Development of mouse embryos in hanging drop culture

Mouse blastocysts were cultured in hanging drops for up to 6 days in order to study development under conditions that avoid the distortion of embryos typically seen when they are allowed to attach to a glass or plastic surface. The survival rate of embryos in hanging drops was equal to that of embryos attached to culture dishes and superior to that of embryos suspended in gyrating flasks. Development of the embryonic portion was similar to that in vivo and on culture dishes but slower than in vivo; the egg cylinder stage was reached after 8-10 equivalent gestation days (4 to 6 days in culture), while that stage is reached at 5.5 to 6 days in vivo. The trophectoderm, however, developed in a unique manner. The cells migrated away from the inner cell mass (ICM), similar to embryos on a culture dish, but without a surface on which to spread they clustered distal to the ICM. In vivo, trophectoderm remained covering the ICM. By 5 days in hanging drop culture the embryos had developed a segmented appearance with trophoblast giant cells at the abembryonic pole, extraembryonic cells not covered by vacuolated endoderm in the central region, and embryonic endoderm surrounding a developing proamniotic cavity in embryonic ectoderm at the embryonic pole. These observations suggest that the trophectoderm is able to follow a developmental program independent of that in the embryonic portion and that its behavior is dominated by the different adhesive properties of the trophoblastic and embryonic cells.