Vitrification of mouse embryos with super-cooled air

Abstract

To develop a closed vitrification device (i.e., one that requires no direct contact with liquid nitrogen) for successful cryostorage of embryos. Prospective laboratory research study. University-based research laboratory. F1 mice and mouse embryos. Mouse embryos were vitrified using two methods and compared with nonvitrified controls. Embryos were vitrified on a device by either [1] presealing it within a straw before plunging into liquid nitrogen or [2] placing the straw into liquid nitrogen so that the air inside the straw is super-cooled before inserting the device holding the embryos. Survival, subsequent embryo development, and cell number were determined. Embryos were also cryopreserved for 12 months to assess long-term storage. Synchronized ETs were performed to compare viability with nonvitrified control embryos. All embryos survived with both techniques. Day-4 and -5 embryo development was comparable between the two vitrification methods. Use of the presealing method resulted in a significantly lower mean cell number than the postsealing method and control. Long-term storage did not affect subsequent embryo development or cell number. The implantation and fetal development rates of embryos vitrified with super-cooled air were comparable to those for nonvitrified control embryos. These data demonstrate that a closed vitrification device (Rapid-i), which does not require direct liquid nitrogen contact for vitrification, is appropriate for vitrification and long-term storage of mouse embryos.