X-Message-Number: 2497
From:	 (Ben Best)
Date:	Mon, 20 Dec 1993 00:50:00 -0500

  [The following article appeared in the Autumn 1993 issue of
CANADIAN CRYONICS NEWS. Subscriptions are Cdn$10/year in Canada,
US$10/year in the US and US$14/year elsewhere from The Cryonics
Society of Canada; Box 788 Station "A"; Toronto, Ontario; M5W 1G3
CANADA. The article is reproduced  at the request of Alcor President
Steve Bridge, who wanted to see what CryoNet people think is wrong or
right about Skrecky's ideas. -- BB]

          HIGH TEMPERATURE CRYONICS By Douglas Skrecky

   Longterm storage of biological materials has traditionally been at
liquid nitrogen (-196C) temperatures. This may have been a costly
mistake. It now appears that dry ice (-78C) temperatures are all
that is required.

   Deterioration of frozen tissue has been regarded as a common
feature at all temperatures above the glass transition temperature.
This varies depending on how quickly freezing is carried out, as well
as the nature of the cryoprotectant solute. When a solution first
freezes it forms a mixture of ice crystals and a freeze concentrated
liquid which holds a higher concentration of the solute than the bulk
unfrozen solution. If the temperature is decreased slowly, the liquid
phase will become maximally freeze concentrated so that its viscosity
rises to the maximum possible for a given solute, irrespective of the
original concentration in the bulk solution. This process, which is
called annealing, raises the glass transition to the highest
temperature possible. As the temperature further decreases to this
glass transition, the viscosity increases to the point where the liquid
becomes a glass and the solution is only then considered to be
completely frozen.

    The glass transition for pure water is -135C, while that for
most slowly frozen foods varies from -45 to -15. The use of glycerol
as a common cryoprotectant lowers this transition since glycerol
solutions form glasses only below -65C. *1 The glass transition for
sucrose solutions was originally believed to be -32C, but recently a
more critical examination yielded a temperature of -46C. *2 This
advantage of sucrose over glycerol also extends to temperatures above
the glass transition as sucrose has been found to be more effective
than glycerol in inhibiting protein denaturation in frozen tissue
stored at -20C. *3 Would dry ice temperatures be sufficient to
preserve tissue indefinitely? It seems so. Low density lipoprotein
treated with sucrose, sodium chloride and EDTA and stored at -70C
showed no signs of either oxidative or proteolytic deterioration over
an 18 month period and when thawed retained functionality similar to
fresh LDL. *4

    What would be the best cryoprotectant solution? Sodium chloride
depresses the glass transition, so replacing it with potassium
chloride might be a good idea. EDTA is an effective antioxidant when
the storage temperature is -20C, but its value at below the glass
transition remains to be proved. *5 Dietary supplementation with
vitamin E improves the resistance of postmortem tissue to oxidation.
*6 In any case, long term storage must be in an oxygen free environment
as even glasses can oxidize. Adding egg yolk to sucrose improves the
survival of sperm by stabilizing membranes during freezing and
thawing, so this would appear to be a desirable addition. *7

   The replacement of glycerol by sucrose could do much to reduce
the costs of cryonic storage by enabling the replacement of liquid
nitrogen refrigerant with dry ice, but why stop here? Unlike
glycerol, sucrose is an effective anhydroprotectant in addition to
being a cryoprotectant. Partially drying tissue by pumping dry gas
through the cardiovascular system could eliminate all damage due to
ice crystal formation during freezing since 80% sucrose/20% water
mixtures do not freeze, but instead vitrify directly to a glass at
-46C. With further desiccation the glass transition is increased to
29C for 96.5% sucrose and 62C for anhydrous sucrose. *2 Thus the
replacement of glycerol by sucrose might very well eliminate any need
for refrigeration. *8

                p.115-360 (1991)

                p.553-566 (1991)

   *3 JOURNAL OF FOOD SCIENCE, Vol.55,No.5, p.1222-1227 (1990)

   *4 JOURNAL OF LIPID RESEARCH, Vol.33, p.1551-1561 (1992)

   *5 CRYOBIOLOGY, Vol.29, p.668-673 (1992)

   *6 JOURNAL OF ANIMAL SCIENCE, Vol.71, p.1812-1816 (1993)

   *7 CRYOBIOLOGY, Vol.30, p.32-44 (1993)

   *8 The glass transition for anhydrous glycerol is very low -93C. *1

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