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


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