An efficient and mass reproducible method for vitrifying mouse embryos on a paper in cryotubes

To date, the most successful and popular vitrification method is based on the minimum volume cooling (MVC) concept, in which embryos are vitrified in a very small volume of vitrification solution (VS) and then stored in cryotubes in liquid nitrogen (LN₂). Unfortunately, these methods need special devices and may not be suitable for vitrifying a large number of embryos. Theoretically, more embryos in VS on a paper (MVC concept) in cryotubes can be vitrified effectively. Therefore, this study directly vitrifies mouse embryos on a Kimwipes tissue in an 1.8 mL cryotube. The ICR 2-celled to blastocyst embryos were used for testing this procedure. In Treatment 1, embryos transferred with 1-2 μL of VS into a cryotube. Treatment 2 was similar to Treatment 1 except that the cryotube was filled with LN₂. Treatment 3 was identical to Treatment 1 except that a small piece (5 mm²) of a sterilized Kimwipes tissue was placed on the top of VS. Treatment 4 was identical to Treatment 3 except for the cryotube being filled with LN₂. After each treatment, the cryotubes were capped and transferred to a LN₂ tank. After warming, the recovered embryos were cultured in KSOM+AA for 1-3 days. There were no differences in the recovery rate, overnight survival rate, blastocyst rate, and birth rate after embryo transfer among all treatment groups. Our results demonstrated an alternative simple, efficient, and mass reproducible method for vitrifying mouse embryos using papers as a vehicle and cryotubes as a container.