Small-volume vitrification and rapid warming yield high survivals of one-cell rat embryos in cryotubes
Small-volume vitrification and rapid warming yield high survivals of one-cell rat embryos in cryotubes
复制标题
小体积玻璃化和快速升温可提高冷冻管中单细胞大鼠胚胎的存活率
DOI：
10.1093/biolre/ioab059
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发表时间：
2021
期刊：
影响因子：
3.6
通讯作者：
Seki S
中科院分区：
文献类型：
--
作者：
Fukuda Y;Higashiya M;Obata T;Basaki K;Yano M;Matsumura K;Ono K;Ohba T;Okamoto Y;Nishijima K;Seki S
为了低温保存细胞，必须在冷却和加热过程中避免细胞内结冰。实现这一目标的一种方法是将细胞内的水转化为非结晶玻璃。目前认为，为了实现这种玻璃化，细胞必须悬浮在非常高浓度（20-40%）的玻璃化溶质中，然后非常迅速地冷却。在此，我们报告，这种信念是错误的，就单细胞大鼠胚胎的玻璃化。在本研究中，用5 μL EFS10（10%乙二醇（EG）， 27% Ficoll和0.45 M蔗糖的混合物）在低温管中以中等冷却速率玻璃化单细胞大鼠胚胎，并以不同速率加热。根据细胞发育成囊胚和发育至足月的能力来评估存活率。以2613°C/min的冷却速率将胚胎玻璃化，并加入1 mL蔗糖溶液（0.3 M, 50°C），以18467°C/min的升温速率解冻，58.1±3.5%的efs10玻璃化胚胎发育为囊胚，50.0±4.7%发育为足月。这些比率与未处理的完整胚胎相似。使用传统的冷冻管，即使使用低浓度（10%）的细胞渗透冷冻保护剂和低冷却速率，我们也能通过快速加热获得与完整胚胎相当的单细胞大鼠胚胎的发育能力。
To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to achieve this is to convert the water inside the cells into a non-crystalline glass. It is currently believed that to accomplish this vitrification, the cells must be suspended in a very high concentration (20–40%) of a glass-inducing solute, and subsequently cooled very rapidly. Herein, we report that this belief is erroneous with respect to the vitrification of one-cell rat embryos. In the present study, one-cell rat embryos were vitrified with 5 μL of EFS10 (a mixture of 10% ethylene glycol (EG), 27% Ficoll, and 0.45 M sucrose) in cryotubes at a moderate cooling rate, and warmed at various rates. Survival was assessed according to the ability of the cells to develop into blastocysts and to develop to term. When embryos were vitrified at a 2613 °C/min cooling rate and thawed by adding 1 mL of sucrose solution (0.3 M, 50 °C) at a warming rate of 18 467 °C/min, 58.1 ± 3.5% of the EFS10-vitrified embryos developed into blastocysts, and 50.0 ± 4.7% developed to term. These rates were similar to those of non-treated intact embryos. Using a conventional cryotube, we achieved developmental capabilities in one-cell rat embryos by rapid warming that were comparable to those of intact embryos, even using low concentrations (10%) of cell-permeating cryoprotectant and at low cooling rates.