X-Message-Number: 11010
From: "Brian Wowk" <>
Subject: Vitrification and Cracking
Date: Fri, 1 Jan 1999 22:52:47 -0800

	Freezing in the presence of cryoprotectants (such as glycerol)
preserves blood and other isolated cells because growing ice
crystals push cells into residual unfrozen areas of concentrated
cryoprotectant that eventually vitrify (turn into a glass).  Freezing
destroys tissues and organs because unlike cells in blood, the
cells in organs cannot move in response to growing ice crystals.
Vitrification seeks to overcome this problem by preventing ice from
forming anywhere, essentially turning an entire organ into a single
glassy blob similar to the millions of tiny glassy blobs that contain
blood cells when blood is successfully frozen.

	As Mr. Dugue alludes to, the price paid for turning an
organ into a single piece of glass is that glass tends to break.
Above the glass transition temperature (usually between
-110'C and -130'C, depending on the cryoprotectant), this is not a
concern because organs are more like a thick syrup than a
glass.  As the temperature is lowered below the glass transition
temperature, fracturing becomes more likely.  The risk becomes
significant about 20 degrees below the glass transition, and
becomes certain at liquid nitrogen temperature for large samples.
See the article by Fahy, et al:

Physical problems with the vitrification of large biological systems. 
     Cryobiology. 1990 Oct;27(5):492-510. 

	Mr. Ettinger has written about CI's research showing
that slow cooling to liquid nitrogen temperatures prevents fracturing
of frozen sheep brains.  Unfortunately this gives us no reassurance
about vitrified brains because frozen brains, consisting of a sintered
aggregrate of ice and glass, are less likely to fracture than a
single monolithic block of glass.  Essentially a frozen brain is already
fractured in millions of places so it's more difficult for new thermal
stress cracks to propagate macroscopically.  Ironically, the better
you cryoprotectant an organ against ice, the more likely it is to
fracture macroscopically at very low temperatures.

	The limited data available does suggest that very slow
cooling through the glass transition reduces fracturing in both
frozen and vitrified samples.  This is probably due to an "annealing"
effect in which the glass is given sufficient time to relax into a
more stable form.  It's even possible that with sufficient annealing
time (months?  years?) large vitirified samples could be taken
all the way to liquid nitrogen temperature without fracturing.  This
is a subject for future research.

	In the meantime, the most expedient and effective solution
to the fracturing problem is simply to store at temperatures
near -130'C instead of liquid nitrogen temperature (-196'C).
Laboratory freezers that operate in this temperature range
are available commercially.

---Brian Wowk

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