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

Abstract

The germplasm of mutant mice is stored as frozen oocytes/embryos in many facilities world-wide. 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 hour to three months, and warmed to 25 °C at one of three rates. With the highest warming rate (2,950 °C/min), survival remained at 75% for the first month, but then slowly declined to 40% over the next two 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 2,950 °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-months storage at −80 °C. From the practical point of view, this protocol yields sufficient stability to make it feasible to ship oocytes world-wide in Dry Ice.

Generation of Live Piglets from Cryopreserved Oocytes for the First Time Using a Defined System for In Vitro Embryo Production

“…Consecutive insertion of several vitrified objects in warming media may, however, gradually reduce the medium temperature and thus the warming speed. During the warming of vitrified embryos or oocytes, intracellular ice can form around −80°C which is detrimental for their viability [9] . Recently it has been confirmed in mice that high-and-stable warming rates are crucial to pass through this dangerous temperature zone and are essential to provide high survival rates for oocyte vitrification [10] – [12] .…”

Storage Temperatures for Cold-Chain Delivery in Cell Therapy: A Study of Alginate-Encapsulated Liver Cell Spheroids Stored at −80°C or −170°C for Up to 1 Year

“…25 In this study, vitrification techniques with high CPA concentrations were applied, which differ from the slow-cooling protocol used for the ELS. Nevertheless, these authors showed a significant fall-off in viability by 3 months when the oocytes were stored at -80°C, and this was more pronounced at slower warming rates.…”

Equilibrium vitrification of mouse embryos at various developmental stages using low concentrations of cryoprotectants

“…Similar results were obtained for two-cell mouse embryos in a previous study [9]. This suggests that EDFS10/10a damaged embryos based on toxicity during long-term storage at -80 o C. Seki and Mazur reported that mouse oocytes can be cryopreserved using EAFS10/10a (containing 10% (v/v) EG, 10% (v/v) acetamide, and 0.4 M sucrose) in LN2 and then stored at -80°C for one to three months, but developmental ability decreased gradually during storage [25]. Therefore, with storage at -80°C, water in the embryos was devitrified without recrystallization because of the high degree of dehydration.…”