Fracture damage of embryos and its prevention during vitrification and warming
Fracture damage of embryos and its prevention during vitrification and warming
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DOI：
10.1006/cryo.1996.0046
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发表时间：
1996-08-01
期刊：
影响因子：
2.7
通讯作者：
Edashige, K
中科院分区：
文献类型：
--
作者：
Kasai, M;Zhu, SE;Edashige, K
通过玻璃化冷冻和加温的重复循环检查小鼠囊胚中断裂损伤的频率。将悬浮在乙二醇、Ficoll和蔗糖吸管溶液中的小鼠囊胚直接（快速）或在液氮蒸汽中保持3分钟或更长时间（适度）后投入液氮中。通过将玻璃化样品立即（快速）或在空气中保持5-30秒（适度）后浸入25 ℃水中来加热玻璃化样品。加热的吸管被重新冷却和重新加热多达9次，以夸大冷却和加热的效果。当胚胎快速冷却和加热一次时，胚胎损伤的发生率仅为1.2%，并且91%的回收胚胎在培养中重新扩增。然而，随着反复快速冷却和升温，玻璃化冷冻损伤的发生率增加，在10次玻璃化冷冻后达到75%;存活率也下降。当胚胎在空气中悬浮15或30 s，进行10个循环的适度冷却和适度加热时，100%的胚胎具有完整的透明带。另一方面，在10次玻璃化冷冻后，适度冷却然后快速升温或快速冷却然后适度升温，分别有41%和16%的胚胎的胚胎受损。因此，在冷却和升温期间都会发生断裂损坏，但可以通过采用稍微较慢的冷却和升温速率来完全防止断裂损坏。此外，在空气中悬浮15秒的10个循环的适度冷却和适度加热后的高存活率（88%）表明玻璃化、熔化或温度波动本身不影响胚胎存活。(C)出版社：Academic Press，Inc.
The frequency of fracture damage in mouse blastocysts was examined by repeated cycles of vitrification and warming. Mouse blastocysts suspended in a solution of ethylene glycol, Ficoll, and sucrose in a straw were plunged into liquid nitrogen either directly (rapidly) or after holding them in liquid nitrogen vapor for 3 min or more (moderately). Vitrified samples were warmed by plunging them into 25 degrees C water either immediately (rapidly) or after holding in air for 5-30 s (moderately). Warmed straws were recooled and rewarmed up to 9 times, to exaggerate the effect of cooling and warming. When embryos were cooled and warmed rapidly once, the incidence of the zona damage was only 1.2%, and 91% of recovered embryos reexpanded in culture. However, with repeated rapid cooling and warming, the Incidence of zona damage increased, reaching 75% after 10 vitrifications; survival also dropped. When embryos were subjected to 10 cycles of moderate cooling and moderate warming with 15 or 30 s of suspension in air, 100% of the embryos had intact zonae. On the other hand, with moderate cooling followed by rapid warming or with rapid cooling followed by moderate warming, 41 and 16% of embryos had damaged zona, respectively, after 10 vitrifications. Therefore, fracture damage occurs during both cooling and warming, but it can be prevented completely by employing somewhat slower rates of cooling and warming. Furthermore, a high survival rate (88%) after 10 cycles of moderate cooling and moderate warming with 15 s of suspension in air indicates that vitrification, melting, or temperature fluctuation per se do not affect embryonic survival. (C) 1996 Academic Press, Inc.