Optimal temperature ranges for control of cooling rate.

Survival of hamster fibroblasts following cooling at 1 °C/min to various subzero temperatures in the presence of penetrating or nonpenetrating cryoprotective agents was examined. In the presence of nonpenetrating agents maximum recovery was obtained when the cooling rate was controlled between ?5 and ?20 °C followed by rapid cooling to ?196 °C. For penetrating agents recovery was maximal in samples cooled at 1 °C/min to ?30 °C or lower. These different temperature ranges for maximum recovery indicate different modes of actions of penetrating and nonpenetrating cryoprotective agents. The action of penetrating agents appear to be based on their colligative properties. Nonpenetrating agents may promote electrolyte leaks out of the cell and a corresponding osmotic efflux of cell water during slow cooling, thereby reducing the amount of intracellular ice present at ?196 °C.