X-Message-Number: 3393 Subject: SCI.CRYONICS: HIGH PRESSURE CRYONICS REPLIES From: (Ben Best) Date: Thu, 10 Nov 1994 08:17:00 -0500 I was at the Ontario, California Cryonics Conference (along with many others who are frequently on CryoNet), so these High Pressure Cryonics replies are somewhat delayed. I am glad I have flushed-out some thoughts on the use of pressure in cryonics, even if it is mostly of the "it has all been thought of before and tried before" dismissive variety. If the idea is so simplistic and standardized, why are the responses so varied? I now discern five potential uses of pressure in cryonics: (1) Use of pressure to lower the boiling point of liquid nitrogen (2) Rapid application of pressure to force vitrification (3) Rapid release of pressure to force vitrification (4) Use of pressure to reduce cryoprotectant toxicity (5) Use of pressure as an adjunct to cryoprotectants in vitrification I will discuss each of these uses in turn: (1) Use of pressure to lower the boiling point of liquid nitrogen While I favor the lowest possible storage temperature, I suspect that the technical difficulties and/or costs of long-term storage at both high temperature and pressure would be prohibitive. I have toyed with the idea of maintaining high pressure after vitrification, but not very seriously. Primarily my interest has been the use of pressure to achieve vitrification. Once vitrification is achieved, it is probably more reasonable to rely on low temperature (liquid nitrogen temperature) than on high pressure to maintain vitrification. It would probably be far cheaper and easier to use liquid helium than to attempt to use pressure at liquid nitrogen temperature and below. (2) Rapid application of pressure to force vitrification This is Hergenhahn's idea. Unlike (3) to (5), which rely on ice being less dense than water, this method presumes the formation of a vitrification solution which is more dense than water. Hugh Hixon entirely misses the point when he refers to "ice crystals formed at high pressure". The whole point of this procedure is to apply pressure so rapidly that the solution does not have time to form ice crystals. Moreover, a vitrified solid of tissue could be formed in this way WITHOUT the need for (and toxicity of) cryoprotectant. However, this requires pressures in the order of 14,000 atmospheres, which could be quite technically challenging, as Hugh Hixon points out. Also, even though a vitreous solid (rather than ice) is formed, I think Hugh makes a very good point about the metastable state -- a factor I had not considered. The vitrified solid would be potentially "explosive" due to its high density, and yet brittle and vulnerable to cracking (just like glass). Possibly the cohesive forces could adequately exceed the explosive forces, and there would be no problem. Otherwise, it would be necessary to maintain the pressure. Since the solid is super-dense, it could maintain the inertness of a liquid nitrogen-stored solid at higher temperatures. But liquid nitrogen would probably be the most economical means of cooling, which returns us to the problems already mentioned in (1). I must also acknowledge that the extreme high pressures required for this technique are bound to cause tissue damage and result in loss of viability. The differential compressibility of tissue substances and conformational changes with some enzymes and proteins are inevitable. Nonetheless, I believe that the structural damage would still be considerably less than the damage due to cryoprotectant toxicity and freezing damage. Nonetheless, quantifying and comparing this damage should be attempted experimentally, not simply on the basis of speculation. (3) Rapid release of pressure to force vitrification The freezing point of water at 1,000 atmospheres is -10 degrees Celcius and at 500 atmospheres is probably -4 or -5 degrees Celcius. The freezing point of (saline) biological tissue will be lower, but the freezing point depression should be comparable. 500 atmospheres is the maximum pressure Dr. Greg Fahy gives for no loss of viability due to denaturization of proteins (although he apparently arrived at this figure in experiments where pressure was applied to tissues containing cryoprotectants). Under this scheme, a patient could be cooled under high pressure and then vitrified by a sudden release of pressure. The problem with this approach is that heat of fusion would prevent the entire patient from vitrifying -- and the result would probably be heating, liquification or recrystallization. (4) Use of pressure to reduce cryoprotectant toxicity If freezing point can be lowered, cryoprotectant can be introduced at a lower temperature with the tissue still liquid, but with the cryoprotectant less toxic. I would like to see work on this, in fact, this was work I had hoped that Dr. Greg Fahy would do. But he had no interest in this. He introduced cryoprotectant near zero degrees Celcius and THEN applied pressure. He found that pressures greater than 500 atmospheres reduced viability (by denaturing proteins). Because 500 atmospheres only lowers freezing point by 5 degrees Celcius or so, he didn't think the cost was worth the effort. It is undoubtedly also more technically difficult to introduce cryoprotectants under high pressure than to introduce cryoprotectants and then apply pressure. Since Dr. Fahy was achieving such great success with his cryoprotectant cocktail, it's not surprising that he lost interest in the (technically difficult) high pressure methods. (5) Use of pressure as an adjunct to cryoprotectants in vitrification Since ice is more dense than water, the application of pressure during cooling can resist ice formation. As was mentioned, this is the approach Dr. Fahy evidently experimented-with. As was also mentioned, the benefits of this approach did not justify the costs for him -- especially given the fruitfulness he was seeing with improvement of the cryoprotectant cocktail and the re-warming protocol. *IF* this is true, I would agree with Hugh Hixon (and Brian Wowk) that "the resources that would have to be devoted to [high pressure methods] would almost certainly be better expended in other directions." -- Ben Best () Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=3393