X-Message-Number: 3150
Date: 16 Sep 94 18:56:05 EDT
From: Mike Darwin <>
Subject: SCI.CRYONICS why mammals

I recently received the following message via private e-mail.  I thought the
questions were good ones and also thought others might be interested in my
answer (ans perhaps wish to elaborate upon it).  I have posted the e-mail query

I received minus the sender's name.  The sender is, of course, not only free to,
but encouraged to claim credit for his/her post. I have deleted any identifying
references from the sender's text but otherwise left it unedited.


>Mike I read your paper on your cat experiments. What I wonder is why start
>of with mammals.  Would it not be more informative to first freez amphibians
>that are over-Wintering at -30C (under controled conditions) to -196C. Then
>raise the temperature back to -30C and then wait for Spring or thawing under
>controled conditions. Then observe what damage occured to these animals that
>has the natural ability survive at low temperatures.
>     I do not do research anymore. I am teaching High School science. If
>this research on amphibians has not been done I might manage to do it.
>First I need to know if research has been done with amphibians. I know
>Thomas Donaldson implied that it was done. Yet I can trace no record of it.
>I really feel it is imperative to successfully thaw these animals in order
>to understand the effect of LN temperature on other animals.

The questions you raise are good ones.  Unfortunately,  the answers are not
simple one to give, and in order to give them I will have to subject you to a
bit of a tutorial about basic cryobiology.  I hope you will not be insulted by
this. I have taken the liberty of posting your questions (minus your name) and
my answer to Cryonet and SCI.CRYONICS because I feel it would be worthwhile to
others.

At the outset, I should point out that we know a fair bit more about the
mechanisms of cryoinjury and cryoprotection than many people in the cryonics

community have any inkling of. Indeed, if they had more of an inkling they would
probably be considerably less enthused about cryonics patients' prospects...

I am greatly simplyfying here, but fundamentally there are three major
mechanisms of cryoprotection: colligative cryoprotection, thermohysteresis
compounds, and membrane cryoprotection.  Colligative cryoprotection consists of
the use of so-called anti-freeze chemicals to not only depress the freezing
point but to limit the total amount of ice formed.  In overwintering animals
what happens is that some of the water in the animals' bodies is converted into
ice (pure water crystals) while the rest of the water is kept in a liquid state
as a fairly concentrated solution of water-cryoprotectant.  In the case of
mammalian cells which are cryopreserved, the increase in the anti-freeze
concentration versus the decrease in the temperature has to be tightly
controlled to avoid toxicity and to avoid intracellular freezing: thus the
critical importance of "freezing" rate or more accurately, cooling rate in
freezing cells to cryogenic temperatures.


In the case of cells viably cooled to -196xC the end-result is that the unfrozen
portion of the system is no longer a liquid (as with our overwintering friends)

but rather a glassy solid which has a more-or-less amorphous molecular structure
(we call this state "vitreous").

Now, for individual cells all that is required is that their interiors be ice
free (vitreous) and that they not be poisoned by exposure to too high a
concentration of cryoprotectant at too high a temperature.  What this means in
practice is that you can turn a lot of  the system into ice and still have the
cells survive.  And, if all you are interested in is cells surviving (as in a
tissue culture) that is all well and good.  A modest amount of cryoprotectant

(5-15% glycerol or DMSO) will usually work, combined with a fairly rapid cooling
rate (say 1xC per minute).


However, tissues, organs and organisms are organized at a higher level than that
of individual cells.  They are complex *arrangements* of cells and the
relationship of the cells to each other and to noncellular structures (like
basement membrane to which capillary endothelial cells are attached) is

critical.  So, it is not sufficient to just have the cells survive freezing, the

cells' relationships to each other and to structures like basement membrane must
also survive.

When I was in the 5th grade I began freezing red eared slider turtles.  I found
that they could tolerate exactly 30 minutes wrapped in aluminum foil in a -20xC

freezer.  They would recover just fine after such exposure.  Later, I learned of
Audrey Smith's work with golden hamsters and found out that they too could
tolerate freezing, again with certain limits.  Smith's hamsters had a maximum
tolerable limit of % of body water converted into ice of roughly 50%.  When I

did calorimetery on my turtles for my Science Fair project I found that they too
could only take about 50% of their body water being converted into ice.

Smith also reckoned that because the head was thinner in cross-section than the

body it would freeze more completely in the time alloted and she calculated that
roughly 60% of the water in her hamsters' brains was converted into ice.  While
I never did any calculations (and turtles retract their heads into their shell
upon cooling) I would guess that my turtles also tolerated about 60% of their
brain water converted into ice.

While I was in grade school working on my Science Fair project  I also worked
with the pupae of an insect called the Cecropia moth.  I lived in Indiana and
these moths have a large pupae (about an inch long by 1/2" in diameter and very
exoctic looking) which overwinters at temperatures at least down to -14xC.  I
collected a number of these moths and frozen them to -20xC for months at a time
and found they could survive it.  They d not survive cooling to dry ice (an

experiment which I tried) although interestingly they are not immediately killed
by it either; they just fail to go onto the next stage of development and die a
short while after freezing.  When I did calorimetery on these insects I found
they could tolerate about 60% of their body water being frozen. (I became an
expert at finding their papery gray-brown cocoons  on tree branches (which are
about 3 inches long, but devilishly difficult to spot).

When Ken Storey and others began investigating overwintering vertebrates they
discovered that much like the other examples I cite these creatures limited the
amount of ice formed to the range of 50-60% (although I think the tree frogs
could take more).  Attempts at cooling these animals to lower temperatures were

fatal because too much ice was formed (and probably also ice in the wrong places
as well).  This was probably lethal for two reasons at a minimum:

1) Exceeding the 50-60% mark probably results in mechanical injury to the

animal's tissues at the histological (cell-to-cell) level.  Cells are torn apart
from each other, tears occur in tissue, cells are stripped away from basement
membrane, etc.

2) As the v/v % of ice formed increases so does the concentration of

cryoprotectant. (Remember, ice will freeze out as *pure* water.)  Cryoprotectant
is not innocuous stuff: even glucose and glycerol are toxic if they are present

at a high enough concentration at a high enough temperature.  So, in addition to
mechanical injury to tissues from ice you also have the probability of chemical
injury from the cryoprotectant

3) Per #2 above the chemical injury can take several possible forms.  Simple
dehydration (shrinkage) of the cells is associated with injury for reasons not
fully appreciated.  The degree of shrinkage tolerated can be quite nicely

predicted for most cells with a mathematical tool known as the  Boyle-Van't Hoff
equation.  (I will give some references at the end here which can serve as
foundation for better understanding these effects.)  I am greatly simplifying
here, but suffice it to say that generally there is a "minimum volume" beyond
which cells cannot generally be reduced (shrunken) without injury.  Cooling to
below the protective temperature conferred by colligative cryoprotectants
(anti-freeze compounds) results in excess cell shrinkage and damage.  And, as I

mentioned previously,  forming added ice means a higher overall concentration of
cryoprotectant.  Beyond its impact on cell volume reduction, the increased
cryoprotectant concentration may also cause injury due to biochemical effects:
it may change the shape of key enzymes or dissolve or distort key proteins or
lipids, rendering the cells nonfunctional.

So, where does this leave us in regard to your questions?  The simple answer is
probably that no one has tried to cool these vertebrates to liquid nitrogen
temperature.  The *reason* is that attempts to cool them to higher subzero
temperatures have failed, for the reasons outlined above.

As to why we in the cryonics community are working with mammals rather than
overwintering vertebrates, again,  the answers are complex:

1) We are mammals and the problems we encounter will necessarily be different
than those of overwintering vertebrates; for one reason because we must cool to
much lower subzero temperatures and either a) convert more of ourselves into
ice, or b) use more colligative cryoprotectant.

2) We understand broadly how freezing injures and we have one viable path to
avoiding this injury while still achieving the thermodynamic benefits of
ultracold temperatures: we can vitrify the whole system and simply avoid
freezing by taking colligative cryoprotection to its logical conclusion: don't

form any ice at all by using a high enough concentration of antifreeze.  This is
a bit of an art since such high concentrations of antifreeze can be toxic.

However, it has been achieved for a wide range of tissues and embryos and it has
been achieved for organ slices such those from the rabbit kidney and liver.

3) All of this is not to say that we should not pay close attention to what
these overwintering vertebrates are doing.  From the preliminary data of
Rubinsky (using MRI) and from the work of Ken Storey and others it seems likely
that these creatures use techniques to control *where* ice forms which might
well be applicable to human cryopreservation.

Finally, you mention a willingness to do experiments.  That is VERY good and
there are some very simple experiments I can suggest to you involving
vertebrates (goldfish) that are inexpensive and might answer some important
questions.  For instance, goldfish  can tolerate equilibration with 7% DMSO at

about 17xC.  I know this because I've done it as a kid.  However, I have not had
the time to set up careful experiments to freeze animals so treated and do lots
of related studies that would be of interest.

I hope I have answered your questions to your satisfaction.

A brief bibliography on relevant aspects of cryobiology follows:

Perhaps the best overall introduction and tutorial to what we know about
cryoinjury and cryoprotection is Peter Mazur's:

Freezing of living cells: mechanisms and implications. Am. J. Physiol. 247(Cell
Physiol.. 16):  C-125-C142,  1984.

Mazur, P. Kinetics of water loss from cells at subzero temperatures and the
liklihood of intracellular freezing. J. Gen. Physiol. 47;347:1963.

Mansoori, G.A. Kinetics of water loss from cells at subzero temperatures.
Cryobiology 12;34-45:1975.

Fahy, G.M. Simplified calculation of cell water content during freezing and
thawing in nonideal solutions of cryoprotective agents and its possible
application to the study of solution effects injury. Cryobiology
18;473-482:1981.


Meryman, H.T. Cryopreservation of blood cells and other tissues: current status.
Hematologia  15;337-350:1982.


Fahy, G.M.  Analysis of "solution effects" injury: rabbit renal cortex frozen in
the presence of dimethylsulfoxide.  Cryobiology 17;371-388:1980.

Fahy, G.M. Vitrification: a new approach to organ cryopreservation in
Transplantation Approaches to Graft Rejection, H.T. Meryman, Ed., Alan R. Liss,
Inc, 1986.

Fahy, G.M. The relevance of cryoprotectant "toxicity" to cryobiology.
Cryobiology 23;1-13:1986.


Fahy, G.M., et al Cryoprotectant toxicity and cryoprotectant toxicity reduction:
in search of molecular mechanisms. Cryobiology  27;247-268:1990.

An excellent lay-level introduction to cryoprotection by thermohysterisis
(glycoprotein anti-freeze compounds) is:

Eastman, J.T., and De Vries, A.L., Antarctic Fishes.  American  Scientific
American (I do not have the cite for this article as SA did not put dates or
volume #'s on their pages at that time!)

Good introductions to overwintering mechanisms in vertebrates have been posted
to Cryonet by Ken Storey.  A very brief precis which I highly recomment is:

Storey, K.B., and Storey, J.M.  Frozen and Alive.  Scientific American,
December, 1990.

Storey, K.B. Life in a frozen state: adaptive strategies for natural
freeze-tolerance in amphibians and reptiles. Am. J. Physiol. 258 (#3, Part
II);R559-568:1990.

Mike Darwin

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