by Gregory M. Fahy, PhD
Cryobiology -- usually thought of as the study of the effects of subfreezing temperatures on biological systems -- stands at the interface between physics and biology. The physical principles of cryobiology are universal, which provides some coherence to the field, but the biosphere contains many surprises and twists, which adds endless fascination.
The present very abbreviated overview is primarily directed at living systems exposed to temperatures below zero degrees Celsius, but it should be printed out that the cryobiology tent covers all branches of low-temperature biology, including the effects of temperatures above freezing. Technically, cryobiology is actually the study of living systems at any temperature below the standard physiological range. This includes, for example, human hypothermia (both deliberate and accidental) and even natural hibernation, which is a physiological modification of sleep that has allowed the physiological temperature range to be stretched to include temperatures that were previously fatal. Above-freezing temperatures can be just as lethal as sub-freezing temperatures, a fact that has significant ecological and agricultural significance.
Some cryobiological topics involving temperatures below zero Celsius are: plant, insect, and vertebrate natural cold hardiness and sensitivity; freeze-drying; supercooling; cryosurgery; frostbite; and deliberate cryopreservation.
One reason natural cryobiology is interesting is that nature has had millions of years to adapt organisms to the stresses of low temperatures. Understanding how nature has reconciled the principles of physics with those of biology is potentially quite illuminating.
There are, for example, trees whose twigs can survive direct immersion in liquid nitrogen after suitable pre-conditioning. It turns out they achieve this by manufacturing proteins and sugars that allow the cytoplasm to turn into a glass at temperatures about 30 to 40 degrees below zero (1); once the plant cells vitrify, they are immune to most low-temperature excursions. Certain lichens are even more dazzling, vitrifying in toto upon cooling and warming, without previous crystallization (2).
Another and even more prominent example is the freezable frog, a vertebrate that, along with certain turtles, snakes, and salamanders, has found a way of exposing all of its internal organs to severe freezing at relatively high temperatures (about -6(C) for weeks at a time with spontaneous recovery upon thawing (3). It turns out that major elements in the success of these creatures are the elaboration of natural cryoprotective agents (especially glucose and glycerol, plus a plethora of less-significant agents) and the ability to control the location of ice, typically depositing most of it external to rather than inside the major organs (4). Certain plants achieve the same control of ice by elaborating a physical barrier between sensitive areas (the apical meristems) and the loci of ice formation, such that water can leave the meristems and deposit on ice in the ice-tolerant area, but ice cannot grow through the barrier to invade and thereby to kill the meristem. The meristem can survive the dehydration, and so it survives the winter.
Perhaps the real champions of natural low-temperature survival (with the possible exception of bacteria and the like) are the insects, some of which can survive freezing to at least the temperature of dry ice (about -79 degrees C) (5). There are tales of insects that slowly digest food over weeks of frozen storage, that "prefer" to remain frozen rather than to thaw, whose brains yield evoked potentials when their eyes are exposed to light at -20 degrees C (6), and that supercool (cool to temperatures below their freezing point without freezing) to the lower limits of temperature compatible with the physics of water.
On the other hand, many insects make proteins that specifically cause them to freeze at the highest possible temperature during cooling. Other insects make "antifreeze" proteins that can prevent ice from growing within the insect even when the insects are below their freezing points and contain ice! Polar fish are the most famous for making "antifreeze" proteins, in part because to these very large fish, living about 1 degree below their freezing points without freezing is a constant way of life (7), but the insects have actually mastered this art to a higher degree. Curiously, less is probably known about the physical mechanisms of survival in insects than in any of the other natural freeze-tolerant species.
Freeze-drying has occasionally been reported to yield viable cells. Meryman reported the survival of freeze-dried bull sperm, but this observation could not be duplicated. The LifeCell Corporation invented a technique by which cells are "vitri-dried": the cells are cooled so rapidly that ice either does not form or forms such small crystals that they do not damage the cells, after which space-quality vacuums are drawn to distill off the water at very low temperatures (8). After the cells are stored at room temperature for a short time, they can be rehydrated and, allegedly, recover life functions. They are not, however, able to divide.
Freeze-drying works best for bacteria and other prokaryotes, and extensive work has gone on to develop freezing media that optimize survival. Freeze-drying as a future technology for storing some cells more conveniently than storage in liquid nitrogen gains some credibility from the existence of anhydrobiotic organisms which, as their name implies, survive practically complete drying in the absence of freezing. The tardigrades are perhaps the most famous creatures in this group, since they are highly complex, with heads, limbs, and internal body parts like those of many insects (9). It turns out that anhydrobiosis is made possible in large part through the elaboration of a sugar, trehalose, which happens to have the right geometry to support membrane structure against collapse by substituting for water at the polar head groups of the lipids (10), and some laboratories have reported that trehalose has cryoprotective effects.
As we have noted, whole-body supercooling allows many insects and fish to survive the winter. Surprisingly, this is also true for some mammals, including bats and a particularly adroit Alaskan ground squirrel that can cool without freezing to about -3 degrees C while it hibernates, despite nominal blood freezing points (determined by freezing samples using an osmometer) above -0.6 degrees C (11).
Supercooling has recently formed the basis of a British company, Pafra, Ltd., which preserves enzymes and even whole cells by cooling them in tiny droplets of water to temperatures several degrees below their freezing point (12). Because the probability of spontaneous freezing is small in such small volumes, and because the droplets are prevented from touching each other by being dispersed in a non-aqueous phase as an emulsion, stable supercooling of great magnitude (e.g., -10 to -20 degrees C) can be attained for months. This allows the advantages of low temperature to be attained without the damage associated with freezing. Supercooling can cause enzyme denaturation, since cooling weakens the hydrophobic interactions that give rise to protein folding and membrane self-assembly, but such denaturation is usually readily reversed spontaneously when the proteins are warmed up.
Supercooling has been tried for mammalian organ preservation, but has not yielded storage times longer than above-zero storage techniques, perhaps in part because of factors such as protein denaturation, which give rise to "chilling injury," an injury associated with low-temperature exposure per se. Supercooling is also the basis of organ vitrification, a topic we will touch on again below.
Cryosurgery is considered a branch of cryobiology even though the object of cryosurgery is to kill cells rather than to preserve them. Cryosurgery works by exposing cells in the patient to very rapid cooling to deep subzero temperatures. Rapid cooling, for basic physical reasons, causes water inside cells to freeze, whereas the slow cooling found in nature and usually used for cryopreservation causes intracellular water to leave cells and freeze extracellularly. Intracellular freezing tends to be lethal, and its lethality is enhanced by slow warming, which allows intracellular ice to rearrange itself into a simpler structure, in the process literally grinding up the cellular interior. Cryosurgery essentially involves localized rapid cooling to lethal temperatures followed by relatively slow warming and is used to eliminate unwanted cells such as tumor cells.
A major advantage of cryosurgery is cryoimmunology, i.e., the stimulation of the immune system by the damage-induced liberation or better presentation of target cell antigens to the immune surveillance mechanisms. Dramatic reductions in cancer persistence have been attained through cryosurgery in comparison to conventional surgery alone, and a new company, CryoMedical Sciences, has developed excellent new cryosurgical technology that will be of great value in this respect (13).
A form of slow freezing that can be as lethal as cryosurgery is freezing of extremities as a result of unintended exposure to low temperatures. Here the ice remains extracellular, but because there is no cryoprotective agent available, the extent of freezing exceeds tolerable limits. The injury is mostly mechanical in nature but does not necessarily imply the death of cells in the frozen extremity. This has been shown by a number of interesting experiments. For example, Alaskan investigators found that severely frozen extremities could often be saved by the simple expedient of slicing them open to allow accumulated edema fluid to exit, upon which the blue, unperfused limb may suddenly pink up with the inrush of fresh blood. Freezing damages blood vessels, making them tend to weep fluid to the interstitium; this edema increases tissue pressure, which collapses the blood vessels externally and halts blood flow. Given a release of tissue pressure, sufficient blood may flow back into the limb to prevent the need for amputation (14). Low molecular weight dextran has also been helpful in preventing blood from clogging the damaged vessels in frostbite cases.
Another set of fascinating experiments were the hamster freezing experiments of Audrey Smith in the 50's (summarized in 15). She found that 80% of the water in the skin could be frozen without damage, and that between 50 and 80% of the water in extremities could be converted to ice without frostbite provided the limb was not bent while frozen.
Others have similarly found that rather fantastic freezing stresses, involving core limb temperatures of -10(C or even -30(C (14), can be tolerated amazingly well. Experiences such as these indicating that complex tissues can withstand massive distortion by ice encouraged the belief that even internal organs such as the kidney and liver could be frozen for long periods and retrieved for transplantation.
It is certainly true that organized tissues that do not require vascular support for their function can be frozen and thawed with success. In fact, it is hard to think of an organized mammalian tissue that can't be successfully frozen and thawed when proper technique is used, and thousands of different cell lines exist in frozen repositories where they have been deposited by various investigators to make them accessible to others for future needs. The transplantation of formerly-frozen human heart valves, major blood vessels, and knee components has been made into a multi-million dollar industry by CryoLife, Inc. (16), and there are major industries based on frozen human skin, semen, and embryos.
On the other hand, there are also some mammalian cells such as platelets, granulocytes, and oocytes which are very sensitive and hard to freeze adequately. Military applications that require cells that can be frozen, thawed, and transfused without removing any cryoprotective additives also represent a difficult challenge. Furthermore, it is evidently difficult to cryopreserve certain types of plant tissue, and the cryopreservation of small marine organisms often proves problematic in part because of their adverse reaction to cryoprotectants.
There is clearly still plenty of work to do in cryobiology, both from a practical and from a theoretical point of view, but to me at least it seems that the greatest challenge is the cryopreservation of mammalian organs.
It is not true to say, as many often do, that no success has been achieved in this area. Quite the contrary. Several labs have reported freezing dog intestines in liquid nitrogen with recovery after thawing, although massive damage was apparent and success was dependent on major regeneration and self-repair in combination with a tolerance of severe vascular injury (17). Livers have regained partial function after freezing to -60(C (18), dog spleens (19) and ureters (20) have survived deep freezing and transplantation, and lungs have survived major freezing stresses at high subzero temperatures (21). Even hearts and kidneys have been consistently reported to survive partial freezing, but not sufficient freezing for long-term preservation. The problem is a matter of degree: partial success is not a useful substitute for complete success.
Given that nature seems to tell us that ice is best avoided when possible, and given well-documented physical damage to the non-living connective tissues found in organs that ruin capillaries and cell-cell relationships in a way that renders organs useless whether they contain living cells or not, it seems logical to pursue vitrification rather than freezing as a solution to the problem. Vitrification involves an extreme elevation of viscosity on cooling, resulting ultimately in a liquid that has the same lack of internal motions as a crystalline solid, and thus has no capacity for change over time, yet lacks the molecular rearrangements of crystallization that do so much damage (22).
I have been working on this approach since late 1980, and every year that has elapsed since then has brought important new progress toward this goal. In early 1995, after more than 14 years of effort, there are better reasons than ever to continue to hope that this problem can be solved. The result could be not only victory over a long-standing scientific challenge, but also significant improvements in transplantation medicine.
In my career as a cryobiologist, I have found it possible to add a new dimension to the physics of cryobiology by finding practical ways to eliminate ice crystals. Additional dimensions will one day be added by changing the physics of ice in a fundamental way. The ability to manipulate both the physical and the biological aspects of living systems during cooling to and warming from cryogenic temperatures ensures that cryobiology will remain a lively field for some time to come. Even after currently-possible manipulations of physics and biology have all been explored, nanotechnology will come into play, allowing someone to enter the field from a wholly new perspective and change the rules of the game in more radical ways than most cryobiologists living today can imagine.
The future of cryobiology seems secure.
1. Hirsh, A. Vitrification in plants as a natural form of cryoprotection. Cryobiology 24: 214-228, 1987.
2. Robert Williams, personal communication; Dr. Williams is currently with the Naval Medical Research Institute, Building 29, 8901 Wisconsin Avenue, Bethesda, MD 20889 (USA).
3. Storey, K.B., and Storey, J.M. Natural freeze tolerance in ectothermic vertebrates. Annu. Rev. Physiol. 54: 619-637, 1992.
4. Lee, R.E., Jr., Costanzo, J.P., Davidson, E.C., and Layne, J.R., Jr. Dynamics of body water during freezing and thawing in a freeze-tolerant frog (Rana sylvatica). J. therm. Biol. 17: 263-266, 1992.
5. Miller, L.K. Physiological studies of arctic animals. Comp. Biochem. Physiol. 59A: 327-334, 1978.
6. John Baust, personal communication; Dr. Baust is currently with Cryomedical Sciences, Inc. (see address below)
7. Feeney, R.E., and Burcham, T.S. Antifreeze glycoproteins from polar fish blood. Ann. Rev. Biophys. Chem. 15: 59-78, 1986.
8. Linner, J.G., and Livesley, S.A. Low temperature molecular distillation drying of cryofixed biological samples. in: Low Temperature Biotechnology: Emerging Applications and Engineering Contributions. J.J. McGrath and K.R. Diller, Eds. Amer. Soc. Mech. Engineering, New York, 1988, pp. 147-157.
9. Crowe, J.H., and Cooper, A.F., Jr. Cryptobiosis. Scientific American 225: 30-36, 1971.
10. Crowe, J.H., and Crowe, L.M. Water and carbohydrate interactions with membranes: studies with infrared spectroscopy and differential scanning calorimetry methods. Methods Enzymol. 127: 696‑703, 1986.
11. Barnes, B.M. Freeze avoidance in a mammal: body temperatures below 0(C in an arctic hibernator. Science 244: 1593-1595, 1989.
12. Pafra, Ltd., Cambridge, England
13. Cryomedical Sciences, Inc., 1300 Piccard Drive, Rockville, MD 20850-4303 (-417-7070).
14. Personal communication to H.T. Meryman from W.J. Mills, Jr., but see also: Franz, D.R., Berberich, J.J., Blake, S., and Mills, W.J., Jr. Evaluation of fasciotomy and vasodilator for treatment of frostbite in the dog. Cryobiology 15: 659-669, 1978.
15. Smith, A.U. Biological Effects of Freezing and Supercooling. Edward Arnold, Ltd. London, 1961.
16. CryoLife, Inc., Marietta, Georgia, USA 30067.
17. Hamilton, R., Holst, H.I., and Lehr, H.B. Successful preservation of canine small intestine by freezing. J. Surg. Res. 14: 313-318, 1973.
18. Zimmermann, G., Tennyson, C., and Drapanas, T. Studies of preservation of liver and pancreas by freezing techniques. Transpl. Proc. 1: 657-659, 1971.
19. Barner, H.B., and Scheck, E.A. Autotransplantation of the frozen-thawed spleen. Arch. Pathol. 82: 267-271, 1966.
20. Barner, H.B., Rivers, R.J., Cady, B., and Watkins, E. Survival of canine ureter after freezing. Surgery 53: 344-347, 1963.
21. Okaniwa, G., Nakada, T., Kawakami, M., Fujimura, S., Arakaki, Y., Chiba, S., Yonechi, M., Kagami, Y., and Suzuki, C. Studies on the preservation of canine lung at subzero temperatures. J. Thoracic Cardiovasc. Surg. 65: 180-186, 1973.
22. Fahy, G.M. Vitrification: A new approach to organ
cryopreservation. Prog. Clin. Biol. Res. 224: 305-335, 1986.