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14 October 2009 Equilibrium Vitrification of Mouse Embryos
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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°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°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°C for 10 days. When embryos of ICR and C57BL/6J mice were vitrified with EFS35c or EFS40c and then kept at −80°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.

Bo Jin, Keiji Mochida, Atsuo Ogura, Eri Hotta, Yukiko Kobayashi, Kaori Ito, Go Egawa, Shinsuke Seki, Hiroshi Honda, Keisuke Edashige, and Magosaburo Kasai "Equilibrium Vitrification of Mouse Embryos," Biology of Reproduction 82(2), 444-450, (14 October 2009).
Received: 22 March 2009; Accepted: 1 October 2009; Published: 14 October 2009

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