The submarine incubation system, a new tool for in vitro embryo culture: A technique report

Abstract In vitro culture of sensitive structures such as oocytes and preimplantation embryos requires a specific, stable environment (temperature, gas atmosphere and humidity levels). Most available carbon dioxide thermostates are not adequate for this purpose, as their large interior area is undivided and frequent opening of the doors required by the daily work disturbs equilibrium of the cultures kept inside. A new approach for overcoming this problem is described here. Tissue culture dishes containing embryos are individually wrapped in laminated foil bags nearly impermeable to carbon dioxide, oxygen and nitrogen and are filled with the desired gas mixture, then heat-sealed and submerged into a circulating temperature-adjusted water bath for the required culture period (up to 7 d). In this way, all wrapped culture dishes function as individual incubators (“submarines”). The advantages of this system are stability of temperature, humidity and gas mixture; quick recovery of these parameters after opening; flexibility in using different gas mixtures; safety; cost efficiency; reduced contamination risks; few problems with cleaning; easy transport. When transparent foil is used for wrapping, frequent microscopic observations are also possible without disturbing the gas atmosphere and humidity. Slightly elevated atmospheric pressure inside the foil bags (between 1 and 9 water cm) has no apparent deleterious effects on embryo development. Expired, sterile filtered human lung air containing 4% carbon dioxide and 16 to 17% oxygen expired into the bags was also found to sustain bovine embryo development. Thus, the culture system is also suitable for use under field conditions. To prove the efficiency of this incubation system, in vitro matured and fertilized bovine zygotes were cultured in TCM199 and calf serum on a granulosa cell monolayer using expired lung air in the submerged foil bags. The cumulative result of 25 identical replicates was 517 blastocysts from 1,052 oocytes (49%) at 8 d after fertilization. The Submarine Incubation System provides an alternative method for in vitro embryo production and/or culture of other sensitive tissues or cells.