Structural Changes in Mammalian Cells Associated with Cooling to -79°C

Suspensions of HeLa and S37 cells, with and without added glycerol, were cooled in stages to -79 degrees C. and held at that temperature for 30 minutes. After warming to room temperature the cells were fixed, sectioned, and compared by phase contrast and electron microscopy with similar specimens kept at room temperature. Correlated viability tests were made. Abnormal cytological characteristics, visible with the phase contrast microscope, were clearly related to the sequence of freezing and thawing, and the proportion of altered cells was highest in specimens cooled without glycerol. Electron microscopy showed that even in the presence of glycerol all cells were markedly altered, with distinctive vesiculation and disruption of the various intracellular membranes. There is evidence that much cytoplasmic damage is compatible with survival, but it seems likely that separation of the two layers of the nuclear envelope and rearrangement of the nuclear contents are signs of irreversible damage. The findings lend some support to the belief that cell death on cooling is due largely to denaturation of semipermeable membranes, caused by the increasing concentration of electrolytes.