X-Message-Number: 2497 Subject: HIGH TEMPERATURE CRYONICS 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 *1 CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION, Vol.30,No.2-3, p.115-360 (1991) *2 INTERNATIONAL JOURNAL OF FOOD SCIENCE AND TECHNOLOGY, Vol.26, 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 Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=2497