From Cryonics, March 1988
The Cryobiological Case For Cryonics
[Note: This article was written in 1988. It summarizes much indirect evidence that supported the practice of cryonics at that time. More direct evidence of the efficacy of freezing and vitrification for preserving brain structure is now available. It should be noted that the hypothesis that ice formation of less 60% at cryogenic temperatures is compatible with recovery of neurological function ("Smith Criterion") is now known to be incorrect.]
Any casual newspaper reader will have decided quite confidently by now
that cryonics has no chance whatever of success, due to the systematic
misinformation contained in all media coverage of this subject to date.
Not only has the scientific evidence supportive supportive of cryonics
not been presented, but the unchallenged, supposedly scientific criticisms
of cryonics presented in the media have been as harsh as they have been
vapid and without merit. In reality, it seems that no supposedly scientific
criticism of cryonics has ever addressed the real issues involved or ever
been based on a grasp of them. The purpose of this discussion is to provide
a summary of the extensive cryobiological evidence which exists to support
cryonicists' premise that existing freezing techniques preserve the molecular
basis of human memory and personality and thus offer a reasonable chance
of allowing future restoration of cryonics patients to life.
Why has this evidence not been presented previously? The reasons are
largely political. Also it should be appreciated that even a neutral position
with respect to the emotionally charged subject of cryonics is hazardous
for a cryobiologist because of hardened opposition on the part of many
key scientists who control job availability and grant support. This opposition
is generally based on a gut reaction and/or philosophical objections that
do not invite further consideration. Unfortunately, almost no one ever
seriously asks whether anything as seemingly outrageous as cryonics could
have any compelling scientific foundation, despite the fact that it does.
The problem is that the relevant scientific facts are far from obvious
or readily available, and that no well-established scientist has ever
dared or even been able to enunciate them.
The result has been the suppression of discussion, the creation of anxiety,
the propagation of gross misinformation among the general public, and
the censorship of valid scientific observations: in short, the antithesis
of what science is supposed to be all about. It is time to consider the
scientific facts and to show that what is really outrageous is not cryonics
but the notion that there is no scientific basis for cryonics or that
cryonics cannot possibly work.
A. Premises and their scientific evaluation
What are the cryobiological issues? Another way of asking this question
is: What is the minimum cryobiological requirement for "success"
with the cryonics endeavor? Since the one indispensable goal of cryonics
is restoration of the brain, we can limit our attention to the cryobiological
requirements for the achievement of this goal. Questions concerning maintenance
of the brain after restoration are not cryobiological and can therefore
be neglected here.
What then would be required for the brain to be restorable? First, the
brain must be preserved well enough to repair, i.e., it must be possible
today to preserve with some reasonable fidelity the basic biological components
of the brains of humans shortly after clinical death. Second, repair technology
must be available to carry out any repairs required.
The two indispensable premises of cryonics, then, are reasonable brain
preservation and the development of advanced molecular scale (nanotechnological)
biological repair devices. Both premises are fully open to scientific
scrutiny and falsification by experiment or calculation and, in fact,
both seem at present to withstand such scrutiny, as the experimental evidence
which is presented in this paper as well as the work of others on the
problems of biological repair (see K. Eric Drexler's book, "Engines
of Creation," and his technical papers) should show. If both premises
are valid (assuming cryonic suspension is done under reasonable conditions
and nonscientific problems do not intervene), then in principle cryonics
should work to at least some extent.
As noted above, this article is about the cryobiological basis of cryonics
rather than the cell repair aspect. But because the cryobiological premise
of cryonics loses significance without the nanotechnological premise of
cryonics, it is necessary to comment at least briefly on nanotechnology
in order to clarify the relevance of the evidence to be presented about
cryobiology. There appear to be no significant flaws in K. Eric Drexler's
concepts of molecular scale cell repair devices, and this judgment is
supported by the absence of even a single significant and coherent objection
to his concepts. The concepts involved are powerful enough to make it
easy to imagine the technology not only for repairing the fine structure
of the brain but also the technology for transplanting a brain into a
new body. It seems not only possible but inevitable that such technologies
will be developed. A person waiting in liquid nitrogen should remain changeless
for centuries if need be while such developments proceed.
B. Summary of general conclusions
It can be stated quite firmly that cell bodies, cell membranes, synapses,
mitochondria, general axon and dendrite patterns, metabolites such as
neurotransmitters, chemical constituents such as proteins and nucleic
acids, and general brain architecture are preserved reasonably well or
excellently with current techniques. The brain can withstand severe mechanical
distortion by ice without impairment of subsequent cognition, and a glycerol
concentration of less than 4M-a concentration achieved in current cryonics
procedures--can be shown to limit ice formation to quantities currently
thought to be consistent with good functional recovery of the intact brain.
Information is lacking about the ultrastructure of frozen-thawed brains,
but much can be inferred from the customary observation of a high level
of functional recovery of frozen-thawed brains, brain tissue, or brain
cells which depends on a high degree of both local and long-range ultrastructural
integrity. Absolute proof is lacking about the quality of preservation
in each and every brain region, since not all brain regions have been
examined by neurobiologists to date. However, in the experience of those
who have histologically examined entire cross-sections through the frozen-thawed
brain at many different levels, no clear differences in preservation quality
from one brain region to another have ever been apparent.
A reasonable way of summarizing the world literature on this subject at
present is to say that wherever either brain structure or brain function
has been evaluated after freezing to low temperatures and thawing, robust
preservation has almost always been demonstrable provided at least some
minimal attention was paid to providing cryoprotection, and in some cases
good preservation has been documented in the complete absence of reasonable
cryobiological technique. The implication of these findings is that structures
and functions not examined to date will also respond in a favorable way
to freezing and thawing.
C. Detailed review of relevant current cryobiological
1. General Cryobiological Background
Freezing is not a process of total destruction. It is well known that
human embryos, sperm, skin, bone, red and white blood cells, bone marrow,
and tissues such as parathyroid tissue survive deep freezing and thawing,
and the same is true for systems of animal origin. In 1980 a table was
published listing three dozen mammalian organized tissues and even a few
mammalian organs which had been shown to survive cooling to low temperatures
(1), and this list could now be expanded due to additional experiments
on other systems. Such survival could not occur if the molecules comprising
biological systems were generally altered by freezing and thawing. In
general, freezing does not cause chemical changes or protein denaturation
Contrary to popular imagination, cells do not burst as a result of intracellular
freezing. The expansion of water as it is converted to ice causes less
than a 10% increase in volume, whereas cells can withstand far larger
increases in volume, e.g., 50-100% increases. The primary misconception
here is the idea that ice forms in cells at all under ordinary conditions
of slow freezing: it does not. Instead, ice forms between cells, and water
actually travels from the interior of the cell to the ice outside the
cell, causing shrinkage rather than expansion of the cell.
Cell death during slow freezing may be related to changes in the cell
membrane produced by cell shrinkage, or to toxicity of cryoprotectants
as they are progressively concentrated as a consequence of the formation
of pure ice in initially dilute solutions. Both of these putative causes
of death are relatively mild on the molecular level and are certainly
not irreversible in principle. But whatever the cause of death, cells
examined in the frozen state appear to be structurally intact even when
they are known to be nonviable upon thawing (with very few exceptions
on the part of non-mammalian systems not relevant to the brain). This
is true both for single plant and animal cells and for cells that comprise
animal tissue. Hence, lack of functional recovery after thawing is not
proof of lack of structural preservation in the frozen state before thawing,
and it is the latter that is relevant to cryonics.
A truism of cryobiology is that different types of cells require different
protocols of cryoprotectant treatment, cooling and warming rates, and
cryoprotectant washout in order to exhibit maximal survival. All of these
differences can be minimized greatly by using high concentrations of cryoprotectant,
provided such concentrations can be tolerated. Nevertheless, other than
a few generalizations such as those described above, it is difficult to
extrapolate from one biological system to another in terms of predicting
the details of its cryobiological behavior.
For this reason, if we wish to understand what happens to the brain when
it is frozen, we can't argue on the basis of results obtained with kidneys
or plant cells or embryos or granulocytes, but must, instead, focus specifically
on the brain. Herein lies one of the largest errors cryobiologists and
other scientists have made in dismissing the prospects for cryonics: the
making of sweeping negative statements without sufficient knowledge about
the cryobiology of the brain (or, for that matter, the primacy of the
brain or the concepts of nanotechnology).
In order to examine the scientific evidence bearing on the only indispensable
cryobiological premise of cryonics, then, the balance of this article
will be devoted to an extensive review of the contents of a large number
of scientific papers on the freezing of brains, brain tissue, and/or brain
cells. As extensive as the following remarks are, it should be understood
that they are not exhaustive. No attempt has been made to obtain the complete
scientific literature describing the state of brains after freezing in
ways which are relevant to the issue of cryonics. This review simply reflects
the relevant information currently at hand.
2. Living Adult Animal Brains
Dr. Robert J. White, chairman of the Dept. of Neurology at Case Western
Reserve University's School of Medicine, has favorably discussed the prospects
for the eventual successful cryopreservation of human brains (2,3,4).
(Dr. White is also an expert on cephalic transplantation and hypothermic
brain preservation, and has published several scientific papers on these
subjects.) However, it is clearly impossible to experiment with entire
living human brains, so the closest we can come to evaluating the degree
of total brain preservation achieved in best-case cryonics procedures
is to review the results of freezing the brains of animals.
The earliest observations of this sort were made by Lovelock and Smith
(5,6) in 1956. These investigators froze golden hamsters to colonic temperatures
between -0.5 degrees C and -1 degrees C and quantitated the amount of
ice formed in the brain, allowing them to determine how much ice formed
in the brains of animals which made full neurological recoveries. They
determined that at least 60% of the water in the brain could be converted
into ice without damaging the ability of the hamsters to regain normal
behavior after thawing. Considerably more ice was consistent with restoration
of breathing, a complex neural function. However, the exact quantity of
ice (above 60%) consistent with full neurological recovery could not be
clearly determined, because of death due to intestinal, pulmonary, and
renal bleeding. Nevertheless, tolerance of at least 60% ice by the brain
shows that this organ is considerably more tolerant of freezing than is
The prospects for successfully avoiding damage due to the formation of
ice at much lower temperatures can be assessed to a first approximation
based on this finding of Lovelock and Smith. The quantity of glycerol
required in theory to prevent mechanical injury from ice (Cgr) can be
calculated from the equation (derivable from reference 7):
Cgr = 9.3 -.093Vt
where Vt is the percentage of the liquid volume of the brain which can
be converted into ice without causing injury. Assuming Vt = 60%, Cgr is
The work of Lovelock and Smith was followed up by Suda and his associates
(8,9,10), who made a number of critical observations on frozen glycerolized
cat brains. Their first publication, in 1966, demonstrated that cat brains
gradually perfused with 15% v/v glycerol at 10 degrees C and frozen very
slowly for storage for 45-203 days at the very unfavorable :temperature
of -20 degrees C regained normal histology, vigorous unit (individual
cell) activity in the cerebral cortex, hypothalamus, and cerebellar cortex,
and strong if somewhat slowed EEG activity (8) after very slow thawing.
These results are remarkable in a number of ways. First, it is clear that
no other organ would be capable of the same degree of activity after such
prolonged storage at such a high subfreezing temperature. Second, Suda
et al. made no attempt to supplement their perfusion fluid (diluted cat
blood) with dextrose, which must have become depleted fairly rapidly,
worsening the EEG results. Third, Suda and colleagues did not wash the
glycerol from the brain carefully, and this may have caused injury during
brain reperfusion. Fourth, the presence of EEG activity implies preservation
of long-range neural connections and synaptic transmission, and unit activity
indicates preservation of cell membrane integrity, energy metabolism,
and sodium and potassium pumping capability. In short, these brains appeared
to be basically viable based both on function and on structure. "Pial
oozing" was noted (though not described adequately) after about an
hour of blood reperfusion, but this defect seems minor.
Their second publication, in 1974 (9), went considerably farther. After
7.25 years of storage at -20 degrees C, "well synchronized discharges
of Purkinje cells were observed" (i.e., normal cerebellar unit activity)
as well as "spontaneous electrical activity...from the thalamic nuclei
and cerebellar cortex," and short-lived EEG activity from the cerebral
cortex. Another brain stored for 777 days showed cortical EEG activity
for 5 hours after reperfusion. In both cases, EEG activity was of lower
quality than EEG activity of fresh brains, but the existence of any activity
at all after such extraordinary conditions is amazing. Cell loss after
7.25 years and hemorrhage after reperfusion of brains stored for 5-7 years
is not surprising.
More important was a comparison of the frequency distribution of EEG activity
in a fresh brain before perfusion and after storage at -20 degrees C for
5 days. The EEG pattern before freezing and after thawing was very nearly
the same (9). It should be noted that in a typical cryonics operation,
the time spent near -20 degrees C is measured in hours rather than days
or years and, based on the work of Suda et al., should not therefore involve
appreciable deterioration of the brain.
It is noteworthy that in both reports of Suda's group, the brains were
successfully reperfused with diluted cat blood after thawing. The quality
of reperfusion was not documented in detail, but the autocorrelogram comparing
the EEG of the 5-day cryopreserved brain to the EEG of the same brain
before freezing could not have been as good as it was without relatively
complete restoration of cerebral circulation. This is an important question
not only with respect to viability and functional recovery, but also with
respect to the accessibility of the brain to nanotechnological repair
devices which might be administered via the vascular system.
Also relevant were unpublished results mentioned in passing (9) on storage
at -60 and -90 degree C and on the effectiveness of other cryoprotectants
[dimethyl sulfoxide (DMSO) or polymers]. Evidently, EEG activity could
be obtained after freezing to -60 degree C and storage for weeks, but
not after freezing to -90 degrees C, and dimethyl sulfoxide was effective
but not as effective as glycerol. This is confirmed in an unpublished
manuscript by Suda (10), which reveals also that unit (single cell) activity
can still be recorded in brains frozen to -90 degrees C. This unpublished
paper (written in Japanese) also shows that brain reperfusion was better
after thawing when glycerol rather than DMSO was used.
These results can be evaluated with respect to the information obtained
previously by Lovelock and Smith. For protection against mechanical injury
at -90 degrees C, as noted above, the results with hamsters suggest that
3.72 M glycerol, or 27.2% glycerol by volume, might be required, whereas
Suda and colleagues used only 15% glycerol by volume. It can be calculated
(11) that at Suda's storage temperature of -20 degrees C, 62% of the liquid
content of the brain was converted into ice, while at -60 degrees C, 77%
of the liquid volume of the brain was converted to ice, a quantity which
equals or exceeds the tolerable degree of distortion by ice in the hamster
brain. Therefore, the finding by Suda and his colleagues of no injury
at -20 degrees C for 5 days but of injury after freezing to -60 degrees
C and especially to -90 degrees C is entirely consistent with predictions
from the work of Lovelock and Smith and is also entirely consistent with
an absence of any such mechanical injury in the brains of cryonic suspension
patients perfused with more than 3.72M glycerol.
The work with hamsters and with cat brains demonstrates that extensive
freezing of the brain at high temperatures is compatible with its full
functional recovery and that at least partial functional recovery from
low temperatures is a reasonable prospect, but these studies do not describe
the histological effects of freezing brains to the low temperatures required
for truly long-term preservation. This information was provided by Fahy
and colleagues (12-14a). They reported that with either 3M or 6M glycerol,
excellent histological preservation of the cerebral cortex and the hippocampus
was observed after slow freezing to dry ice temperature (-79 degrees C).
In fact, there was no difference in structure between brains which had
been perfused with glycerol only and brains which had been perfused, frozen,
and thawed. Although Fahy et al. did not report it formally, this finding
was also true in every other region of the brain examined, such as the
cerebellum and the area of the ventral brain containing giant neurons
and well-organized axonal bundles. It is of interest that Fahy et al.
observed brain shrinkage if the perfusion temperature was held constant
below room temperature (142). But Suda and his colleagues also observed
the same degree of brain shrinkage (10), yet this did not prevent apparent
survival of their frozen cat brains.
One report (14b) has appeared which briefly documented the ultrastructural
effects of a now-obsolete cryonics procedure on the brain. A single dog
was perfused directly with 15% DMSO for 55 minutes at 10-17 degrees C.
The head was then cooled at O.1 degrees C/min to -14 degrees C and then
cooled at 0.5 degrees C/min to lower temperatures. The brain was estimated
to have reached -79 degrees C after 3 hours, after which it was shipped
cross-country for thawing, fixation, and examination by light and electron
microscopy. Histochemical staining of undefined nature showed evidence
of appreciable enzymatic activity and cellular retention of histochemical
reaction product, i.e., intact cell membranes. Ultrastructure, as documented
in a single electron micrograph, revealed intact cell bodies, an intact
double nuclear membrane, intact myelin sheaths around small myelinated
fibers, recognizable organelles (mitochondria and endoplasmic reticulum),
and recognizable synapses. Extensive damage was also apparent, but it
was not clear whether this was due to freezing and thawing, perfusion
with DMSO in one step as opposed to gradual addition, or abrupt dilution
of DMSO upon fixation. No details were provided as to DMSO washout and
fixation procedures. Significantly, the concentration of DMSO employed
was not sufficient to prevent mechanical damage according to "the
Smith criterion" mentioned earlier. The presumption would be that
current cryonics procedures, employing the preferred cryoprotectant glycerol
in higher concentrations, better preserve ultrastructure. Nevertheless,
it is not obvious from the published micrograph that the original brain
structure could not be inferred.
3. Living Adult Human and Animal Brain Tissue
In 1981, Haan and Bowen (15) reported that they had collected sections
of cerebral cortex from living human patients (from brain operations requiring
removal of cortex to allow access to deep tumors), and frozen them using
10% v/v dimethyl sulfoxide) as the cryoprotectant. The DMSO was added
and removed essentially in one step each, with some agitation of tissue
samples to promote equilibration in the short times allowed for equilibration
at 4 degrees C. Freezing was accomplished by a two-step method in which
the tissue was placed at -30 degrees C for 15 min (5 min required to reach
-30 degrees C, for a cooling rate of about 6 degrees C/min, and 10 min
of equilibration at -30 degrees C) and then transferred directly to liquid
nitrogen. Thawing was rapid. For comparison, rat brain tissue was obtained
by decapitating rats and removing their brains (probably involving a warm
ischemic insult of 5-10 min), and this rat brain tissue was equilibrated
with dimethyl sulfoxide and frozen in the same way.
The results? Norepinephrine uptake was 94-95% of control uptake for both
rats and humans. Incorporation of glucose-derived carbon into acetylcholine
was 89-100% of control incorporation for rats and 85% of control for humans.
Incorporation of glucose-derived carbon into CO2 was 86-100% of control
for rats, 78% of control for humans.
Haan and Bowen noted that their tissue prisms are mostly synapses, so
their results imply that synapses of both rats and humans survive freezing
by their technique. This agrees with inferences noted above that synapses
survive in whole brains frozen with completely different techniques. Although
not strictly brain tissue, the superior cervical ganglion, considered
pan of the central nervous system, also demonstrated 100% recovery of
synaptic function after freezing to dry ice temperature in 15% glycerol,
according to Pascoe's report in 1957 (16). It was noteworthy that Pascoe's
ganglia also showed 100% recovery of action potential amplitude and conduction
velocity after thawing from dry ice temperature (16).
In 1983, Hardy et al. (17) confirmed the extreme survivability of synapses
in human brain tissue beyond any doubt. Once again, normal living adult
human cerebral cortex was removed during operations on deep brain structures
and compared to viable rat forebrains in terms of freeze-thaw recovery.
The best results were obtained by freezing 1-5 gram pieces of human brain
(or 1 gram rat forebrains), as opposed to freezing homogenates. The cooling
rate to -70 degrees C was slow but was not measured or controlled; the
thawing rate was fast but not measured or controlled; the sole cryoprotectant
was 0.32 M sucrose. (Far from an optimal regimen!) After thawing, synaptosomes
were prepared from the tissue samples and tested for functional recovery.
A summary of the results is shown in Table A-I.
As Hardy et al. stated, it is apparent that both human and rat brain tissue
frozen to -70 degrees C with almost no cryoprotection has synapses "closely
comparable to [those from]... fresh tissue."
As if this were not demonstration enough, Welder (18) has shown that not
even cryosurgery destroys synapses. He applied a -60% cryoprobe to the
brain of cats for 5 min and examined the resulting lesions in the electron
microscope. Not only were well preserved synapses found, but also cell
bodies, organelles, and neuronal processes could be identified, despite
considerable damage to the organization of the neuropil and to astrocyte
4. Living Fetal Human and Animal Brain Tissue
In 1986, Groscurth et al. reported the successful freezing of human fetal
brain tissue(19). 1x2x2 mm brain fragments from a 9-14 week abortus were
treated with 10% DMSO and 20% fetal calf serum and placed into a -30 degrees
C environment for 3 hours or overnight, then stored at -80 degrees C for
several weeks, then finally transferred to liquid nitrogen. After storage
for 3-12 months, the samples were "thawed at room temperature,"
trypsinized, and seeded on glass cover slips for 2-4 weeks of tissue culture
at 37 degrees C. The brain cells were found to be alive and to grow in
culture: "Twenty-four hours after trypsinization the cells formed
clusters of variable size.... During further cultivation numerous fiber
bundles were found to grow from the margin of the clusters. Single fibers
showed varicosities as well as growth cones at the terminal projection.
Bipolar spindle-shaped cells with a smooth surface were regularly apposed
along the bundles." The first reports of attempts to freeze fetal
animal brain tissue seem to be those of Houle and Das in 1980 (20-22).
These attempts were fully successful, the frozen-thawed transplanted cerebral
cortex being indistinguishable from non-frozen brain tissue transplants
in every way. Das et al. have more recently described their technique
in finer detail (23). Briefly, they use 10% DMSO, a cooling rate of 1
degrees C/min, storage at -90 degrees C, and rapid thawing. Survival was
best if the tissue was not dissociated or minced before freezing.
Although a variety of conditions allowed for 100% success rates for 16
and 17-day neocortex, brainstem tissue from 16-day fetuses showed at best
a 50% survival rate, and Das et al. suggested that these more differentiated
cells, which have a low transplant survival rate even in the absence of
freezing and thawing, might be more damaged by freezing and thawing. On
the other hand, it should be kept in mind that, as should be clear from
the earlier discussion of cryoprotectant concentrations necessary for
protection at low temperatures, 10% DMSO is a rather low concentration
of a possibly suboptimal cryoprotectant (Suda indicated that glycerol
was superior to DMSO for brain), and better survival might well have been
obtained using the more gentle freezing/thawing conditions employed in
Jensen and colleagues (24) reported their work on freezing fetal hippocampal
tissue in 1984, again using 10% DMSO, a cooling rate of 1 degrees C/min,
storage in liquid nitrogen, and rapid thawing. Treatment with DMSO at
4 degrees C was for 2 hours, with rapid washout at room temperature (not
necessarily an innocuous approach; unfortunately, no DMSO controls were
done). Although 21% of the cryopreserved hippocampi showed ideal structural
preservation after development in oculo, in general there was some structural
alteration compared to nonfrozen control hippocampal transplants. It was
felt that this may have been due to the extra manipulations of the cryopreserved
tissue (controls were not washed in DMSO solutions, etc.). Only half of
the cryopreserved transplants at most were found to be present after 20-68
days in oculo, survival rate being dependent upon fetal age. It was felt
that this once again may have been due to loosening of the hippocampal
structure by the experimental manipulations.
This tended to be confirmed by transplants into the brain rather than
into the eye (24b): the brain provides more confinement to transplanted
hippocampi, helping to prevent disintegration of the grafts, and, in fact,
100% of hippocampi transplanted to the brain survived. (It should be obvious
that the hippocampus of a frozen intact brain will of course receive support
from all surrounding structures and will thus be more analogous to the
intracerebral transplants noted by Jensen et al. than to the intraocular
transplants, in addition to being spared from disruptive manipulations
Frozen-thawed hippocampi grown in oculo were smaller than control grafts,
and frozen-thawed hippocampi transplanted either to the eye or to the
brain showed a loss of dentate granule cells (a 35% loss was seen in oculo).
In several other ways, this complex brain structure important for encoding
and decoding memories appeared to be unaffected by freezing and thawing.
Moreover, freezing in 10% DMSO, as noted above, might not be an ideal
procedure. It should be noted that Fahy et al. were not impressed by any
loss of dentate cells in whole adult rabbit brains after freezing and
Jensen's group followed up this work with more extensive work on many
different subregions of the fetal rat brain, i.e., the neocortex, habenula,
septum and basal forebrain, cerebellum, and retina (25). All of these
regions showed good survival and preservation of normal structural organization
after transplantation into an adult recipient's cerebral cortex, despite
wide, uncontrolled variations in cooling protocol from run to run, The
only exception was the cerebellum: Only 2 of 7 grafts were found at the
time of sacrifice, although they were structurally normal. The numbers
involved are too small for adequate statistical analysis, and no control
cerebellar grafts were performed to determine if this rate of takes is
normal for this tissue. All in all, then, this paper tends to confirm
the impression from other studies that tissue from many quite different
brain areas survives freezing and thawing quite well.
5. Living Human and Animal Isolated Brain
Silani et al. (26) dissociated human fetal cerebral cortex into cells
and froze the cells at 1 degrees C/min in 7% DMSO plus 20% fetal calf
serum. After more than 12 months in liquid nitrogen, the cells were thawed
rapidly. Immediately after thawing, the cell recovery was 96.5+/-2.1%,
showing that brain cells are not physically destroyed by freezing even
under rather severe conditions. After 72 hours of culture, 53% of the
total cell population was alive, but only 24% of the neurons were alive.
The surviving neurons were, however, morphologically and functionally
normal, as were astrocytes. Silani et al. considered their yield of human
neurons to be high. These results show unequivocally that human brain
cells can survive freezing and thawing and imply that, as was the experience
of Hardy et al. (17) and Das et al. (23) (and as is suggested by the experience
of Jensen et al. (24)), it is best to use undissociated tissues (analogous
to the intact brain in cryonics procedures) rather than dissociated cells
to obtain optimal results.
Kim et al. (27) isolated living oligodendrocytes and astrocytes from the
white matter of brains of human cadavers aged 62, 86, and 93 years after
5, 14, and 6 hours of clinical death, respectively. These cells were cultured
for 2-28 days, then scraped from their substratum, exposed abruptly to
10% DMSO, frozen to -70 degrees C at an unknown and uncontrolled, exponentially
decreasing rate, immersed in liquid nitrogen for 1-3 weeks, thawed rapidly,
and abruptly diluted to 1970 DMSO, further washed, and recultured. The
excellent morphology of the cultured cells after thawing and the robust
presence of membrane markers was not different from what existed before
freezing. 70%, 60%, and 55% survival was obtained after 2, 7, and 28 days
of culture before freezing, respectively.
Kim et al. (27) also reported internally the following. "Recently,
we have frozen various types of neural tissue cultures and found that
the recovery of frozen neurons and glial cells was excellent. The neural
cultures tested were: (a) dissociated chick embryo spinal cord and dorsal
root ganglia; (b) dissociated newborn mouse cerebellum and dorsal root
ganglia; (c) dissociated adult mouse dorsal root ganglia, and; (d) dissociated
or explant fetal human brain cultures."
Kawamoto and Barrett (28) froze rat fetus striatal (including overlying
cortical) and spinal cord cells by dissociating these tissues in 5-10%
DMSO and placing them into uninsulated boxes in a -90 degrees C freezer
and leaving them there for up to 88 days. They were then thawed rapidly
and exposed immediately to DMSO-free solution, a procedure these scientists
found to be damaging. Nevertheless, they observed "neuronal survival
rates comparable to those of brain tissues plated immediately after dissection."
Preliminary results indicated similar survival of neuroglia frozen in
the same way. Survival was roughly independent of DMSO concentration above
5%. Increased sensitivity of the cells to mechanical forces was observed
after thawing or after simple cold storage, but this was reduced by using
cryoprotectant carrier solutions low in sodium. Beautiful morphology was
seen after thawing, and vigorous regrowth of cellular processes occurred
after thawing, to yield mature cultures indistinguishable from controls.
Surprisingly, dissociated cells survived freezing and thawing better than
cells embedded in undissociated tissue.
Scott and Lew (29) gradually exposed undisturbed cultured adult mouse
dorsal root ganglion cells to 10% DMSO, placed them in a -15 degrees C
environment for 30 min, then placed them in liquid nitrogen vapor. Thawing
took 5 min, after which the DMSO was removed gradually. Other cultured
neurons were dissociated and frozen and thawed similarly as a cell suspension.
The relative number of surviving neurons was not quantified in this study,
although there was evidently considerable cell death (probably due to
the high cooling rate below -15 degrees C, which would be expected to
induce intracellular freezing and cell death). Nevertheless, many neurons
survived and were capable of basically normal electrical activity as well
as regeneration of new nerve fibers.
6. Post-Mortem Human and Animal Brains
Human brain banks are now in existence for investigators interested in
understanding human brain biochemistry and pathology (30-33). Sections
or subregions of post-mortem human brains, frozen rapidly several hours
after death, are sent to medical researchers who analyze these brains
for neurotransmitters, proteins, enzyme activity, lipids, nucleic acids,
and even histology. There would be no reason for such banks if no molecular
or structural preservation were achieved by freezing.
Haberland et al. (34) isolated synaptosomes after freezing the nucleus
accumbens of rats and of 72 (plus or minus 5) year old humans. The humans
were dead 15 +/- 5 hours before this brain structure was removed and frozen.
Previous studies indicated that dopamine uptake by synaptosomes could
still achieve 55% of the values of fresh brains even 24 hours after death.
In this study, the humans were not refrigerated until 3-5 hours after
death. Freezing was done with varying concentrations up to 10% DMSO, 1.2
degrees C/min to -25 degrees C, and subsequent immersion in liquid nitrogen.
Experiments on rat nucleus accumbens (NA) removed 5-10 min after decapitation
of the rat indicated that freezing to -25 degrees C caused no measurable
reduction of dopamine uptake. When rat NA was frozen to -196 degrees C,
survival ranged from 96% of control using 0.07 M DMSO to 99.7% of control
using 0.7 M DMSO. Human NA frozen to -196 degrees C as described in the
presence of 0.7 M DMSO (5% v/v) yielded dopamine uptakes equaling 102.9+/-5.2%
of unfrozen control uptakes.
Stahl and Swanson (35) looked at the fidelity of subcellular localization
of 6 brain enzymes and total brain protein after guinea pig or post-mortem
human brain tissues were frozen to -70 degrees C without a cryoprotectant
simply by being placed into a freezer. Their conclusion: "Subcellular
fractionation of brain material is possible even with post-mortem tissues
removed from the cranial cavity some hours after death." Two other
groups have subsequently fractionated human post-mortem brain and have
come to a similar conclusion: "Our present study further shows that
even after freezing and prolonged storage, human and guinea pig brains
can be separated into biochemically distinguishable subcellular fractions....frozen
storage for several months did not strikingly modify the fractionation
characteristics of freshly homogenized cerebral cortex."
Schwarcz (36) subjected rat brains to post-mortem conditions comparable
to those experienced generally by humans: 4 hours of storage in situ at
room temperature followed by 24 hours of storage in situ at 4 degrees
C followed by brain isolation and freezing of brain regions by placement
in a -80 degrees C freezer for 5 days. Glutamate uptake by striatal synaptosomes
prepared from striata frozen in this way amounted to 26% of control uptake
by fresh tissue synaptosomes, an amazing degree of preservation. (Schwarcz
noted, however, that glutamate uptake processes may be more resistant
than serotoninergic, dopaminergic, and cholinergic uptake mechanisms.)
Brammer and Ray (37) confirmed that it is possible to isolate intact,
if not living, oligodendroglial cells from bovine brain white matter after
freezing to -30 degrees C without any cryoprotective of the central nervous
system. A human cryopreserved by now-obsolete cryonics procedures was
decapitated while frozen, the body thawed, and the spinal cord and spinal
nerves examined histologically after aldehyde fixation and osmication.
The basic finding was that myelin sheaths were intact, and shrunken axoplasm
could be seen within the myelin sheaths, conceivably indicating intact
axolemmas. Large neuronal cell bodies were observed which appeared intact
and normal in shape. In general, the histological preservation was impressive.
Apparently intact blood vessels were observed within the spinal cord.
(Other, non-neuronal tissues were also examined and were found to be surprisingly
intact, with the exception of the liver and, to a lesser extent, the kidney.)
The scientific literature allows no conclusion other than that brain structure
and even many brain functions are likely to be reasonably well preserved
by freezing in the presence of cryoprotective agents, especially glycerol
in high concentrations. Thus, cryonics' premise of preservation would
seem to be well supported by existing cryobiological knowledge. This is
not to say that cryonics will inevitably work, but it is to say that cryonics
may work and that it is a reasonable undertaking.
General Cryobiological Background
1. Fahy, G.M., Analysis of "solution effects" injury: rabbit
renal cortex frozen in the presence of dimethyl sulfoxide., Cryobiology,
Living Adult Animal Brains
2. White, R.J., Brain, In: Organ Preservation for Transplantation, A.M.
Karow, Jr., G.J.M. Abouna, and A.L. Humphries, Jr., Eds., Little, Brown,
St Company, Boston, 1974. pp. 395-407.
3. White, R.J., Brain In: Organ preservation for Transplantation, Second
Edition, A.M. Karow, Jr. and D.E. Pegg, Eds., Marcel Dekker, New York,
1981. pp. 655674.
4. White, R.J., Cryopreservation of the mammalian brain, Cryobiology,
16, 582 (1979).
5. Smith, A.U., Revival of mammals from body temperatures below zero.
In: Biological Effects of Freezing and Supercooling, A.U. Smith, Ed. Edward
Arnold, London, 1961.pp.304-368.
6. Lovelock, J.E., and A.U. Smith, Studies on golden hamsters during cooling
to and rewarming from body temperatures below 0%. III. Biophysical aspects
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7. Fahy, G.M., D.I. Levy, and S.E. Ali, Some emerging principles underlying
the physical properties, biological actions, and utility of vitrification
solutions, Cryobiology, 24, 196-213 (1987).
8. Suda, I., K. Kite, and C. Adachi, Viability of long term frozen cat
brain in vitro, Nature (London), 212, 268-270 (1966).
9. Suda, I., K. Kite, and C. Adachi, Bioelectric discharges of isolated
cat brain after revival from years of frozen storage, Brain Res, 70, 527-531
10. Suda, I., Unpublished Japanese language manuscript (including figures)
based on a talk given by Dr. Suda (President of Kobe University) in Japan
and reportedly being prepared for publication in English.
1l. Fahy, G.M., Analysis of "solution effects" injury: Equations
for calculating phase diagram information for the ternary systems NaC1-dimethylsulfoxide-water
and NaCI- glycerol-water, Biophys J, 32, 837-850 (1980).
12. Fahy, G.M., T. Takahashi, A.M. Crane, and L. Sokoloff, Cryoprotection
of the mammalian brain, Cryobiology, 18, 618 (1981).
13. Fahy, G.M., T. Takahashi, and A.M. Crane, Histological cryoprotection
of rat and rabbit brains, Cryo-Letters, 5, 33-46 (1984).
14a. Fahy, G.M., and A.M. Crane, Histological cryoprotection of rabbit
brain with 3M glycerol, Cryobiology, 21, 704 (1984).
14b. Gale, L., Alcor experiment: Surviving the cold, Long Life Magazine,
2, 58-60 (1978).
Living Adult Human and Animal Brain Tissue
15. Haan, E.A., and D.M. Bowen, Protection of neocortical tissue prisms
from freeze-thaw injury by dimethyl sulphoxide, J Neurochem, 37, 243-246
16. Pascoe, J.E., The survival of the rat's superior cervical ganglion
after cooling to 76 degrees C, Proc. Roy. Sec. (London) B, 147, 510-519(1957).
17. Hardy, J.A., P.R. Dodd, A.E. Oakley, R.H. Ferry, J.A. Edwardson, and
A.M. Kidd, Metabolically active synaptosomes can be prepared from frozen
rat and human brain, J Neurochem, 40, 608-614 (1983).
18. Welder, H.A.D., The effect of freezing and rewarming on feline brain
tissue: an electron microscope study In: The Frozen Cell, G.E.W. Wolstenholme
and M. O'Connor, Eds., J. & A. Churchill, London, 1970. pp. 251-266.
Living Fetal Human and Animal Brain Tissue
19. Groscurth, P., M. Erni, M. Balzer, H.-J. Peter, and G. Haselbacher,
Cryopreservation of human fetal organs, Anat Embryol, 174, 105-113 (1986).
20. Houle, J.D., and G.D. Das, Cryopreservation of embryonic neural tissue
and its successful transplantation in the rat brain, Anat Rec, 196, 81A
21. Houle, J.D., and G.D. Das, Freezing of embryonic neural tissue and
its transplantation in the rat brain, Brain Res, 192, 570-574(1980).
22. Houle, J.D., and G.D. Das, Freezing and transplantation of brain tissue
in rats, Experientia, 36, 1114-1115 (1980).
23. Das, G.D., J.D. Houle, J. Brasko, and K.G. Das, Freezing of neural
tissues and their transplantation in the brain of rats: technical details
and histological observations, J Neurosci Methods, 8, 1-15 (1983).
24. Jensen, 8., T. Sorensen, A.G. Moller, and J. Zimmer, Intraocular grafts
of fresh and freeze-stored rat hippocampal tissue: comparison of survivability
and histological and connective organization, J Comp Neurol 227, 558-568
24b. Sorenson, T., S. Jensen, A.G. Moller, and J. Zimmer, Intracephalic
transplants of freeze-stored rat hippocampal tissue, J. Comp. Neurol.,
252, 468-82 (1986).
25. Jensen, S., T. Sorensen, and J. Zimmer, Cryopreservation of fetal
rat brain tissue later used for intracerebral transplantation, Cryobiology,
24, 120-134 (1987).
Living Human and Animal Isolated Brain Cells
26. Silani, V., A. Pizzuti, O. Strada, A. Falini, et al, Human neuronal
cell cryopreservation, (abstract from unidentified literature source)
27. Rim, S.U., G. Moretto, B. Ruff, and D.H. Shin, Culture and cryopreservation
of adult human oligodendrocytes and astrocytes, Acts Neuropathol (Berlin),
64, 172-175 (1984).
28. Kawamoto, J.C., and J.N. Barrett, Cryopreservation of primary neurons
for tissue culture, Brain Res, 384, 84-93 (1986).
29. Scott, B., and L. Lew, Neurons in cell culture survive freezing, Exp
Cell Res, 162, 566-573 (1986).
Post-Mortem Human and Animal Brains
30. Itabashi, H.H., W.W. Tourtellotte, B. Baral, and M. Dang, A freezing
method for the preservation of nervous tissue for concomitant molecular
biological research and histopathological evaluation, J Neuropath Exp
Neurol 35, 117-119 (1976).
31. Tourtellotte. W.W., R.C. Cohenour, J. Raj, A. Morgen, R. Warwick,
J. Sweeder, et al, The NINCDS/NIMH human neurospecimen bank, Neuro-Psychopharmaco1.
32. Bird, E.D., Brain tissue banks, Trends in Neurosci, 1(5), I-II (1978).
33. Tourtellotte, W.W., H.H. Itabashi, I. Rosario, and K. Berman, Notional
neurological research bank: A collection of cryopreserved human neurological
specimens for neuroscientists, Ann Neurol, 14, 154 (1983).
34. Haberland, N., L. Hetey, H.A. Hackensellner, and G. Matthes, Characterization
of the synaptosomal dopamine uptake from rat and human brain tissue after
low temperature preservation, Cryo-Letters, 6, 319-328 (1985).
35. Stahl, W.L., and P.D. Swanson, Effects of freezing and storage on
subcellular fractionation of guinea pig and human brain, Neurobiology,
5, 393-400 (1975).
36. Schwarcz, R., Effects of tissue storage and freezing on brain glutamate
uptake, Life Sci, 28, 1147-1154 (1981).
37. Brammer, M.J., and P. Ray, Preservation of oligodendroglial cytoplasm
in cryopreservative-prepared frozen white matter, J Neurochem, 38, 1493-1497
38. Iqbal, K., et al., Oligodendroglia from human autopsied brain. Bulk
isolation and some chemical properties, J Neurochem, 28, 707-716 (1977).
39. Morrison, M.R., and W.S.T. Griffin, The isolation and in vitro translation
of undegraded messenger RNAs from human post-mortem brain, Anal. Biochem,
113, 318-324 (1981).
40. Tower, D.B., S.S. Goldman, and O.M. Young, Oxygen consumption by frozen
and thawed cerebrocortical slices from warm-adapted or hibernating hamsters:
the protective effects of hibernation, J Neurochem, 27, 285-287 (1976).
41. Tower, D.B., and O.M. Young, The activities of butyrylcholinesterase
and carbonic anhydrase, the rare of anaerobic glycolysis. and the question
of a constant density of glial cells in cerebral cortices of various mammalian
species from mouse to whale, J Neurochem, 20, 269-278 (1973).
42. Tower, D.B., and O.M. Young, Interspecies correlations of cerebral
cortical oxygen consumption, acetylcholinesterase activity and chloride
content: studies on the brains of the fin whale (Balaenoptera physalus)
and the sperm whale (Physeter catodon), J Neurochem, 20, 253-267 (1973).
Spinal Cord and Spinal Nerves
43. Anonymous, Histological study of a temporarily cryopreserved human,
Cryonics, #52, 13-32 (Nov, 1984)