Equilibrium vitrification of mouse embryos.
For the cryopreservation of embryos, vitrification has various advantages, but it also has disadvantages because embryos are vitrified with a considerable supercooling (i.e., in nonequilibrium). Here, we tried to develop a novel method in which embryos are vitrified in near-equilibrium. The extent of equilibrium was assessed by examining whether vitrified embryos survive after being kept at -80 degrees C. Two-cell embryos of ICR mice were vitrified with ethylene glycol (EG)-based solutions, either EFSa or EFSc solutions, which were mixtures of EG (30%-40%) and an FSa or FSc solution, respectively. The FSa and FSc solutions were PB1 medium containing 30% Ficoll plus 0.5 or 1.5 M sucrose, respectively. In vitro survival rate was high when embryos vitrified with 30%-40% EG (EFS30a, EFS40a, EFS30c, and EFS40c) were warmed rapidly. When embryos were vitrified and then kept at -80 degrees C for 4 days, large proportions survived with EFS30c and EFS40c. When embryos were vitrified with EFS35c or EFS40c, the survival rate was high even for those kept at -80 degrees C for 10 days. When embryos of ICR and C57BL/6J mice were vitrified with EFS35c or EFS40c and then kept at -80 degrees C for 4 days, the survival rate was high even after recooling in liquid nitrogen; a high proportion (75%) of C57BL/6J embryos vitrified with EFS35c developed to term after transfer. In conclusion, we have developed a novel method by which embryos are vitrified in near-equilibrium. This will be a supreme method for cryopreservation, retaining the advantages of both current vitrification and equilibrium slow freezing.
Jin
Bo
B
Laboratory of Animal Science, College of Agriculture, Kochi University, Nankoku, Kochi, Japan.
Mochida
Keiji
K
Ogura
Atsuo
A
Hotta
Eri
E
Kobayashi
Yukiko
Y
Ito
Kaori
K
Egawa
Go
G
Seki
Shinsuke
S
Honda
Hiroshi
H
Edashige
Keisuke
K
Kasai
Magosaburo
M
D000818:Animals; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D004622:Embryo, Mammalian / Q000502:physiology*; D019855:Ethylene Glycol; D005260:Female; D005362:Ficoll; D006358:Hot Temperature; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D012996:Solutions; D013395:Sucrose
D012996:Solutions; D005362:Ficoll; D013395:Sucrose; D019855:Ethylene Glycol