X-Message-Number: 4879
Date: Sun, 17 Sep 1995 07:17:37 -0700 (PDT)
From: Doug Skrecky <>
Subject: desiccation as cryonic insurance
DESSICATION AS CRYONIC INSURANCE
(From Longevity Report 38 April 1993)
By Doug Skrecky
(Note: The British spell the word "desiccation" as "dessication".)
If financial concerns preclude continued liquid nitrogen suspension
at some point in the future it would be helpful to have a backup plan in
place ahead of time so as to prevent the destruction of the cryonics
"patient". The only alternative backup technique capable of long term
preservation is complete desiccation. This must be achieved with a
minimum of damage to cryopreserved tissues in order to keep reanimation
chances alive, yet be inexpensive enough to be feasible on a limited
budget.
Freezing requires cryoprotectants to preserve tissue. Similarly
desiccation requires anhydroprotectants to preserve tissue. Fortunately
there do exist two cryoprotectants which can also serve as
anhydroprotectants. One of these is the sugar trehalose. Recently
trehalose has been found to outperform all other cryoprotectants in its
ability to enable 80% of frozen organisms (tardigrades) to revive. *1 No
other tested cryoprotectant comes even close to this performance. Thus
trehalose would appear set to replace all other cryoprotectants, except
for one significant defect. It is very, very expensive. Fortunately the
other dual cryoprotectant/anhydroprotectant is sucrose or common table
sugar. So as a starting point we will assume that a large amount of
sucrose in used in the cryo-perfusiate so that desiccation without
massive tissue destruction would then be a possibility.
Desiccation requires that all water be removed from tissue. This
could be achieved by thawing the patient, embalming and then air drying
at room temperature at a negligible expence. Unfortunately the chances
for reanimation would then also appear to be negligible. Vaccuum freeze
drying could be used instead, but then the cost would be prohibitive.
Solvents such as methanol or acetone have been used to thaw small frozen
tissue blocks at -80 C with good results. *2 *3 Although at room
temperature these solvents dissolve lipids and fix proteins, at low
temperatures these effects are largely prevented, although some DNA
denaturation still occurs. Unfortunately when larger tissue blocks are
used, which require longer substitution times tissue deterioration does
occur, which requires fixatives to prevent. *4 DMSO/water mixtures have
preserved tissue quite well for an extended period at low temperatures,
but have virtually no ability to extract moisture from tissue. *5
Dimethyformamide has looked promising in a preliminary experiment, but
this solvent has a high boiling point, so it would be of little use as
ALL solvents must be removed before tissue could be said to be truly
desiccated and inert. *6 Tetramethylsilane is an inert liquid, has a low
.3 C boiling point and it has recently been proved to be superior to
acetone as a substitution medium. *7 However the best medium overall may
well be any dry inert gas.
A vaccuum increases drying rates because it (like solvents)
eliminates the barrier to diffusion that meniscus effects create during
air drying. However diffusion is only a problem with large blocks of
tissue. *8 By using the cardiovascular system as a drying surface the
problem of diffusion through tissue is avoided and simultaneously the
total surface area available for sublimation is increased by over 300
times. Internal desiccation with an inert gas would have the same result
as vaccuum freeze drying, but at a tiny fraction of the price. Of the
inert gases desiccation has been found to proceed most rapidly with the
lightest -helium.
The patient could be prepared for such a possible future internal
desiccation by removing all of the liquid cryoperfusiate from the
cardiovascular system immediately prior to cryonic suspension. If this
system is intact infusion with a mildly pressurised inert gas would
force out the any fluids and simultaneously prevent collapse of veins
and arteries. If the cardiovascular system is not intact any nontoxic
liquid with a low freezing point could be used to replace the
cryoperfusiate. The tissue could then be frozen at a temperature which
is still above the freezing point of this liquid, which would then be
drained off before nitrogen suspension itself. What about those
already frozen without anhydroprotectants? Could these patients also be
desiccated in the future without massive tissue damage? The answer to
this will require some further research, but the provisional answer
appears to be yes. Provided the patient is first thawed at close to
freezing temperatures and immediately perfused with a sugar based
transplantation solution only a modest amount of deterioration should
occur. Then the body could be refrozen before drying or even desiccated
without further freezing (or freezing damage). Drying at close to but
above freezing temperatures occurs more rapidly since liquid water can
evaporate much faster than ice can sublimate. *9
*1 "Survival of the Cryptobiotic Eutarigrade Adorybiotus Coronifer During
Cooling to -196 C:Effect of Cooling Rate, Trehalose Level and Short-term
Acclimation" 125-130 Vol.29 1992 Cryobiology
*2 "Freeze-Substitution Without Aldehyde or Osmium Fixatives:
Ultrastructure and Implications for Immunocytochemistry" 355-363 Vol.158
1990 Journal of Microscopy
*3 "Immunoelectron Microscopy of Tissues Processed by Rapid Freezing and
Freeze-substitution Fixation Without Chemical Fixatives: Application to
Catalase in Rat Liver Hepatocytes" 617-623 Vol.38 No.5 1990
*4 "Freeze-substitution Techniques for Preparing Nematodes for Scanning
Electron Microscopy" 187-196 Vol.164 1991 Journal of Microscopy
*5 "Effects of Electrolyte Composition and pH on the Structure and
Function of Smooth Muscle Cooled to -79 C in Unfrozen Media" 82-100 Vol.9
1972 Cryobiology
*6 "Improved Cryoprotection and Freeze-substitution of Embryonic Quail
Retina: A TEM Study on Ultrastructural Preservation" 348-356 Vol.1 1990
Journal of Electron Microscopy Technique
*7 "An Evaluation of the Usefulness of Air-Drying Biological Samples From
Tetramethysilane in Preparation for Scanning Electron Microscopy" 198-202
Vol.40 1991 Journal of Electron Microscopy
*8 "Freeze Drying Without Vaccuum: A Preliminary Investigation 94-96
January 1962 Food Technology
*9 "Controlled Low-Temperature Vaccuum Dehydration- A New Approach for
Low-Temperature and Low Pressure Food Drying" 1573-1579 Vol.54 No.6 1989
Journal of Food Science
.....I now find a variety of faults in the above article. Perhaps from
the standpoint of cryonicists the most serious deficiency is my
implication that trehalose is a superior cryoprotectant. Unfortunately
although excellent results have been obtained with tardigrades this does
imply that similar results would be obtained with human tissue treated
with trehalose. The difference is that tardigrade cell membranes have an
active transport mechanism for ferrying sugars from the inside to the
outside of cells. In human cells trehalose and sucrose are almost
completely impermeable to the cell membranes. There do however exist
other anhydroprotectants that are slightly permeable such as mannitol. I
will have something more to say about this later....
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