X-Message-Number: 1545
Date: 05 Jan 93 03:27:26 EST
From: Mike Darwin <>
Subject: CRYONICS Organ Prefusion Fluids & Brain Cryopreservation
> To: Edgar Swank
> From: Mike Darwin
> Re: Organ Perfusion Fluids
> Date: 4 January, 1992
I have been away on holiday and thus have been unable to reply to net
queries and communications sooner.
Neither DCS 200 or FC-75 are water soluble or water miscible. This is
an *advantage* because toxicity is minimized and the desirable physical
properties of the material are not likely to be affected by dilution with
water.
I do not know offhand what the vapor pressure of these agents are,
however, the point I wish to make is that these fluids are not supposed to
be used as intermediaries for helium or other gas perfusion but rather are
designed to *replace* said gas perfusion. The principal objective of gas
perfusion is to clear the circulatory system of fluid that will turn into
ice. These agents will not freeze or vitrify until very low subzero
temperatures and will not EXPAND when they do. Thus, they would serve to
substitute for gas perfusion.
The problem of large biomasses fracturing during cooling after
reaching the glass transition point is not one of "uneven" cooling.
Rather it is a complex property of weak glasses interacting with stresses
which occur during the cooling of inhomogenous materials below the glass
transition point of the solution which they are embedded in/comprised of.
To translate: Let's use a simple analogy that's easy to grasp. If
you have two dissimilar materials such as a sheet of steel and a sheet of
glass and you cool both of these materials very evenly and homogeneously
the fact still remains that steel contracts at a different rate than
glass. Indeed, the fact remains that almost ANY material will contract as
it is cooled. This is why we have expansion joints in bridges and other
large structures. If the steel and the glass cool they will contract at
different rates. This is not a problem providing they are not attached to
each other or are in some kind of frame or restraint which prevents
contraction during cooling. However, if we glue the glass to the steel
(leaving aside the extra set of problems introduced by the glue!) we now
have a situation wherein materials with different coefficients of
contraction and expansion are *bonded to each other*. As they try to
contract at different rates they will be unable to slide over each other.
Forces will begin to build up and at some point the material with the
lower tensile strength will break -- or fracture. Thus, the *eveness*
of cooling has little to do with the ultimate occurrence (or lack thereof)
of fractures.
Similarly, large masses of structurally weak glasses may accumulate
internal stresses during (even very slow) cooling which will subsequently
cause them to shatter. I have cooled 100 cc volumes of 60% (v/v) DMSO or
75% (v/v) glycerol very slowly to -190xC. Just lightly tapping the
container or warming it slightly by lifting it up out of cold vapor is
enough to cause "instantaneous" and massive fracturing of the solution.
With rapid cooling below the glass transition point (TG) you see
infrequent large fractures that occur at relatively high temperatures.
With very slow "differential free" cooling of bulk solutions you see
literally hundreds or thousands of smaller fractures. The same seems to
be true of biological material such as cats which are cooled either
rapidly or slowly.
As to the possibility of "voids" of water based perfusate remaining
in the circulatory system after perfusion with oils or fluorocarbons; I
can't really say. I have inadvertently administered DCS 200 intravenously
to a rabbit during intraperitoneal toxicity testing and I can tell you
that a small volume of this solution (ca. 5 cc) resulted in immediate
death of the animal from massive pulmonary embolism. Of course, that was
with the material being given into circulating blood rather than
introduced as the sole circulating fluid (where one would expect less
creation of blebs of oil that would act as emboli).
As to the issue of my remarks about using these agents to cool to
below -80xC I have the following to say:
From a cryobiological standpoint most of the events causing injury
which you are trying to avoid by using gas, silicone, or fluorocarbon
perfusion will have already occurred or been avoided. Here I refer to ice
formation and its subsequent effects. If you are going to freeze the
system it will almost certainly be frozen by -80xC and thus you will
either have reduced or eliminated ice mediated damage by gas perfusion or
you will have failed.
Gas or "inert" liquid perfusion below -80xC is not going to effect
this kind of damage one bit, and increasing the eveness of cooling to
below these temperatures isn't going to help you avoid fracturing.
Indeed, delaying fracturing until very low temperatures seems to result in
more numerous fractures which would seem to present a greater barrier to
subsequent repair.
As to the issue of rates of reaction, storage temperature and so on,
it is important to realize that the Arrhenius equation is predictive and
useful only in aqueous or gaseous systems where the two products can get
at each other and react. If an enzyme cannot reach its target substrate
because it is embedded in glass, then no reaction will occur. Also,
enzymes, unlike inorganic chemicals, are very complex and depend upon
their shape (among other things) i.e., stereospecificity, in order to be
able to engage in a reaction. Many enzymes have "energies of activation"
below which there is a sharp drop-off in their activity, if not their
complete inactivation.
What is the relevance of this to cryopreservation of biological
systems? The answer is simple: Below the glass transition point the
Arrhenius equation breaks down. When enzyme and substrate become
immobilized in glass, chemistry is, for all intents and purposes, brought
to a halt. This "theoretical" observation is confirmed in fact. Most
cells and tissues are stored not *in* liquid nitrogen, but rather in
liquid nitrogen *vapor* at about -135xC to -150xC. Higher temperatures,
such as -80xC have been tried, but do not work probably because many
enzymes are still somewhat active at this temperature and *much more
importantly, because a substantial fraction of the cell volume is still in
the liquid state.* It is very important to realize that biochemistry is
largely *diffusion driven* and if you stop the diffusion you stop the
chemistry. Several companies manufacture *mechanical* refrigerators which
take advantage of this fact and deliver a temperature of -135xC. These
freezers are advertised and successfully sold and used to store viable
biological systems such as sperm, embryos, heart valves, and so on at -
135xC. Queue Systems is one such company and a great deal of thought has
been given to using this kind of technology for storage of suspension
patients.
The crux of all this is that if you do not cool much below TG you
should not experience fractures. Furthermore, a large body of both
theoretical and practical evidence indicates that storage at temperatures
not far below TG should be biologically acceptable. TG for glycerol-water
systems is around -100xC. For DMSO water systems it is probably closer to
-130xC (I'd have to check to be sure). Based on my limited experience,
solutions tend to fracture about 10xC to 20xC below TG.
I have no ideas on the cost of a vacuum chamber to carry out the
experiment you outline.
I have no comment on the DMSO-trehalose idea because I don't know
what the result would be. I know that Greg Fahy did some experiments in
this area and found the results disappointing, but I do not recall the
specifics.
I have tried trehalose alone with my own RBCs and found a modest
increase in survival with slow cooling. This work and the work of Crowe
and Anchordougy was responsible for Alcor switching from mannitol to
sucrose as the impermanent osmotic agent in the base perfusate.
Conversations with membrane cryobiologist Tom Anchordougy indicated that
sucrose was almost as effective as trehalose, but it costs many times
less.
Keep in mind however that trehalose and these other agents cannot
protect against mechanical damage from ice. THAT is the major kind of
injury we appear to dealing with right now, and in any event it is
certainly a major factor. Trehalose and related sugars and amino acids
such as glycine provide membrane cryoprotection: they do not provide
colligative cryoprotection by significantly reducing ice formation. For
that we need a penetrating agent which alters the total amount of ice
formed and/or its location.
>To: Ben Best
>From: Mike Darwin
>Re: Brain cryoprotection
>Date: 4 January, 1992
We are in complete agreement: theorizing is THE BEST way to have your
research ask the right questions. Indeed, it is the essential first step
in any research: create a hypothesis. I did not mean my remarks to be in
any way disparaging of your comments or suggestions. I just wished to
point out that we need to have laboratory verification before we go out
and change human protocol.
I will ask Greg to comment on a nondehydrating protocol for
introducing 6M glycerol. I seem to recall that what was required was to
perfuse at 20xC or above.
Per the paragraph above I have the following information to relay:
the nondehydrating protocol consisted of starting glycerol perfusion at
normothermia and then cooling down to room temperature. He wasn't sure
how long was spent during cooldown or if the whole procedure was carried
out at room temperature or whether further cooling during glycerolization
was employed.
Alas, things are not as simple as they seem. Molecular weight is not
everything in terms of cryoprotectant penetrability. Charge, and other
properties are also critical. There also exist substantial variations
between species. Rat brains are almost completely dehydrated by agents
that appear to freely penetrate rabbit brains.
In fact, formamide is a significant candidate for causing the CNS
dehydration that Greg observed with VS. solutions. Also, formamide makes
a lousy CPA since it does not depress the freezing point very well and it
is poor at forming glasses (i.e., supporting vitrification). Greg
includes formamide in the mix largely to counteract the toxicity of the
DMSO. Given a choice it would be far better to replace the formamide mole
per mole with DMSO in terms of reducing mechanical damage from ice
formation.
"Greg sounds so awesomely confident in CRYOMSG 486 when he says "I
may or may not be able to work out a technique for vitrifying the brain,
but I can certainly reduce ice crystal growth enough to preclude
structural damage which would be good enough." I concur completely.
Furthermore, I think that that is an objective that we can achieve in this
laboratory as well. However, I would like to aim higher (i.e., for
substantial or complete preservation of viability) and for that I believe
Greg is essential. Greg has made a major commitment to work with us,
giving us as a minimum of at least 1/3rd of his time. We definitely have
the physical plant and personnel to support such an operation.
Why isn't implementing such a program the number ONE priority of
cryonics research? I don't know? You tell me! A major reason for my
dissatisfaction with Alcor was lack of attention to this area. I spoke up
more and more vociferously and was ignored. Indeed, several people have
actually accused me of being the reason the work hasn't been done.
Well, we now have an independent capability to do this kind of work,
indeed we have the ONLY capability that I know of. Now the question is,
will the cryonics community support it both in terms of dollars and other
efforts?
Keith Henson's inappropriate remarks discouraging involvement in
Greg's efforts, my efforts and the efforts of a growing number of others
to achieve this end is not the kind of response we need. I had hoped that
despite division in other areas, this whole community could get together
on the need to do this kind of work. To this end, I have tried to
minimize my participation in the many areas of contention.
A comprehensive approach to improved or perfected brain
cryopreservation will demand enormous amounts of financial and other
support. Fragmentation in this area will lead to FAILURE.
Greg has been working on the master research proposal for brain
cryopreservation for several months now. A careful program is being
mounted to present this proposal to the entire cryonics community in a
thoughtful and orderly way.
If you (Ben) or others are interested in knowing more about this
and/or helping, please feel free to call me at (909)824-2468 M-F after
3:00 P.M. PST.
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