Injury of larger 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 the fertilized killifish egg was observed under a microscope with a cooling rate from 0.1 to 10°C/min. In glycerol-water, 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 hatching rate. As a result, when the egg shell defectively buckled due to dehydration of perivitelline, the hatching rate was more than 80% in glycerol-water solution. Hatching rate was maximum at the glycerol concentration of 7.5%. Nearly identical results were obtained in DMSO-water solution (maximum at 15%). In distilled water, hatching rate was very low. Transformation of egg shell and injury of the egg are not correlated. Optimum concentration of cryoprotectant minimizes injury of larger cells and tissue due to extracellular freezing.