X-Message-Number: 7668
From: Brian Wowk <>
Date: Sat, 8 Feb 1997 13:51:54 -0600
Subject: Partial Vitrification

Doug Skrecky <> wrote on CryoNet:
>   Here's yet another possible explanation for the variability of Visser's
> results, again with a simple solution. When the techinque works, it may
> be due to partial vitrification of the rat heart. To achieve this a
> period of slow cooling by exposure to nitrogen gas must preceed the fast
> cooling that accompanies immersion in liquid nitrogen. Variations in the
> length of exposure to nitrogen gas before immersion could then account
> for the variations in the results of the Visser technique.
> For partial vitrification to happen the heart would have to be frozen
> slowly at least initially, so that the cryoprotectant has time to become
> what is called freeze concentrated as ice forms between cells. Rat hearts
> are able to survive partial ice formation. (Cryobiology 29: 470-477 1992)
> Once this ice has formed the remaining extracellular cryoprotectant is so
> concentrated that it vitrifies when the heart is quickly cooled.
> Intracellular freezing is avoided because cells have time to dehydrate
> during the slow cooling phase so that they too are vitrified. This works
> well for mouse embryos for example. (Cryobiology 31: 423-433 1994)
	The problem is that to freeze concentrate an initially
dilute cryoprotectant to vitrifiable concentrations will require the
formation of a lot of ice.  The presence of this ice will then
further drive up the concentration needed to vitrify the residual
solution to far above CNV (the minimum cryoprotectant concentration 
needed to vitrify in absence of ice).  In fact, you will end up with 
a quantity of ice that is not much different than what would be 
obtained during equilibrium freezing all the way down.
	This interpretation is borne out in the reference you cite.
They started with a 1.5M cryoprotectant concentration, slowly cooled to
a target temperature, and then rapidly cooled to liquid nitrogen
temperature.  The highest target temperatures that worked were
were -35'C for propylene glycol, and -50'C for DMSO.  These are 
the approximate freezing points of 55% concentrations of these agents,
which is an almost unfreezable concentration well above CNV.  They
thus found it necessary to essentially maximally freeze the samples
to an equilibrium concentration of ice before the LN2 plunge, or
the embryos wouldn't survive slow rewarming (20'C per minute).
	That's not to say that your suggestion is of no utility.
It's a very clever idea that might be useful *if* the initial
cryoprotectant concentration was already close to CNV (so that the ice
formed in the freeze concentration was minimal), and the cryoprotectant
was particularly resistant to ice growth.
	But that's not the regime we are in with these Visser
experiments, where the DMF concentration is far below CNV (as it 
must be to avoid toxicity).  In short, there doesn't seem to be any 
cryobiological way to perfuse organs with dilute DMF solutions that 
will result in less than 50% of the organ turning to ice.  This is a 
quantity of ice that is absolutely fatal to kidneys and livers, 
borderline for recovery of cardiac contractility, and out of the 
question for transplant viability.
Brian Wowk          CryoCare Foundation               1-800-TOP-CARE
President           Human Cryopreservation Services   

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