X-Message-Number: 5455 Date: Wed, 20 Dec 1995 11:51:03 -0800 (PST) From: Doug Skrecky <> Subject: Re: just a question >Sorry if this is a dumb question but it didnt seem to be covered >in the FAQ. >If at this point of time we are succesful in freezing animals to a >temperature of below zero and most damage from freezing comes from >the actual use of the liquid nitrogen to bring the temperature down >to way below zero, my question is this how cold do we need to make it? >Is the not another method that would keep us cold enough so we dont >deteriorate but not so cold that we get the "fractures" (a term I've >heard but don't really understand). In other words do we have to go >so cold? Thanks Brett Corlett No we don't, but we do have to reduce the temperature enough so that the frozen tissue has completely solidified. Hold your breath - here comes the explanation: The water in tissue is composed of ice crystals (solid) and an 80% solute 20% water mixture below a certain temperature (called Tm). The solute/water mixture does not crystalize, but instead vitrifies to a rigid glass (solid) at a temperature called the annealed glass transition temperature (or Tg'). For frozen tissue to be completely inert/solid it has to be stored at a temperature below Tg'. This Tg' varies depending on what cryoprotectant is used. For glycerol this is below even dry ice (-78 C) temperature, so liquid nitrogen (-195 C) is used instead. Glycerolized bone marrow cells stored on dry ice retain normal morphology after 1 year of storage, begin to deteriorate after 2 years after 3 years are almost completely destroyed. (Cryobiology 65-69 1970) Cryonicists use glycerol primarily because cryobiologists do. There is a lot of research that has been done on cryopreservation using glycerol. Cryobiologists are not much interested in reducing storage costs as they are interested only in short term storage. In theory liquid nitrogen storage could be dispensed with if a cryoprotectant with a Tg' above -78 C was used. Moreover freeze-drying would also serve to further increase possible storage temperatures. For example the latest research has assigned a Tg' of -64 C to sorbitol. So dry ice should be sufficient if sorbitol was substituted for glycerol. When completely dried sorbitol (also called glucitol by the way) has a Tg of -9 C, so storage in a standard refrigerator (or even permafrost?!) should be sufficient for freeze-dried sorbitolized tissue. (Carbohydrate Research 39-48 Vol.238 1993) Other cryoprotectants with higher Tg's could be tried, but tissue penetration starts to become a problem here. Recently dry lotus seeds stabilized naturally with sucrose (dry Tg = 62 C) were successfully germinated after as much as 1288 years of storage at ambient temperatures. (American Journal of Botany 1367-1380 Vol.82 No.11 1995 Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=5455