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.
Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, Tennessee 37996-0840, USA.
Shinsuke
S
Seki
Peter
P
Mazur
D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D003080:Cold Temperature / Q000009:adverse effects; D015925:Cryopreservation / Q000379:methods*; D003460:Crystallization; D005260:Female; D005615:Freezing / Q000009:adverse effects*; D007053:Ice / Q000009:adverse effects*; D042541:Intracellular Space; D051379:Mice; D008813:Mice, Inbred ICR; D009865:Oocytes; D013997:Time Factors; D058989:Vitrification
D007053:Ice
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