Humidified plastic chamber eliminates media evaporation and osmolality increase during uninterrupted culture in a dry incubator.

Media evaporation during uninterrupted embryo culture in a dry incubator may lead to harmful osmolality increase and result in compromised embryo development. Before new sophisticated incubators are more widely used, a cost-effective, safe, and efficient solution to mitigate media evaporation is needed to provide a proper environment for uninterrupted embryo culture. Five specially designed embryo culture devices were tested to examine their ability to control media evaporation during uninterrupted embryo culture in a modern dry IVF incubator. In the first experiment, a newly designed 3D printed dish with a water reservoir combined with three novel dish lid designs aimed at avoid evaporation and related issues were tested. Dish 1 contained an inner lid; 2 had a lid thickened center area; and 3 had a slanted lid aimed to minimize condensation and avoid water dripping in the embryo culture areas. These were compared to a commercially available Humdish® with a water reservoir in the outer ring and a 5th humidified chamber with a water retention area. All dishes were filled with milliQ water in the water reservoir and six to seven 20 ul embryo culture medium drops covered with 4ml mineral oil were placed in the embryo culture region. To test the 5th humidified plastic chamber, a Mini GPS® culture dish with 20 ul embryo culture medium drops covered with 3.5ml mineral oil were used. All devices were kept in a dry incubator (G185, K-systems) for a six-day incubation period and a regular culture dish without water reservoir was used as a control. Water condensation on the lid over the culture area and osmolality of culture drops were assessed. In the second experiment, CF1 mouse one-cell embryos were cultured with or without the humidified plastic chamber for 5 days in a dry incubator. Day 4 blastocyst formation, and day 5 hatching were assessed. Developmental data (nine replicates with a total of 528 2PNs) were analyzed by chi-squared test, and osmolality (three replicates) were analyzed by one-way ANOVA and pairwise comparisons. Data are presented as the mean± SEM, P<0.05. All four humidified dish designs resulted in water condensation on the lid and dripping into the embryo culture area and were not tested for embryo development. However, the 5th humidified chamber was able to avoid media evaporation inside the culture dish and maintained culture media osmolality throughout six day incubation period (256.4 ± 0.8 mOsm with chamber, versus 272.3 ± 1.2 mOsm without chamber, P<0.05). Day 4 blastocyst formation (79.4% ± 3.6% with chamber, versus 76.2% ± 2.8% without chamber) and day 5 hatching (80.6% ± 2.9% with chamber, versus 76.7% ± 3.3% without chamber) were not significantly different. The combination of the humidified chamber with Mini GPS® culture dish was able to eliminate media evaporation during uninterrupted embryo culture in a dry incubator.