X-Message-Number: 30106
Date: Thu, 6 Dec 2007 09:44:47 -0800 (PST)
Subject: types of cryoprotectant toxicity Part 1

    [Cryoprotectant dependant toxicity can be divided in three types of
damage: osmotic, intrinsic, and extrinsic. Osmotic damage occurs when cells
shrink too much, after being exposed to too high a concentration of
permeating cryoprotectants too quickly. An effective means for completely
eliminating this type of damage, is to reduce the rate of increase in the
molarity of cryoprotectants, so cells never shrink beyond a certain
threshold, which will vary by cell type. In the addition to direct cellular
shrinkage, indirect shrinkage can be triggered by an intact blood/brain
barrier, which can allow water to pass out of the brain too quickly, while
cryoprotectants only slowly enter the brain to replace the lost fluid.
Unfortunately the price paid for gradually ramping up molarity, is increased
length of time of exposure to cryoprotectants, which can in turn increase
both intrinsic as well as extrinsic toxicity. The addition of small amounts
of rapidly permeating methanol has been used to limit osmotic damage.
    Intrinsic damage is time dependant direct damage to cellular
constituents by concentrations of cryprotectants beyond a certain threshold.
Examples of intrinsic damage include high molar propylene glycol
destabilizing cell membranes, while DMSO, and formamide directly denature
DNA. Intrinsic damage can be effectively eliminated by reducing the period
of exposure. Such rapid freezing techniques can work for individual cells,
but are impossible to impliment for organ systems, due to the prolonged
period of time required for cryoprotectant equilibration, as well as high
thermal inertia. However not all cryoprotectants show significant
intrinsic toxicity. Glycerol appears to be virtually free of this type of
toxicity, with ethylene glycol in second place. However glycerol only slowly
diffuses across cell membranes, and so greatly increases the risk of osmotic
damage. Ethylene glycol's major limitation is in the area of extrinsic
    Extrinsic toxicity is time dependant damage to cellular constituents by
either metabolites of cryoprotectants, or to free radicals generated during
the process of their metabolism. Extrinsic damage can be eliminated by
blocking metabolism of cryoprotectants. For example cooling from room
temperature tends to reduce metabolism, and thereby helps to limit this type
of damage. There are other options to reduce metabolism of cryoprotectants
such as inhibiting an enzyme called alcohol dehydrogenase. The main
destructive metabolite of ethylene glycol is known to be calcium oxalate.
Ethanol has been used to reduce the metabolism of ethylene glycol. IMHO, the
reason ethylene glycol has seen use in vitrification solutions despite a
high extrinsic toxicity may be due to a similar reduction of ethylene glycol
metabolism by the other cryoprotectants.]

Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=30106