Innocuous biological freezing during warming

Abstract

Intracellular freezing during the cooling of living cells is reported to reduce or abolish their recovery on thawing1. In microscopical and microprobe studies, low-temperature techniques may induce intracellular ice artifacts2. The amount of intracellular ice can be reduced either by selecting a slow cooling procedure that allows extracellular crystallization and cellular dehydration before the intracellular ice forms or by the ultrarapid cooling of very small samples to minimize the nucleation and growth of ice3. However, a few reports suggest that in some conditions intracellular ice may be either innocuous4 or even sometimes beneficial5 for the survival of function. We have therefore used here the technique of low-temperature light microscopy to examine the relationship between intracellular freezing and cell injury. We followed the morphological appearance of eight-cell mouse embryos during cooling and warming in medium containing dimethyl sulphoxide (DMSO; 1.5 M) and correlated it with their functional survival after thawing. Unexpectedly, we found that ice formed in the cytoplasm of embryos during slow warming in conditions when it did not form during cooling. Death of embryos, however, did not correlate with the temperature during slow warming at which intracellular ice appeared (−85 °C). The occurrence in some conditions of freezing at these low temperatures during warming has important implications both for the preservation of function of cells and for the preservation of morphology in electron microscopy.