Theoretic considerations regarding slow cooling and vitrification during cryopreservation

This review presents the methodology of using theoretic models for development of cryopreservation protocols by designing specific cooling profiles and selecting appropriate external conditions to optimize cryopreservation survival. Biophysical events during the processes of cryopreservation were examined and corresponding theoretic equations were used to simulate cryopreservation procedures under various slow cooling conditions for rat zygotes in the presence of DMSO, using a 0.25-mL plastic straw as the container. Simulation revealed three regions with their own characteristics and cryopreservation relevance. In addition, this review discusses vitrification cryopreservation using two-step additions. The effects of exposure durations and exposure temperatures on cell survival and subsequent development rates were examined in a series of cryopreservation experiments. Values of accumulative osmotic damage were used to quantitatively examine the magnitude of the associated osmotic damage during cryoprotective agent (CPA) additions and dilutions. In these investigations, oocyte blastocyst rates were highly correlated with the values of accumulative osmotic damage in the processes of CPA additions/dilutions. This review emphasizes the most essential step of the selection of the cell container in the process of cryopreservation, and provides practical suggestions and guidelines for optimizing slow cooling protocols. The review stresses that conducting CPA addition steps at 25 °C would be preferable for vitrification. It also suggests that the final dilution process needs more systematic research to optimize vitrification procedures.