Cell-cell contact affects membrane integrity after intracellular freezing

The response of cells to freezing depends critically on the presence of an intact cell membrane. During rapid cooling, the cell plasma membrane may no longer be an effective barrier to ice propagation and can be breached by e xtracellular ice resulting in the nucleation of the supercooled cytoplasm. In tissues, the formation of intracellular ice is compounded by the presenc e of cell-cell and cell-surface interactions. Three different hamster fibro blast model systems were used to simulate structures found in organized tis sues. Samples were supercooled to an experimental temperature on a cryostag e and ice nucleated at the constant temperature. A dual fluorescent stainin g technique was used for the quantitative assessment of the integrity of th e cell plasma membrane. A novel technique using the fluorescent stain SYTO was used for the detection of intracellular ice formation (IIF) in cell mon olayers. The cumulative incidence of cells with a loss of membrane integrit y and the cumulative incidence of IIF were determined as a function of temp erature. Cells in suspension and individual attached cells showed no signif icant difference in the number of cells that formed intracellular ice and t hose that lost membrane integrity. For cells in a monolayer, with cell-cell contact, intracellular ice formation did not result in the immediate disru ption of the plasma membrane in the majority of cells. This introduces the potential for minimizing damage due to IIF and for developing strategies fo r the cryoprotection of tissues during rapid cooling. (C) 2000 Academic Pre ss.