Optimised CO2-containing medium for in vitro culture and transportation of mouse preimplantation embryos without CO2 incubator

Conventional in vitro culture and manipulation of mouse embryos require a CO2 incubator, which not only increases the cost of performing experiments but also hampers the transport of embryos to the other laboratories. In this study, we established and tested a new CO2 incubator-free embryo culture system and transported embryos using this system. Using an Anaero pouch, which is a CO2 gas-generating agent, to increase the CO2 partial pressure of CZB medium to 4%–5%, 2-cell embryos were cultured to the blastocyst stage in a sealed tube without a CO2 incubator at 37°C. Further, the developmental rate to blastocyst and full-term development after embryo transfer were comparable with those of usual culture method using a CO2 incubator (blastocyst rate: 97% versus 95%, respectively; offspring rate: 30% versus 35%, respectively). Furthermore, using a thermal bottle, embryos were reliably cultured using this system for up to 2 days at room temperature, and live offspring were obtained from embryos transported in this simple and very low-cost manner without reducing the offspring rate (thermal bottle: 26.2% versus CO2 incubator: 34.3%). This study demonstrates that CO2 incubators are not essential for embryo culture and transportation and that this system provides a useful, low-cost alternative for mouse embryo culture and manipulation.

­­Highly Conducive Ex utero Mouse Embryogenesis from Pre-Gastrulation to Late Organogenesis

In mammals, morphogenesis and organogenesis take place after the embryo implants into the uterus, which makes it relatively inaccessible for observation and manipulation. While methods for in vitro culture of pre- and peri-implantation mouse embryos are routinely used, approaches for efficient and stable culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. We recently developed highly robust ex utero post-implantation mouse embryo culture platforms, that enable appropriate and faithful development of embryos before gastrulation (E5.5) until the hind limb formation stage (E11). In these protocols, late gastrulating embryos (E7.5) are grown in 3D rotating bottles settings, while extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle culture protocols. These systems support stable growth of normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis.