Mouse Embryo Cryopreservation by High-Osmolality Vitrification

Abstract

In conventional vitrification methods, the embryos are vitrified under considerable supercooling (i.e., under nonequilibrium conditions). This protocol is a refinement of newer equilibrium vitrification methods. The equilibrium vitrification solution contains a high concentration of cryoprotectants that increase its osmolality and allows vitrified embryos to survive not only during storage in liquid nitrogen (LN2) at -196°C but also during temporary holding at -80°C (for at least 5 mo). This high-osmolality vitrification (HOV) method is as simple as conventional vitrification and provides essentially the same high postwarming survival rate. It has several advantages over conventional vitrification: (1) Cryopreserved embryos are less likely to be damaged during handling for warming; (2) samples can be temporarily evacuated to a -80°C freezer and can be successfully recovered after 5 mo at -80°C; (3) samples can be arranged and sorted at -80°C; and (4) vitrified embryos can be transported using dry ice. Also included here is an alternative protocol that describes the use of straws instead of cryotubes.