X-Message-Number: 3393
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

    (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 ()

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