Hollow fiber vitrification: a novel method for vitrifying multiple embryos in a single device.
Current embryo vitrification methods with proven efficacy are based on the minimum volume cooling (MVC) concept by which embryos are vitrified and rewarmed ultrarapidly in a very small amount of cryopreserving solution to ensure the high viability of the embryos. However, these methods are not suitable for simultaneously vitrifying a large number of embryos. Here, we describe a novel vitrification method based on use of a hollow fiber device, which can easily hold as many as 40 mouse or 20 porcine embryos in less than 0.1 μl of solution. Survival rates of up to 100% were obtained for mouse embryos vitrified in the presence of 15% DMSO, 15% ethylene glycol and 0.5 M sucrose using the hollow fiber vitrification (HFV) method, regardless of the developmental stage of the embryos (1-cell, 2-cell, morula or blastocyst; n = 50/group). The HFV method was also proven to be effective for vitrifying porcine in vitro- and in vivo-derived embryos that are known to be highly cryosensitive. For porcine embryos, the blastocyst formation rate of in vitro maturation (IVM)-derived parthenogenetic morulae after vitrification (48/65, 73.8%) did not decrease significantly compared with non-vitrified embryos (59/65, 90.8%). Transfer of 72 in vivo-derived embryos vitrified at the morula/early blastocyst stages to 3 recipients gave rise to 29 (40.3%) piglets. These data demonstrate that the HFV method enables simultaneous vitrification of multiple embryos while still adhering to the MVC concept, and this new method is very effective for cryopreserving embryos of mice and pigs.
Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571, Japan.
Hitomi
H
Matsunari
Miki
M
Maehara
Kazuaki
K
Nakano
Yuka
Y
Ikezawa
Yui
Y
Hagiwara
Norihisa
N
Sasayama
Akio
A
Shirasu
Hisayoshi
H
Ohta
Masashi
M
Takahashi
Hiroshi
H
Nagashima
D000818:Animals; D001755:Blastocyst; D003433:Crosses, Genetic; D015925:Cryopreservation / Q000295:instrumentation / Q000379:methods / Q000662:veterinary*; D004477:Ectogenesis; D004624:Embryo Transfer / Q000662:veterinary*; D004622:Embryo, Mammalian; D047108:Embryonic Development; D005260:Female; D059471:In Vitro Oocyte Maturation Techniques / Q000662:veterinary; D050498:Live Birth; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D009028:Morula; D010312:Parthenogenesis; D011247:Pregnancy; D018873:Pregnancy Rate; D011897:Random Allocation; D034421:Sus scrofa; D058989:Vitrification; D015053:Zygote