Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02114, USA; Shriners Hospitals for Children, Boston, MA, 02114, USA.
Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA, 02114, USA; Shriners Hospitals for Children, Boston, MA, 02114, USA.
Cryobiology. 2021 Feb;98:233-238. doi: 10.1016/j.cryobiol.2020.10.016. Epub 2020 Nov 1.
Vitrification of mammalian oocytes and embryos is typically a two-step procedure involving two solutions of increasing concentrations of cryoprotectants. In the present study, we report a simple vitrification protocol that uses low cryoprotectant concentration and a single medium (LCSM). This medium, along with the traditional high concentration two media (HCTM) protocol, was used to vitrify mouse oocytes, zygotes, and blastocysts using silica capillary, cryotop, cryolock, and 0.25 ml straws. Survival rates, two-cell rates, and blastocyst formation rates were compared for oocytes and zygotes vitrified using both protocols. Results show that the LCSM protocol was as good as or better than the traditional HCTM protocol for vitrifying mouse MII oocytes and zygotes using silica capillary, cryotop, and cryolock. On the other hand, for blastocysts, only silica capillary using LCSM had comparable results with the traditional HCTM protocol while cryolock and cryotop had significantly lower percentages of re-expanded and hatched blastocysts. Collapsing blastocysts prior to vitrification or longer duration for better cryoprotectant distribution in multicellular embryos may improve the outcome. In conclusion, the LCSM protocol, with one medium of much lower cryoprotectant concentrations and shorter equilibration time, reduces exposure to cryoprotectant toxicity while improves efficiency, consistency and reliability for mammalian oocyte and embryo preservation.
哺乳动物卵母细胞和胚胎的玻璃化通常是一个两步过程，涉及两种浓度逐渐增加的冷冻保护剂溶液。在本研究中，我们报告了一种简单的玻璃化方案，该方案使用低浓度的冷冻保护剂和一种单一的培养基（LCSM）。该培养基与传统的高浓度两种培养基（HCTM）方案一起，用于使用硅基毛细管、 cryotop、 cryolock 和 0.25ml 吸管对小鼠卵母细胞、受精卵和囊胚进行玻璃化处理。比较了使用两种方案玻璃化处理的卵母细胞和受精卵的存活率、二细胞率和囊胚形成率。结果表明，对于使用硅基毛细管、 cryotop 和 cryolock 进行玻璃化处理的小鼠 MII 卵母细胞和受精卵，LCSM 方案与传统的 HCTM 方案一样好，甚至更好。另一方面，对于囊胚，只有使用 LCSM 的硅基毛细管与传统的 HCTM 方案具有可比的结果，而 cryolock 和 cryotop 的囊胚再扩张和孵化率明显较低。在玻璃化处理前使囊胚塌陷或使多细胞胚胎中的冷冻保护剂分布时间更长，可能会提高效果。总之，LCSM 方案使用一种浓度较低的冷冻保护剂和较短的平衡时间的单一培养基，降低了对冷冻保护剂毒性的暴露，同时提高了哺乳动物卵母细胞和胚胎保存的效率、一致性和可靠性。
