research see The Hippocampal Slice Cryopreservation Project .

For further discussion of cryoprotective agents, see Vitrification in Cryonics .

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