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. An article covering more recent advances and evidence is The Arrest of Biological Time as a Bridge to Engineered Negligible Senescence.]

Introduction

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 knowledge


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 kidney.

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 3.72M.

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 cell membranes.


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 cryonics procedures.

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 in vitro.)

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 thawing (12-14a).

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 Cells

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.)


Summary

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.


References

General Cryobiological Background


1. Fahy, G.M., Analysis of "solution effects" injury: rabbit renal cortex frozen in the presence of dimethyl sulfoxide., Cryobiology, 17,
371-388 (1980).

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 and general discussion, Proc. Roy. Sec. B, 145, 427-442 (1956).

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 (1974).

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 (1981).

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 (1980).

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 (1984).

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. 2, 1593-1595(1978).

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 (1982).

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)