From: Mike Darwin <75120.575@compuserve.com>
Subject: hyperbaria
Date: 03 Nov 1995

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Date: 02 Nov 95 12:55: EST
From: Mike Darwin <75120.575@compuserve.com>
Subject: SCI.CRYONICS:hyperbaria

VITRIFICATION:

Bob Ettinger responds to Ben Best's claim that Greg Fahy's current
vitrification approach does NOT require hyperbaria by saying that he is
unaware of this, if it is true.

I wish to confirm that Ben is *correct.*  Greg has not needed to use
hyperbaria for at least two years in order to recover viable kidneys
following loading and unloading with a 1 atmosphere vitrifiable
concentration of cryoprotectants.

This is a BRIEF and very compressed status report:

1) Rabbit kidneys have been loaded with a 1 atmosphere vitrifiable
solution, cooled to -30 C or -40 C (I think Greg recently went from -30 C
to -40 C, but am not sure), been reimplanted into the same animal, and
supported the animal as the SOLE kidney indefinitely. This work has been
repeated many times.

2) WHY then have not kidneys been vitrified since all that is required is
further cooling from -30 or -40 C to -135 C or thereabouts and, since no
ice will form, there will be no cause for added injury except for possible
thermal effects on membranes, proteins, or other cell components (known to
be minimal)?

3) The answer to #2 above is that it is easier to fall down a well than to
climb out of one.  While cooling to and vitrification at -135 C (no ice
formation) using a nontoxic 1 ATM vitrifiable solution can be done in
rabbit kidneys TODAY, what cannot be done is to rewarm them without
freezing.  During cooling, the system steadily loses energy. It also forms
microscopic and biologically insignificant amounts of ice which are
scattered through the organ.  These nidi or crystals are few, far between
and do not propagate during cooling over reasonable time intervals and are
inhibited completely by the glass transition of the solution (VS4 in this
case).

Unfortunately, rewarming at rates achieveable with traditional conductive
methods in something as big as whole kidneys results in the system
FREEZING.  The alternative is to rewarm very rapidly at a rate of 300-400
C/min.  This is theoretically possible using appropriate frequencies of RF
(*not* conventional microwaves as used in kitchen ovens) even for whole
human bodies.  It is certainly possible for masses the size of rabbit
kidneys because it has been done with a previous solution (VS2).

4) WHY THEN HAVE KIDNEYS NOT BEEN VITRIFIED?  The answer here is as simple
as it is perverse.  The expertise and equipment required to do this are
controlled by a researcher employed by the US FDA named Paul Ruggera.  For
the past 5 YEARS Dr. Ruggera and Dr. Fahy have been seeking to get FDA
permission to collaborate (their early collaboration was aborted by
bureacuratic red tape after initial experiments showed the feasability of
the system).  Just recently the CRATA allowing the collaboration was
approved.  However, by this time Greg had moved on to the Naval Medical
Research Facility and left the Red Cross.  The first CRATA between the FDA
and Red Cross took intervention by a Congressman to facilitate plus the
personal approval of FDA Commissioner David Kessler.

As I understand it, efforts are underway to get the approved CRATA
transferred to NAMRI.

5) WHY CAN'T SOMEONE ELSE FILL THE BILL FOR THE TECHNOLOGY RUGGERA IS
NEEDED FOR?

a) Patents on the technology.
b) This work requires extreme expertise and physical proximity of the
investigators.
c) Money; a radio station transmitter, complete with FCC license is
required to rewarm rabbit kidneys (and humans kidneys) fast enough to avoid
freezing upon rewarming.  These things don't come cheap.  Last I heard the
transmitter was sitting in a crate somewhere.  It has been in the crate for
YEARS.

Any Questions?

As you can see from the above, ice free and ultrastructurally injury-free
cryopreservation now exists for rabbit kidneys.  Greg has long ago
completed TEM studies showing tissue at fixed at -30 C in the liquid state
and after loading and unloading with VS4: there is no visible mechanical or
ultrastructural disruption and the tissue at this temperature is virtually
undistinguishable from control (there are some changes to intracellular
structures which appear to be fully reversible on rewarming).  By way of
example, our dogs, when cooled to 4 C lose all the intra-axonal
microtubule structure.  It just falls apart upon simple cooling.  It also
snaps back together via self-assembly upon rewarming.  The dogs seem to be
no worse for wear.

Bob asks about the speed of recrystalization.  It is fast.  So fast that it
is one of the major barriers to recovery of slowly cooled cells and tissues
in the presence of modest amounts of cryoprotectant.  What constitutes slow
cooling?  Temperature descents slower than 0.5 to 1.0 C/min.  The fastest
you could reasonably cool a humam brain would be about (5-10 C an hour).
The rapid loss of viability of sperm frozen with low concentrations of
cryoprotectant to dry ice temperature is due almost exclusively to
recrystalization.

It has for over a decade been a working assumption that humans cooled at
the slow rates their bulk and the laws of thermodynamics require will be
fully or near fully recrystallized by the time they reach -79 C, with our
without cryoprotectant.

Modest concentrations of colligative cryoprotectants slow the rate of
recrystalization but do not stop it or inhibit it sufficiently over the
time scales required for cooling large masses of tissues to stable
temperatures.  Thermohysteriesis proteins do not seem to be a viable
solution to this problem either, much to the chagrin of ice cream
manufactuers who face the same problem at -20 C with loss of "mouth feel",
loss of texture, and the development of "gooiness" in ice cream products
stored over time or rewarmed a little in transit, especially cheaper brands
like ice-milks which have minimal concentrations of fat and sucrose
(sucrose is a good glass former and inhibitor of recrystalization) and are
very susceptible to recrystalization.

Mike Darwin