Freezing of cells; role of ice and solutes in cell damage

The mechanism of cellular damage associated with freezing of biological cells is discussed by summarizing the author's recent studies that consists of four different types of experiments. The "solution effects" that designate the influence of elevated concentration of electrolytes during freezing is examined first by a nonfreezing experiment that exposes cells to hypertonic solutions using a perfusion microscope. The cell damage due to the solution effect is evaluated directly from a pseudo-freezing experiment, where cells were subjected to the milieu that simulated a freeze-thaw process in the absence of ice. Contribution of ice formed in the extracellular solution is then estimated from the difference in cell survival between the pseudo-freezing experiment and a corresponding freezing experiment. The cellular injury by the mechanical stress is also examined independently by a cell deformation experiment, which mimicked the situation that cells are compressed and deformed between ice crystals. This experiment was designed to examine a complex effect of mechanical stress from ice and elevated concentration of electrolytes. Based on all these experiments, the role of concentrated solutes and ice is revealed as a function of freezing conditions.