Evaluation of extracellular ice formation affecting cells in suspended and attached state.

Lethal injury at slow cooling rate was due to excessive cryodehydration caused by elevated solute concentrations and serious compression caused by extracellular ice formation. Human embryonic kidney 293T cells (293T cells) were used as the biological model. We evaluate contribution of extracellular ice and the solution effects to freezing injury at slow cooling rate separately. This was achieved by designing a nonfreezing experiment that exposed cells to various solution concentration at given subzero temperature to simulate the milieu in unfrozen channels during freezing without ice formation. The mechanical stress of extracellular ice was conducted by comparison between the nonfreezing experiments and freezing experiments. The present results suggest that the mechanical injury and solution effect is relative to the temperature. At higher subzero temperature, extracellular ice or solution effect did not influence the cell survival. For modest subzero temperature, cell damage mainly caused by the mechanical stress from extracellular ice crystals. For attached cells, there was more severe mechanical damage than isolated cells.