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

Objective

To store embryos in LN₂ vapor, which eliminates direct contact with LN₂, is presented as an alternative method to protect embryos cryopreserved by vitrification or slow cooling from LN₂-borne pathogens.

Design

Basic animal research.

Setting

University-affiliated hospital.

Animal(s)

Two-cell mouse embryos collected from superovulated ICR mice.

Intervention(s)

Embryos cryopreserved by vitrification or slow cooling were stored in LN₂ or LN₂ 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.

Main Outcome Measure(s)

Survival, apoptosis, and in vitro and in vivo development of freeze-thawed mouse embryos as a function of cryopreservation and cryostorage methods were determined.

Result(s)

After short- and long-term storage of vitrified and slow-cooled embryos, embryonic survival and development rate were the same for embryos stored in LN₂ vapor and in direct contact with LN₂. 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.

Conclusion(s)

The present study shows that embryos stored in LN₂ 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 LN₂.

Key Words: Liquid nitrogen vapor, long-term storage, mouse embryo, slow cooling, vitrification