Stability of mouse oocytes at -80 °C: the role of the recrystallization of intracellular ice.

The germplasm of mutant mice is stored as frozen oocytes/embryos in many facilities worldwide. Their transport to and from such facilities should be easy and inexpensive with dry ice at -79 °C. The purpose of our study was to determine the stability of mouse oocytes with time at that temperature. The metaphase II oocytes were cryopreserved with a vitrification solution (EAFS10/10) developed by M Kasai and colleagues. Two procedures were followed. In one, the samples were cooled at 187 °C/min to -196 °C, warmed to -80 °C, held at -80 °C for 1 h to 3 months, and warmed to 25 °C at one of three rates. With the highest warming rate (2950 °C/min), survival remained at 75% for the first month, but then slowly declined to 40% over the next 2 months. With the slowest warming (139 °C/min), survival was only ∼ 5% even at 0 time at -80 °C. In the second procedure, the samples were cooled at 294 °C/min to -80 °C (without cooling to -196 °C) and held for up to 3 months before warming at 2950 °C/min. Survival was ∼ 90% after 7 days and dropped slowly to 35% after 3 months. We believe that small non-lethal quantities of intracellular ice formed during the cooling and that the intracellular crystals increased to a damaging size by recrystallization during the 3 month's storage at -80 °C. From the practical point of view, this protocol yields sufficient stability to make it feasible to ship oocytes worldwide in dry ice.