A Continuous In Vitro Culture Protocol for Mouse Embryos From the Zygote to the Egg Cylinder Stage

Mouse embryos undergo drastic morphological changes during implantation, but analysis of this process has been challenging because it occurs within the uterus. Various in vitro culture systems supporting development beyond implantation have been established since the 1970s. More recently, 3D-geec, which supports embryos from the peri-implantation to egg cylinder stages while preserving their three-dimensional organization, was developed as a useful platform for studying this critical period. Combining 3D-geec with genome editing in zygotes or experimental manipulation of embryos should further facilitate mechanistic studies of these stages. In this study, to enable such approaches, we established an in vitro culture protocol that supports continuous development of mouse embryos from the zygote to the egg cylinder stage. Embryos cultured from the zygote to the late blastocyst stage in KSOM^AA medium were not compatible with 3D-geec. These embryos exhibited reduced cell numbers, delayed development, and reduced mTORC1 activity, which regulates cell growth and proliferation. The use of Advanced IVC medium supplemented with 10% fetal bovine serum (FBS) during the blastocyst stage substantially rescued these defects, and the resulting late blastocysts underwent further development into egg cylinders in 3D-geec. Furthermore, genome-edited embryos generated from zygotes developed into egg cylinders using this protocol and were successfully subjected to time-lapse imaging during 3D-geec. Thus, this culture protocol enables continuous in vitro development from the zygote to the egg cylinder stage and provides a platform for studying gene function and cellular dynamics during peri-implantation and early postimplantation development.