Use of two-step cooling procedures to examine factors influencing cell survival following freezing and thawing

The two-step cooling procedure has been used to investigate factors involved in cell injury. Chinese hamster fibroblasts frozen in dimethylsulphoxide (5%, $\text{v}\text{v}$) were studied. Survival was measured using a cell colony assay and simultaneous observations of cellular shrinkage and the localization of intracellular ice were done by an ultrastructural examination of freeze-substituted samples.Correlations were obtained between survival and shrinkage at the holding temperature. However, cells shrunken at ?25 °C for 10 min (the optimal conditions for survival on rapid thawing from ?196 °C) contain intracellular ice nuclei at ?196 °C detectable by recrystallization. These ice nuclei only form below ?80 °C and prevent recovery on slow or interrupted thawing but not on rapid thawing. Cells shrunken at ?35 °C for 10 min (just above the temperature at which intracellular ice forms in the majority of rapidly cooled cells) can tolerate even slow thawing from ?196 °C, suggesting that they contain very few or no ice nuclei even in liquid nitrogen. Damage may correlate with the total amount of ice formed per cell rather than the size of individual crystals, and we suggest that injury occurs during rewarming and is osmotic in nature.