Quick freezing of mouse embryos: Freezing of inbred strains and 2- and 4-cell embryos by vitrification

Murine embryos of mice of four different inbred strains and one hybrid strain were evaluated for their ability to survive quick freezing by post-thaw in vitro development. The embryos were transferred to an equilibration medium [10% 1,2-propanediol and 20% glycerol in modified PBS (mPBS)] for 10 minutes and frozen in a vitrification medium (25% glycerol and 25% 1,2-prapanediol in mPBS) by direct lowering into liquid nitrogen. Following thawing at 30°C, dilution in 1 M sucrose in mPBS was washing in mPBS the embryos were cultured, and development was evaluated 24-28 hours later. The number of fertilized eggs obtained by superovulation differed among the strains. The survival rates evaluated by in vitro cultivation of the post-thawed inbred embryos varied from 50-85% depending on the genotype, whereas the normal live offspring from transfer of frozen-thawed embryos to recipient females confirms that the quick freezing method is an applicable method for storage of genetically defined mouse strains and stocks. The quick freezing technique was applied on 4- and 8-cell (day-3) mouse embryos of hybrids. The in vitro development of frozen thawed 4- and 8-cell embryos (23% and 21% respectively) was found to be significantly lower than that of frozen-thawed morulae (89%). Permeation in glycerol-solutions before equilibration significantly increased survival of 4- and 8-cell embryos (66% and 77% respectively). By the use of dimethylsulfoxid (DMSO) in the permeation solutions an even higher survival rate was obtained in the cryopreservation of 8-cell mouse embryos (95%). 16% of 2-cell mouse embryos survived when the embryos were exposed to gradually increasing glycerol solutions with DMSO before equilibration and DMSO was added to the vitrification medium.