X-Message-Number: 7415
Date: 03 Jan 97 23:56:01 EST
From: "Kent, Saul" <>
Subject: SCI.CRYONICS Brain Vitrification

Bob Ettinger writes in Cryonet #7398: 
 
>1. WHY WAIT FOR VITRIFICATION?  
 
>According to relatively recent published and unpublished word
>from Greg Fahy--if I understand it correctly--rabbit kidneys (and
>perhaps other specimens) can already be preserved, using his
>methods, by vitrification, and stored at liquid nitrogen
>temperature without significant damage. The main problem 
>remaining is the RF rapid thawing required, which has also
>already been solved in theory but not yet in practice. 
>From the standpoint of near-future patients, why not use these
>vitrification methods (if applicable to brains)?  

A very good question indeed (although a bit puzzling given that
CI has until now promoted the Visser method as more promising  
than vitrification).  The suggestion of preserving cryonics
patients by vitrification instead of freezing is at least 15 years
old, having been written about by Mike Darwin, Jerry Leaf, Thomas
Donaldson and others since the first seminal papers by Greg Fahy
appeared in the early 1980s. The current state-of-the-art in
vitrification is perhaps best summarized in the technical library
of the Prometheus Project Web site at: 

http://www.prometheus-project.org/prometheus 

   One clarification that should be made is that while recovery
of vitrified organs from deep sub-zero temperatures is 
indeed theoretically possible with RF rewarming, substantial
technical problems may still have to be overcome.  For instance,
injury to the vascular endothelium of the kidney as a result of
_chilling injury_ (quite distinct from _freezing injury_) was a
major obstacle for some years to recovering kidneys which had
been loaded with a vitrifiable amount of cryoprotectant (i.e.,
VS41A) and cooled to, and rewarmed from, relatively high sub-zero
temperatures without vitrification or freezing.  Thus, it remains
to be seen if rates of rewarming of between 100 and 300 degrees C
per minute and the many associated problems, will pose additional
obstacles to recovering viable rabbit kidneys following 
vitrification.  
 
Ettinger continues: 
 
>The thawing
>problem (large licensed RF transmitters etc.) can be solved at
>leisure. We would really then HAVE brain cryopreservation that is
>reversible to a near-certainty.

   This exact approach has been the focus of 21st Century
Medicine's (21CM) and BioPreservation's (BPI) brain 
cryopreservation program for that past 4 years. Careful
readers of Cryonet will note that Mike Darwin has posted that 
such research has been ongoing, and has further stated that he is
cautiously optimistic "that BPI has perfused its last optimally
stabilized patient with glycerol."  In fact 21CM and BPI have
recently commissioned the construction of a sub-zero operating
theatre for the express purpose of deep sub-zero introduction  of
cryoprotectants into human cryopreservation patients with the
objective of vitrifying patients, instead of freezing them. 
(Construction of this room is now complete, and delivery is
awaiting an improvement in L.A. area weather conditions.)  
 
   The creation of a sub-zero perfusion capability for whole
humans raises a number of interesting issues which bear on
Ettinger's post, many of which cannot, regrettably, be discussed
in detail here.  However, a general discussion is possible and
falls under the heading of "Obstacles to application of Fahy's
vitrification solutions to human cryonics patients." 
 
Ettinger notes: 
 
>From the standpoint of public relations, of course there would
>be very limited favorable reaction to e.m. photos showing no
>discernible damage to the vitrified brains.
 
Unfortunately, brain vitrification is not a simple matter, for
direct toxicity to the central nervous system of vitrifiable 
cryoprotective solutions such as VS41A means that methods
developed for kidneys cannot not be expected to be immediately
translatable to the brain.  For instance, the application of
VS41A and a large number of other vitrification mixtures to
brains with subsequent EM examination has yielded results
inferior to those achieved by BPI with glycerol applied under
optimum conditions and in the proper concentrations.  Even
the Visser agent (dimethyl formamide) and other peralkylamides,
while more penetrating than glycerol, were found to be
intensely hemolytic (membrane toxic) at vitrifiable concentrations.
(Peralkylamides were also found to be poor cryoprotectants at
lower concentrations, including 25%, in a variety of relevant
systems.)

   Application of vitrification agents and technology to the
brain and especially to the whole organism with a high degree
of ultrastructural preservation is a complex proposition. 
Indeed, it is a proposition which has occupied 21CM
and BPI for years and is currently consuming nearly $50K per
month in resources (and has been for sometime). 
 
  To the extent that Greg Fahy has had success with VS41A (a
mixture of formamide, propylene glycol, DMSO, and a colloid), it
has been due to the technologically demanding and costly careful
control over the introduction and removal parameters for these
comparatively toxic agents.  It is necessary, for instance, to
terminate loading of rabbit kidneys with VS41A at nearly 30 
degrees below zero C!  But, beyond this, it is also necessary to
carefully control the shape of the cryoprotectant introduction
"ramp" and to carefully control (via computer) many other
parameters as well. 
 
   As 21CM's and BPI's work shows, failure to do this results in
severe membrane toxicity;  in other words the cryoprotectants
being used to achieve vitrification dissolve the cell membranes
and other lipid containing structures of the brain.  This injury
is quite apart from any damage inflicted on cellular metabolic
machinery and is of _primary_ concern to cryonicists, or should
be anyway, since cells without membranes are not cells anymore
and are unlikely to yield recovered cryopreservation (cryonics)
patients with any technology we can now foresee. 
 
   These observations notwithstanding, 21CM and BPI have made
what we believe to be very significant progress in the 
application of vitrification to whole brains and intact humans. 
Many problems with membrane toxicity, temperature control, the
need for sub-zero introduction of cryoprotectants, and the
development of novel cryoprotectants suited to achieving 
excellent ultrastructural preservation of the brain, have now
been largely overcome.  

   The associated technologies and cryoprotective agents are
proprietary, and patents are pending.  Application of this
technology to whole dogs has demonstrated the feasibility of its
use in human cryopreservation patients, and technological tooling
for application to humans is underway.  The sub-zero operating
room/perfusion capability is but one example of this-- there are
many more. 
 
Mike Darwin, Biopreservation, Inc.
Steven B. Harris, M.D., 21st Century Medicine
Brian Wowk, CryoCare Foundation


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