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

Abstract

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.

Development of a new device for manipulating frozen mouse 2-cell embryos on the International Space Station

“…However, the embryo recovery rate obtained by the ETC was lower than that shown in this study (submitted). We tried various improvements right up to launch, such as a good equilibration medium for embryo culture in ETC [20], and identified the most appropriate stage of mouse two-cell embryos for the present project [21]. However, space experiments can be challenging.…”

“…However, none of the embryos developed to the blastocysts, likely because of the bag's impermeability to gas (Table 2). Therefore, we developed a new embryo culture system, in which CZB medium was equilibrated with 5% CO 2 in a CO 2 incubator before use (hereinafter referred to as eCZB medium) (Fig 3d) [20]. Next, fresh embryos were directly placed in the Frozebag, or inserted into the mesh bag on the scoop (Fig 3e), placed in the Frozebag, and then cultured for 4 days in eCZB medium (Fig 3f).…”

“…The embryos were thawed using 0.25 M and 0.75 M sucrose-PB1 (S-PB1) [18]. Equilibrated CZB (eCZB) medium was prepared as previously described [20]. Briefly, CZB medium was used to fill the container, the lid was placed slightly off-center, and the container was then placed in the CO 2 incubator for >24 h to equilibrate the CO 2 concentration of the medium with that of the atmosphere.…”

Live-cell imaging of mouse preimplantation embryos using a simple closed glass capillary method

“…We previously developed the "Optimized Medium with CO2 Pressure and Sealed-tube culture method," which is abbreviated to the "Ops culture method" ("Ops" was a fertility deity and earth goddess in ancient Roman religion) 18 . This is a simple and method for culturing embryos in a sealed container, such as a PCR tube, without a CO2 incubator.…”

“…This is a simple and method for culturing embryos in a sealed container, such as a PCR tube, without a CO2 incubator. It has been used successfully used to transport live embryos to other laboratories using a water bottle 18 and was even used on the International Space Station to determine the effect of microgravity on embryo development 19 . Building on this success, we sought to establish a simple and low-cost live-cell imaging system using the Ops culture method and a time-lapse microscope camera.…”