X-Message-Number: 3898
Date: 23 Feb 95 20:50:34 EST
From: Mike Darwin <>
Subject: SCI.CRYONICS -79xC and glycerol Molarity

Mike Price asks about glycerol molarity currently in use with human

cryopreservation patients.  This varies from group to group: my understanding is
that Alcor is using about 7+M glycerol.  BPI has used that range (between 7-8)
at BPI in the past. CI seem to be getting terminal concentrations in the 5-6M
range in the brain based on their sheep work

Several caveats apply: not all patients will reach target glycerol
concentrations due to case conditions beyond the organization's control such as
ischemic time, cause of death, etc..

Generally the higher the molarity of glycerol used the more stable at dry ice
temperature the cells are.  For instance red cells, which while not nucleated,
still have metabolic machinery which must function if they are to be
transfusable, can tolerate extended storage at -79xC.  In fact, if the freezer
fails and they warm up to say -20xC all you do is cool them down again.  The
difference here is that such high concentrations of glycerol make
recrystalization irrelevant whereas in systems treated with low concentrations
of CPA (5-15%; i.e., less tham 1M) and cooled at the usual 1xC/min. this is a
significant cause of injury at -79xC.


Sperm stored at -79xC rapidly lose motility when treated with low concentrations
of agent, however if treated with higher concentrations they behave much like
red cells in my experience. CPA concentration  (and thus the degree of

colligative cryoprotection) is critical to the stability of the system at higher
temperatures.

Having said all this, clearly, the sooner a patient is cooled to storage
temperature, the better.  However, keep in mind that a human masses more than a
hamster by a lot and simply cooling to -79xC will take a day or nearly so even
for neuro patients.  Whole body patients can take 36-48 hours or longer
depending upon their weight and the amount of fat (which serves as an excellent
insulator).

Function is often rapidly lost at high subzero temperatures for relatively
"simple" reasons such as ion leakage (which continues to occur when the system
is in the liquid state, as it is at -79xC; at least where the cells are
concerned) and from membrane changes such as the lamellar to hex II phase
transition which will occur with increasing probability in the presence of high
CPA concentrations and/or low temperatures above the solidification point for
the membrane/aqueous system of water-cryoprotectant.  These membrane changes
cause the membrane to lose its normally "fluid" lamellar (sheet-like) form and

develop a tubular or crystal-like structure, essentially creating a hole or pore
in the cell which results in ion leakage and/or inability to pump ions.  Ion
sensitive cells like cardiac muscle would be expecially vulnerable to this.

Of course, there is also the issue of cryoprotectant toxicity which is also
temperature dependent.  Red cells are not nucleated mammalian cells and it
should be noted that we (BPI, Alcor, and CI) are currently perfusing (to the
best of my knowledge) patients to terminal concentrations of glycerol which
result in loss of cell viability or organ function *during the course of
perfusion*.  For instance, to my knowledge, David Pegg holds the record for
glycerolizing and deglycerolizing kidneys and he found 3M glycerol to be the
maximum tolerated in the system he used.

BPI is just now completing some ultrastructural studies of storage for one year
at -80xC and we should be able to soon add some information on structural
stability and preservationn vs functional preservation  at these temperatures
the latter of which, given most current cryonics protocols, is lost during
perfusion in any event.


One thing we are certain of:  cracking does not occur at all in 7M+ glycerolized
dogs cooled to -90xC even if the animals are mechanicall stressed across two
rigid supports by loading with over 20 kilos of dry ice at -90xC and, perhaps
more interestingly, even if they are plunged from -90xC to into a fluid bath
with a temperature of 0xC!  In fact, we see excellent reperfusion of virtually
every organ system (including the skin) with carbon particle loaded fixative.
We use carbon particles as opposed to dye because dye diffuses and carbon does
not: it stays where it goes.  Thus the only areas we see turn black are areas

that are perfused at the *capillary* level.  This is further verified by looking
at the carbon black particles in the capillaries with both light and electron
microscopy. Reperfusion failure occurs where glycerol has been diluted by
stomach contents, bladder contents or left ventricle contents (which have low
CPA concentrations).


  The brain turns charcoal black/gray as do all the other tissues.  This is true
  even after 1 year of storage at -80 to -90xC.

Suda stored at -20xC and could get crummy EEGs back after 7 years.  He also did
carbon reperfusion of brains using a variety of techniques and his results were
similar to ours: high concentrations of glycerol preserved capillary integrity
whereas PVP and DMSO in low concentrations did not.  In fact, I have a very
lovely slide from Dr.Suda showing consecutive frames from a motion picture
camera of carbon reperfusion using the three methods outlined above.  The cat
brains that demonstrated EEG activity perfused uniformly (tuned completely

black); the others did not.  Further, brains treated with 15% (v/v) glycerol and
cooled to -79xC also failed to reperfuse well with carbon (a finding we have
verified) and also showed no EEGs althought they did demonstrate some unit
activity (i.e., unorganized cellular electrical activity).

Some people in the cryonics community (I believe Segall, in fact) have stated
that they do "not believe Suda's results, at least with regard to EEGs peerhaps
implying his work was fraudulent or poorly done.  I have see extensive

documentation of Suda's work (which went far beyond what he published) and I for
one am convinced that his work was real and his results meaningful.

I have sent copies of pictures of our carbon reperfusion work to Bob Ettinger.
We should have EMs from these animals in a few weeks, as well as EMs from some
of our vitrification studies.  Unfortunately, these results will have to await
formal write-up for publication so there is no misunderstanding about what
conditions they were obtained under and what they mean.

Mike Darwin


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