Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN, Kobe, Japan.
PLoS One. 2012;7(10):e47512. doi: 10.1371/journal.pone.0047512. Epub 2012 Oct 9.
To culture preimplantation embryos in vitro, water-jacketed CO(2) incubators are used widely for maintaining an optimal culture environment in terms of gas phase, temperature and humidity. We investigated the possibility of mouse embryo culture in a plastic bag kept at 37°C. Zygotes derived from in vitro fertilization or collected from naturally mated B6D2F1 female mice were put in a drop of medium on a plastic culture dish and then placed in a commercially available plastic bag. When these were placed in an oven under air at 37°C for 96 h, the rate of blastocyst development and the cell numbers of embryos decreased. However, when the concentration of O(2) was reduced to 5% using a deoxidizing agent and a small oxygen meter, most zygotes developed into blastocysts. These blastocysts were judged normal according to their cell number, Oct3/4 and Cdx2 gene expression levels, the apoptosis rate and the potential for full-term development after embryo transfer to pseudopregnant recipients. Furthermore, using this system, normal offspring were obtained simply by keeping the bag on a warming plate. This culture method was applied successfully to both hybrid and inbred strains. In addition, because the developing embryos could be observed through the transparent wall of the bag, it was possible to capture time-lapse images of live embryos until the blastocyst stage without needing an expensive microscope-based incubation chamber. These results suggest that mouse zygotes are more resilient to their environment than generally believed. This method might prove useful in economical culture systems or for the international shipment of embryos.
为了在体外培养胚胎，水套二氧化碳培养箱被广泛用于维持气体、温度和湿度的最佳培养环境。我们研究了在 37°C 的塑料袋中培养小鼠胚胎的可能性。体外受精获得的受精卵或从自然交配的 B6D2F1 雌鼠中收集的受精卵被放在一滴培养液滴在塑料培养皿上，然后放在市售塑料袋中。当这些放在烤箱中空气 37°C 下孵育 96 小时时，囊胚发育率和胚胎细胞数量减少。然而，当使用脱氧剂和小型氧气计将 O(2)浓度降低到 5%时，大多数受精卵发育成囊胚。这些囊胚根据其细胞数、Oct3/4 和 Cdx2 基因表达水平、凋亡率以及胚胎移植到假孕受体后的全期发育潜力，被判断为正常。此外，使用这种系统，只需将袋子放在加热板上即可获得正常的后代。这种培养方法成功地应用于杂交和近交系。此外，由于可以通过袋子的透明壁观察到发育中的胚胎，因此可以在不需要昂贵的基于显微镜的孵育室的情况下，拍摄到囊胚阶段的胚胎实时成像。这些结果表明，与普遍认为的相比，小鼠受精卵对环境的适应性更强。这种方法可能在经济的培养系统或胚胎的国际运输中证明有用。
