INTRACELLULAR FREEZING OF GLYCEROLIZED RED-CELLS

The response of glycerolized human red blood cells to freezing was evaluated in terms of the thermodynamic state of the frozen intracellular medium. The physiochemical conditions requisite for intracellular freezing, characterized by the cooling rate and the degree of extracellular supercooling, were altered appreciably by the prefreezing addition of glycerol to the cells. Fresh human erythrocytes were suspended in an isotonic glycerol solution yielding a final cryophylactic concentration of 1.5 or 3.0 M. Subsequently, the cell suspension was frozen on a special low temperature stage, mounted on a light microscope, at controlled constant cooling rates with varying degrees of extracellular supercooling (.DELTA.Tsc). The formation of pure intracellular ice phase was detected by direct observation of the cells. The addition of glycerol produced several significant variations in the freezing characteristics of the blood. As in unmodified cells, the incidence of intracellular freezing increased with the magnitudes of the cooling rate and the extracellular supercooling. The glycerolized cells exhibited a much greater tendency to supercool prior to the initial nucleation of ice. Values of .DELTA.Tsc > -20.degree. C were readily obtained. The transition from 0 to 100% occurrence of intracellular ice covered a cooling rate spectrum in excess of 300-600.degree. K/min, as compared with 10.degree. C/min for unmodified cells. The incidence of intracellular ice formation was significantly increased in glycerolized cells.