Cryobiology

The use of low-temperature environments in the study of living plants and animals. The principal effects of cold on living tissue are destruction of life and preservation of life at a reduced level of activity. Both of these effects are demonstrated in nature. Death by freezing is a relatively common occurrence in severe winter storms. Among cold-blooded animals winter weather usually results in a comalike sleep that may last for a considerable length of time.

In cryobiological applications much lower temperatures are used than are present in natural environments. The extreme cold of liquid nitrogen (boiling at -320^°F or -196^°C) can cause living tissue to be destroyed in a matter of seconds or to be preserved for years and possibly for centuries with essentially no detectable biochemical activity. The result achieved when heat is withdrawn from living tissue depends on processes occurring in the individual cells. Basic knowledge of the causes of cell death, especially during the process of freezing, and the discovery of methods which circumvent these causes have led to practical applications both for long-term storage of living cells or tissue (cryopreservation) and for calculated and selective destruction of tissue (cryosurgery).

The biochemical constituents of a cell are either dissolved or suspended in water. During the physical process of freezing, water tends to crystallize in pure form, while the dissolved or suspended materials concentrate in the remaining liquid. In the living cell, this process is quite destructive. In a relatively slow freezing process ice first begins to form in the fluid surrounding the cells, and the concentration of dissolved materials in the remaining liquid increases. A concentration gradient is established across the cell wall, and water moves out of the cell in response to the osmotic force. As freezing continues, the cell becomes quite dehydrated. Salts may concentrate to extremely high levels. In a similar manner the acid-base ratio of the solution may be altered during the concentration process. Dehydration can affect the gross organization of the cell and also the molecular relationships, some of which depend on the presence of water at particular sites. Cellular collapse resulting from loss of water may bring in contact intracellular components normally separated to prevent destructive interaction. Finally, as the ice crystals grow in size, the cell walls may be ruptured by the ice crystals themselves or by the high concentration gradients which are imposed upon them.

By speeding the freezing process to the point that temperature drop is measured in degrees per second, some of these destructive events can be modified. However, most of the destructive processes will prevail. To prevent dehydration, steps must be taken to stop the separation of water in the form of pure ice so that all of the cell fluids can solidify together. The chief tools used to accomplish this are agents that lower the freezing point of the water. Glycerol, a polyalcohol which is compatible with other biochemical materials in living cells, is frequently used in cell preservation. Besides the antifreeze additive, refrigeration procedures are designed to control the rate of decline in temperature to the freezing point, through the liquid-solid transition, and below, to very low temperatures.

Cryopreservation

The earliest commercial application of cryopreservation was in the storage of animal sperm cells for use in artificial insemination. The microorganisms used in cheese production can be frozen, stored, and transported without loss of lactic acid–producing activity. Pollen from various plants can be frozen for storage and transport, facilitating plant-breeding experiments. Among the most valuable applications of cryopreservation is the storage of whole blood or separated blood cells.

Cryosurgery

Cellular destruction from freezing can be used to destroy tissue as a surgical procedure. One of the significant advantages of cryosurgery is that the apparatus can be employed to cool the tissue to the extent that the normal or the aberrant function is suppressed; yet at this stage the procedure can be reversed without permanent effect. When the surgeon is completely satisfied that he or she has located the exact spot to destroy, the temperature can be lowered enough to produce irreversible destruction. This procedure is of particular assistance in neurosurgery.

A second major advantage of cryosurgery is that the advancing front of reduced temperatures tends to cause the removal of blood and the constriction of blood vessels in the affected area. This means that little or no bleeding results from cryosurgical procedures.

A third major advantage of cryosurgery is that cryosurgery equipment currently employs a freezing apparatus that can be placed in contact with area to be destroyed with a minimum incision to expose the affected area.

Cryobiology

a branch of biology that deals with the effects of low and extremely low temperatures (from 0°C to near absolute zero) on living systems.

The main tasks of cryobiology are to study life under cold conditions, determine the reasons why organisms are resistant to supercooling and freezing, investigate the injurious action of subzero temperatures, and develop methods for protecting cells and tissues against freezing. The problems of cryobiology are of great theoretical importance, because they are related to the determination of the low-temperature boundaries of life, mechanisms of adaptation to cold under natural conditions, the nature of anabiosis, and so forth. From the practical standpoint, cryobiology is concerned with methods of storing and collecting biological objects, the use of cold for therapeutic purposes, the breeding of hardy plant varieties, and the study of the hibernation of crop pests and human activity in the arctic and antarctic. It also is important in space biology.

The scientific foundation of cryobiology was laid at the end of the 19th century by the Russian scientist P. I. Bakhmet’ev, who studied the phenomenon of supercooling in insects and anabiosis in bats. P. Becquerel (1904–36) and the Austrian scientist G. Rahm (1919–24) found that a variety of organisms (microorganisms and such invertebrates as tardigrades, rotifers, and nematodes), as well as spores and seeds, can tolerate extreme chilling (to −269° and −271°C when dried—that is, to temperatures close to absolute zero). It was subsequently shown that certain plants and animals can survive when the water they contain freezes. For example, such highly organized organisms as the caterpillars of some butterflies which were first hardened, that is, adapted to cold, “revived” after prolonged freezing at −78°, −196°, and even −269°C, when the water in their bodies was converted to ice crystals. One of the main objectives of cryobiology is to elucidate the processes that accompany the chilling of living systems and result in irreversible injuries. There are many causes behind injuries produced by chilling and freezing. The rate of chilling and thawing is an important factor. In slow chilling, the water in the fluid surrounding the cell first turns into ice, causing a loss of water by the cell, disruption of the salt balance between the extra- and intracellular fluid, and an increase in concentration of electrolytes in the cell. Some cells die as a result. Very slow chilling is required if plant cells and certain animal tissues are to survive; in so doing, the concentration of substances in the cell does not change abruptly.

Dehydration is particularly dangerous for cells not adapted to cold, because extracellular components that under normal conditions are separate come into contact, rupturing some inter-molecular bonds and forming others, injuring the cell membranes, and so forth. Similar phenomena can also arise when ice crystals form within a cell. They usually form during rapid chilling (over 10° per minute). After the chilling process ends, crystals start to grow (recrystallization) at temperatures above — 120°C. They enlarge substantially during warming. The principal injuries to the cell are thought to occur during warming and thawing. The cell usually dies when ice crystals form in it. However, the cells of certain hardened insects and malignant tumors can withstand intracellular crystallization of water.

With extremely rapid cooling at the rate of several hundred degrees per second (such cooling is possible only in living objects of microscopic size), most of the water is converted to amorphous ice, the structure of which differs little from that of water. Consequently, the cells are not injured, and, regardless of origin, they survive. But after extremely rapid deep cooling, cells remain viable only with very rapid warming (in 3–10 seconds), which can prevent recrystallization. This method of preserving cells is quite impractical because of the impossibility of extremely rapid cooling and warming of comparatively large objects. Protective agents—cryoprotectors—are used to preserve living systems exposed to low temperatures. The most familiar substances of this kind are glycerin, dimethyl sulfoxide, sugar, and glycols, which can penetrate into cells, and some polymers (polyvinylpyrrolidone, polyoxyethylene, and so on), which cannot. Cryoprotectors weaken the effect of crystallization by altering its nature, prevent the adhesion and denaturation of macromolecules, and help to protect the integrity of the cellular membranes. Cryoprotectors are widely used in medicine and livestock raising for long-term preservation at low temperatures of blood, tissues, organs, and the sperm of domestic animals used for artificial insemination.

The resistance of many terrestrial organisms to temperatures below 0°C changes drastically during a life cycle linked to the seasons. For example, insects and plants become highly resistant to cold and frost when entering dormancy (diapause in insects and ticks) even before the onset of frosts. At the start of dormancy, with temperatures a little above 0°C, substantial changes take place in metabolism and the physicochemical state of the cells that increase the resistance of the organism. Fats, glycogen, and sugars accumulate, protective substances are formed, and the state of the water and proteins in the cells changes. Depending on their ecology, insects can withstand extreme chilling, sometimes to −40°C or even lower. Certain insect and plant species hibernate in a frozen state. Many microorganisms (bacteria and yeasts), mosses, lichens, and so forth readily tolerate low and even extremely low temperatures. Their hardiness is usually due to rapid dehydration, increased viscosity of the cytoplasm, the presence of membranes that prevent crystals from penetrating into the cells, and so forth. Organisms (except warm-blooded animals) usually cease to be viable at temperatures somewhat below 0°C, although certain metabolic processes may continue at temperatures of about −20°C (for example, respiration and photosynthesis) and even lower. The little-studied biology of marine organisms inhabiting underwater ice in the antarctic is of interest in this connection.

Special journals are devoted to cryobiology. International symposiums and conferences of cryobiologists are held every year.

REFERENCES

Rey, L. Konservatsiia zhizni kholodom. Moscow, 1962. (Translated from French.)
Smith, O. Biologicheskoe deistvie zamorazhivaniia i pereokhlazhdeniia. Moscow, 1963. (Translated from English.)
Kletka i temperatura sredy. Moscow-Leningrad, 1964.
Lozina-Lozinskii, L. K. Ocherki po kriobiologii. Leningrad, 1972.
“Cellular Injury and Resistance in Freezing Organisms.” Sapporo, 1967. (Proceedings of the International Conference on Low Temperature Science, Aug. 14–19, 1966, Sapporo, Japan, vol. 2.)
Cryobiology. Edited by H. T. Meryman. London-New York, 1966.
The Frozen Cell. London, 1970.
Mazur, P. “Cryobiology: The Freezing of Biological Systems.” Science, 1970, vol. 168, no. 3934, p. 939.

L. K. LOZINA-LOZINSKII

cryobiology

[¦krī·ō·bī′äl·ə·jē] (biology) The use of low-temperature environments in the study of living plants and animals.

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Cryopreservation

Cryosurgery

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REFERENCES

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noun the branch of biology that studies the effects of low temperatures on living tissues or organs or organisms

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