X-Message-Number: 6409
From: 
Date: Fri, 28 Jun 1996 16:40:32 +0700
Subject: Improvements on Cryopreservation

	I was pleased when I found this stuff about Cryonics on the Net. People
who share the same thoughts I have about the opportunities of
Life-extension. It's refreshing to meet this unusual optimism about the
subject. I am used to be met with criticism and a remarkable indifference
when I tell folks about my visions. The Cryonics have rehabilitated my hope
about mankind.
	
	I am a 31 year old Cell Biologist from Sweden, working at the Institute
of Anatomy and Cell Biology in the University of Gothenburg.
I am sharing the great interest in Life with the Cryonics supporters. Time
has no end, but Life has. That can create deep anguish. When I was a child
and couldn't sleep at the nights, I used to think about the Death. Most
children have similar thoughts, but I couldn't stop thinking about Life and
Death. If it was for sure that God exists, and our souls continue to live
after Death everything should be satisfying, but we can definitely not be
sure of that!

Is an eternal life really a possibility? In theory a eternal life can be a
reality, but is this a opportunity also in the practice ? I have my doubts
about that, even if everything works as we hope in the future  (nanotech.
etc.). Probably we have to die anyway, sometime or other in the
never-ending time. Maybe everything's instability have to end the life
anyway, how is it about the decomposition of neutrinos for example ? Rather
a Good Life for 100 years than a worse for 1000 years. Whenever we die
(even if the Life -span should be a million years), it will still be a
snapshot in the never-ending time Maybe we should relax and have more fun
in this snapshot we call Life. 
Or should we do everything in our power to improve the odds ? A good Life
for a thousand years is not so bad, and we can saturate our curiosity about
the future. However I have a compulsion about this, so I have to do
something to give the Life better odds ! (and moderate my anguish?)
Without a thrust in something it's difficult to live. This is giving me
deeper meaning in Life. 

	One problem is how "nanotech" can reestablish our memory if it has been
damaged in the freezing process ? I think that would be very difficult.
Hence, it's important to improve the freezing techniques !
The nerve connections in CNS are important for the personality and the
memory, and we can't allow these to being damaged during the freezing
process.
	I am doing some research about vitrification of whole organs.
I have no money for this project so I have to do this beside my regular
work. Because of that, it will take some time. I have done a few pilots,
cryoperfusing brains from rats down to -70 C with a new mix of cryogenic
fluid. I had some difficulties with, among other things, steering the
freezing rate, because I worked with very simply instruments, but I think
the method has great potential. 

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 ?

I welcome everyone who have comments to this message !
To CryoNet, or to my email address which is 


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