X-Message-Number: 8255
Date: Fri, 30 May 1997 13:31:07 +0200
From: Tom Jonsson <>
Subject: Improvements in cryopreservation

Reply to Brian Wowk, CryoNet #7021
(This is my third message to Cryonet)

>Can you briefly explain the rationale for your CPA mixture?
>and have you tested whether it vitrifies as a pure solution?  Have you
>rewarmed and analyzed any brains yet?

        I am sorry you had to wait for my answer until now. I recently found
your message when I did some scanning through old messages on cryonet. My
name is Tom, not Jon, and that is probably why I missed your message the
first time. However, these are my answers to Brian.

        I gradually add ethanol to the CPA mixture during freezing
(increasing the concentration just before the point of crystallization in
the current mixture). Ethanol makes an organ/body perfusion possible down to
lower temperatures (the freezing point of pure ethanol is -117°C).

        Yes, it vitrified. No cracking was observed (but after repeated
freezing and thawing some cracking could be observed).

        Unfortunately  the freezer (-140°C) where I stored the brains was
out of order and the brains thawed. The temperature was warmer than 90°
below zero (Celsius) for about a week. I did a vitality test on them anyway.
Nevertheless, as expected they show low vitality.

        What I really would like to do is more research on how human tissues
can survive liquid nitrogen temperatures and it's frustrating not to have
the time nor money necessary.

	I made researches on how ethanol as an additive affects the vitality of
frozen nerve cells in brain slices from adult rats. I used fresh tissue as a
positive control, and an ordinary freezing method as a reference (10%DMSO at
a cooling rate of 1°C/minute according to Sörensen T. et.al. Intracephalic
transplants of freeze stored rat hippocampal tissue. J.Comp.Neurol.252
(1986)). The brain slices were stored in liquid nitrogen for two months. Two
different vitality tests on the experiment group showed better results in
comparison with those frozen by ordinary methods. The morphology of the
brain tissues was normal (light microscopy). The good result was confirmed
by electron microscopy. 
It appears that ethanol is a non toxic additive in these temperatures and
concentrations!

I also tried some new antitoxic and cryoprotective agents, but they didn't
show any crucial effects.

        Like I said a year ago, this freezing method, perfusing with
increasing concentration of ethanol during freezing could improve our chance
to "wake up" in equally condition to when we were freezed!    


Tom

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