HIGH IN-VITRO AND IN-VIVO SURVIVAL OF DAY-3 MOUSE EMBRYOS VITRIFIED OR FROZEN IN A NONTOXIC SOLUTION OF GLYCEROL AND ALBUMIN
HIGH IN-VITRO AND IN-VIVO SURVIVAL OF DAY-3 MOUSE EMBRYOS VITRIFIED OR FROZEN IN A NONTOXIC SOLUTION OF GLYCEROL AND ALBUMIN
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DOI：
10.1530/jrf.0.1010681
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发表时间：
1994-08-01
期刊：
影响因子：
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通讯作者：
WOOD, MJ
中科院分区：
文献类型：
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作者：
RALL, WF;WOOD, MJ
检查由 6.5 mol 甘油 l(-1) 和 6% (w/v) BSA 组成的改良 Dulbecco PBS 溶液（指定溶液 VS3a）的玻璃化溶液对 8-12 细胞小鼠胚胎的冷冻保存。当以10-2500℃min(-1)的速率冷却至-196℃时，溶液VS3a玻璃化，并且当在200或2000℃min(-1)加热时，玻璃化悬浮液不结晶。然而，在 5°C min(-1) 下缓慢冷却或在 20°C min(-1) 下缓慢升温会导致溶液 VS3a 出现可见的结晶。胚胎在室温下分三步在溶液VS3a中平衡，并以允许用1mol蔗糖l(-1)进行管内稀释的方式放入0.25ml塑料吸管中。如果暴露于 100% VS3a 溶液的总时间不超过 5 分钟，则在 VS3a 溶液中平衡并立即稀释的胚胎在体外表现出高胚泡发育率 (> 90%)。当在 100% VS3a 溶液中平衡 1 分钟，然后使用三种冷却速率（5200 或 2500 °C min(-1)）和三种升温速率（20 200 或 2000 °C min(-1)）的所有组合进行冷冻保存时，胚胎在体外表现出高发育率（75-97%）。尽管胚胎悬浮液在 5 ℃ min(-1) 缓慢冷却过程中明显结晶，但冷却速率并不是显着的差异来源 (P > 0.26)。然而，研究发现升温速率对胚胎存活率影响虽小但显着（P < 0.05）。玻璃化胚胎移植到养母体内后表现出较高的体内发育率（63%）。一项配对胚胎移植研究比较了 VS3a 中的玻璃化冷冻与 1.5 mol 甘油 l(-1) 中的传统缓慢冷冻，结果显示两种冷冻保存方法后体内发育速率没有差异 (P > 0.12)。这些结果表明，可以通过简单的程序在溶液 VS3a 中玻璃化胚胎，该程序包括迄今为止报道的最广泛的冷却和升温条件。
A vitrification solution consisting of 6.5 mol glycerol l(-1) and 6% (w/v) BSA in a modified Dulbecco's PBS (designated solution VS3a) was examined for the cryopreservation of 8-12-cell mouse embryos. Solution VS3a vitrified when cooled to - 196 degrees C at rates of 10-2500 degrees C min(-1) and vitrified suspensions did not crystallize when warmed at 200 or 2000 degrees C min(-1). However, slow cooling at 5 degrees C min(-1) or slow warming at 20 degrees C min(-1) resulted in visible crystallization of solution VS3a. Embryos were equilibrated in solution VS3a in three steps at room temperature and placed into a 0.25 ml plastic straw in a way that permitted in-straw dilution with 1 mol sucrose l(-1). Embryos equilibrated in solution VS3a and diluted immediately exhibited high rates of development in vitro to blastocysts (> 90%) if the total time of exposure to 100% solution VS3a did not exceed 5 min. Embryos exhibited high rates of development in vitro (75-97%) when equilibrated in 100% solution VS3a for 1 min and then cryopreserved using all combinations of three rates of cooling (5200 or 2500 degrees C min(-1)) and three rates of warming (20 200 or 2000 degrees C min(-1)). Although embryo suspensions visibly crystallized during slow cooling at 5 degrees C min(-1), the rate of cooling was not a significant source of variance (P > 0.26). However, the rate of warming was found to have a small but significant effect on embryo survival (P < 0.05). Vitrified embryos exhibited a high rate of development in vivo after transfer to foster mothers (63%). A paired embryo transfer study comparing vitrification in VS3a with conventional slow freezing in 1.5 mol glycerol l(-1) showed no difference in the rate of development in vivo after either cryopreservation method (P > 0.12). These results demonstrate that embryos can be vitrified in solution VS3a by a simple procedure that includes the widest range of cooling and warming conditions reported to date.