Equilibrium vitrification of mouse embryos using low concentrations of cryoprotectants

Previously, we developed a method for vitrification of mouse embryos in a near-equilibrium state using EFS35c, PB1 medium containing 35% (v/v) ethylene glycol, and 0.98 M sucrose. This method has advantages in both slow freezing and vitrification. However, since the vitrification solution in this method contains high concentrations of cryoprotectants and thus has high osmolality, the solution would injure oocytes and embryos with high sensitivity to chemical toxicity and high osmolality. In this study, we examined whether embryos could be vitrified in a near-equilibrium state using a solution containing low concentrations of cryoprotectants and thus with low osmolality. To investigate whether embryos were vitrified in a near-equilibrium state, 2-cell mouse embryos were vitrified with EDFS10/10a, PB1 medium containing 10% (v/v) ethylene glycol, 10% (v/v) DMSO, and 0.4 M sucrose, in liquid nitrogen, stored at -80 °C for 4-28 days, and warmed in water at 25 °C. The viability of the embryos was evaluated by the appearance of embryos after warming and developmental ability. When embryos were vitrified in liquid nitrogen using EDFS10/10a, the survival and developmental ability into blastocysts after storage at -80 °C for 7 days were high, indicating that embryos were vitrified in a near-equilibrium state. A high proportion of embryos vitrified with EDFS10/10a developed to term after transportation with dry ice, re-cooling in liquid nitrogen, and transfer to recipients. Therefore, new equilibrium vitrification developed in this study may be useful for oocytes and embryos that are highly sensitive to the toxicity of cryoprotectants and high osmolality.