Study on freezing process of killifish egg: utilizing the undercooled state for cryopreservation

The purpose of this study is to find the feasibility of preservation of large cell and tissue by maintaining the undercooled state in a freezing process, leading to avoiding the growth of ice crystals in the intracellular space, which causes destruction of cell and tissue. The fertilized killifish egg was employed to test biological tissue. The cooling system was equipped with Peltier devices and able to decrease the temperature of the test section to -50 degrees C. The cooling rate could be regulated by the electric current supplied to the Peltier devices. In the temperature range 0 to -40 degrees C, the morphology of fertilized killifish egg was observed under a microscope with a cooling rate from 0.1 to 10 degrees C/min. The damage rate to the egg in the intracellular undercooled state was evaluated by hatching rate. As a result, intracellular undercooled states were observed in the freezing process with the extracellular undercooling and the extracellular freezing. Extracellular undercooling proves to preserve the egg, and extracellular freezing frequently damages the egg. Thus the cryopreservation of biological material is achieved by maintaining the undercooled state until the temperature of -40 degrees C, then is instantly frozen by the liquid nitrogen to avoid the growth of ice crystals. The maintaining of the stable undercooled state of biological material is requisite for the initial phase in the freezing process. Therefore, dehydration or maintaining the extracellular stable undercooled state should be desirable to maintain the intracellular undercooled state for cryopreservation of biological material.