Department of Molecular Genetics, Weizmann Institute of Science;
J Vis Exp. 2021 Oct 19(176). doi: 10.3791/63160.
Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.
哺乳动物胚胎植入后的培养方法通常效率低下，并且只能在从子宫中取出后的短暂时间内进行。最近已经开发出了一些平台，可以在体外高度稳健且长时间培养从卵圆柱阶段到高级器官发生阶段的小鼠胚胎。这些平台使胚泡前期（E5.5）的胚胎能够适当且忠实的发育，直到后腿形成阶段（E11）。在这些设置中，晚期囊胚期（E7.5）的胚胎在旋转瓶中生长，而从胚泡前期（E5.5 或 E6.5）开始的延长培养则需要静态和旋转瓶培养的结合。此外，对 O2 和 CO2 浓度、气压、葡萄糖水平以及使用特定的体外培养介质的敏感调节对于胚胎的正常发育至关重要。这里提供了一个详细的分步协议，用于延长体外培养的小鼠胚胎。从原肠胚期到器官发生期在体外生长正常的小鼠胚胎的能力代表了一种有价值的工具，可以用于研究胚胎发育过程中不同实验干扰的影响。
