Physical-chemical aspects of cryoprotection of human erythrocytes and mouse embryos

One theory of freezing damage suggests that slowly cooled cells are killed by being exposed to increasing concentrations of electrolytes as the suspending medium freezes. A corollary to this view is that additives such as glycerol protect cells by acting colligatively to reduce the electrolyte concentration at any subzero temperature. Recently published phase-diagram data for the ternary system glycerol-NaCl-water by M. L. Shepard et al. (Cryobiology 13:9-23, 1976), in combination with data on human erythrocyte survival versus temperature (Souzu and Mazur, Biophys. J. 23:89-100, 1978), permit a precise test of this theory. Appropriate liquidus phase-diagram information for the solutions used in the erythrocyte freezing experiments was obtained by interpolation of the liquidus data of Shepard and his co-workers. The results of phase-diagram analysis of erythrocyte survival indicate that the correlation between the temperature that yields 50% hemolysis and the electrolyte concentration attained at that temperature in various concentrations of glycerol is poor. With increasing concentrations of glycerol, the cells were killed at progressively lower concentrations of NaCl. The data, in combination with other findings, lead to two conclusions: (a) the protection from glycerol is due to its colligative ability to reduce the concentration of sodium chloride in the external medium, but (b) the protection is less than that expected from colligative effects; apparently glycerol may render erythrocytes susceptible to osmotic shock during thawing.

Cells cooled too rapidly are believed to be killed by intracellular freezing. Previously a thermodynamic relationship was developed which

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describes the kinetics of cell dehydration during freezing (Mazur, J. Gen. Physiol. 47:347-369, 1963). This relationship is described by a set of differential equations giving the cell water content as a function of temperature, cooling rate, cell surface area-volume ratio, cell membrane permeability to water, and temperature coefficient of the permeability coefficient. The calculated cell water content permits predictions of the likelihood of intracellular freezing, and these predictions have been confirmed. I have modified this earlier thermodynamic model to allow calculation of the kinetics of cell dehydration for cells frozen by a "two-step' procedure. This procedure has yielded high survival of several cell types after thawing and provides a powerful cryobiological tool (Farrant et al., Nature 249:452-453, 1974). In two-step freezing, rapid cooling to -l96°C is interrupted with a timed exposure at an intermediate temperature (usually ~-25°C). Cell survival has been found to be dependent on the time of exposure at the intermediate temperature. I have calculated the cell dehydration behavior of Chinese hamster tissue culture cells and human lymphocytes during two-step freezing and have predicted the likelihood of intracellular freezing. Both the calculated water contents and predictions of intracellular freezing are consistent with the experimental observations of Farrant and his colleagues. The protective effect of time during the interruptive step of two-step freezing can be explained in terms of sufficient cell dehydration to preclude intracellular freezing.

The temperature at which supercooled cytoplasm spontaneously freezes is an important element in predicting the likelihood of intracellular freezing from thermodynamic model calculations. A cell's nucleation temperature is known to depend on whether a cryoprotective additive is present. I have measured the temperature dependence of intracellular ice nucleation in 8-cell stage mouse embryos equilibrated with 0 to 2.0 M dimethyl sulfoxide (Me₂SO) or glycerol and then rapidly cooled. Two methods were used to assess the nucleation temperature. The first method was to cool embryo suspensions rapidly to various subzero temperatures, thaw them rapidly, and determine in vitro survival and the second method was to observe the microscopic appearance of embryos during rapid cooling and note the temperature at which nucleation occurred. The nucleation temperature depended on the concentration of additive present during cooling; the higher the concentration of additive, the lower the nucleation temperature. For example, embryos in saline were killed over the temperature range -10 to -15°C while embryos in 2.0 M Me₂SO were killed between -42 and -44°C. The experimentally determined nucleation temperatures were compared to predictions based on heterogeneous and homogeneous nucleation theory. The comparison suggests that the depression of the nucleation temperature by additives during rapid cooling is a result of colligative effects on heterogeneous and homogeneous nucleation.