X-Message-Number: 7021
From: Brian Wowk <>
Date: Sun, 6 Oct 1996 00:34:20 -0500
Subject: Reply to Jon Jonsson

	First, I must apologize that no one (publicly) replied to
Mr. Jonsson's CryoNet message in June.  It was evidently lost
among the rush of other events at that time.  In June, Mr. Jonsson
wrote of the following experiments he was conducting privately in Sweden: 

> In brief, 
> Freezing-process
> 1. I relaxed the vessels with Nimotop  (which also protects against ishemic
> brain damage).
> 2. The rat was perfused with a modified Ringer solution down to 4 C.
> 3. do. with added cryopreservative agents : 2.5M DMSO, 60mM Sucrose, 15%
> Egg yolk, L-Glutamine, Glucose.
> 4. do. at a controlled and a relative rapid freezing rate down to -70 C,
> with a media which doesn't freeze at such low temperature (decreasing
> concentration of water and cryoprot. agents during the process).
> 5. do. with Nitrogen in gas form -above the freezing-point of the media.
> 6. Liquid Nitrogen
>
>The brains are showing some shrinkage, caused by the vitrification. This is
>reversible, and probably not on a toxic level. They show no other
>alterations or damages ! (It's very difficult to say how vital the nerve
>cells really are)
>
>So far so good, The brains haven't been thawed and analysed yet. But I have
>outlined it as follows :
>
>Thawing
>1. Put the object in -80 C freezer.
>2. Perfuse with air, for a time enough to prevent recrystalization  . 
>3. Perfuse with a nutrition solution containing decreasing amount of
>sucrose (protection against osmotic stress) and agents protecting against
>brain damage.
>4. Then quickly up to body-temperature 
>
>Analyse
>A. Frequency of neurospecific marker proteins, like S-100 and NSE at a
>certain time after thawing.
>B. Counting the uptake of radioactive thymidin (scintillation counter) in
>the nerve-cells. 
>
>Other opportunities to investigate the vitality of frozen tissue:
>Transplantation of frozen nerve cells, Cell growth in vitro, Vital staining
>EBA(Evan's Blue Albumin) in vivo and Trypan Blue in vitro, but this is only
>demonstrating damage on the cell membranes, TEM and SEM ?
>
>Comparing against controls taken after different steps. 
>
>First I want to see if the cryomedia I use down to -75 C show any toxic
>effects in this low temperature. Which kind and how much of cryoprotective
>agents can be solved in this media ?

	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?

***************************************************************************
Brian Wowk          CryoCare Foundation               1-800-TOP-CARE
President           Human Cryopreservation Services   
   http://www.cryocare.org/cryocare/

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