Injury of biological tissue by extracellular freezing

The purpose of this study is to reduce injury of larger cells and tissue (≥ 1 mm) due to extracellular freezing to achieve successful cryopreservation. In the temperature range of 0 to -40 °C, the morphology of a fertilized killifish egg was observed under a microscope with a cooling rate from 0.1 to 10 °C/min. In glycerol-water, dimethyl sulfoxide (DMSO)-water (cryoprotectant solution), and distilled water, the damage rate to the egg by maintaining extracellular freezing of short duration of various temperatures was evaluated by the hatching rate. As a result, when the egg shell defectively buckled due to dehydration of perivitelline, the hatching rate was more than 80 percent in glycerol-water solution. The hatching rate was maximum at a glycerol concentration of 7.5 percent. Nearly identical results were obtained in a DMSO-water solution (maximum at 15 percent). In distilled water, the hatching rate was very low. Transformation of egg shell and injury of the egg are not correlated. Optimum concentration of the cryoprotectant minimizes injury of larger cells and tissue due to extracellular freezing.