Abstract
For the cryopreservation of embryos, vitrification has many advantages, but it also has disadvantages
because embryos are vitrified with considerable supercooling (i.e., in non-equilibrium state) and thus easily
damaged by intracellular ice formation at over the glass transition point. We tried to develop a novel method
in which embryos are vitrified in near-equilibrium state. The extent of equilibrium was assessed by
examining whether vitrified embryos survive after being kept at -80oC. Mouse embryos were vitrified with
ethylene glycol (EG)-based solutions, EFSc solutions, which were mixtures of EG (30-40% v/v) and an FSc
solution. The FSc solution was PB1 medium containing 30%(v/v) Ficoll plus a high concentration sucrose
(1.5 M). When embryos were vitrified and then kept at -80oC for 4 days, large proportions survived with
EFS35c and EFS40c. When mouse embryos were vitrified with EFS35c, transported with dry ice (-79oC)
from Nankoku to Tsukuba, kept at -80oC for ~ 2 days (at ~80oC for 4 days in total), and recooled with liquid
nitrogen, a high proportion of the embryos developed to term after warming and transfer. In conclusion, we
have developed a novel method by which embryos are vitrified in near-equilibrium, which retains the
advantages of both current vitrification and equilibrium slow freezing.
