Long-term liquid nitrogen vapor storage of mouse embryos cryopreserved using vitrification or slow cooling

Abstract

To store embryos in LN(2) vapor, which eliminates direct contact with LN(2), is presented as an alternative method to protect embryos cryopreserved by vitrification or slow cooling from LN(2)-borne pathogens. Basic animal research. University-affiliated hospital. Two-cell mouse embryos collected from superovulated ICR mice. Embryos cryopreserved by vitrification or slow cooling were stored in LN(2) or LN(2) vapor and then thawed after 1 week, 1 month, or 6 months. Thawed embryos cultured to the blastocyst stage in vitro were evaluated or transferred into the uterus of foster mothers. Survival, apoptosis, and in vitro and in vivo development of freeze-thawed mouse embryos as a function of cryopreservation and cryostorage methods were determined. After short- and long-term storage of vitrified and slow-cooled embryos, embryonic survival and development rate were the same for embryos stored in LN(2) vapor and in direct contact with LN(2). Cell numbers and apoptosis frequency were similarly indistinguishable between the two groups after storage of embryos for 6 months, and there were no differences in delivery rate or litter size after ET. The present study shows that embryos stored in LN(2) vapor retain full developmental potential. This storage system thus represents a useful alternative for safe and effective long-term storage of embryos that avoids direct contact with LN(2).