The establishment of mouse embryonic stem cell cultures on 96-well plates for high-throughput screening.

Embryonic stem (ES) cells can be valuable for monitoring differentiation processes and for improving applications in basic developmental biology. The application of ES cells can be a useful tool for drug discovery and toxicology. Therefore, we suggest the high-throughput screening (HTS) system based on ES cells in this study. Firstly, we optimized the feeder-free condition and seeding cell number which can maintained for at least 7 days without over-confluency. We analyzed the system by cell viability, proliferation activity, RT-PCR and morphologic/immunohistochemical evaluations. The optimal cell seeding number was 30/well that was maintained the typical colonial morphology over 9 d with 1,000 U/ml LIF in the limited space. The cell in optimized condition expressed ALP, SSEA-1, Oct 4 and Nanog and the genetic expressions showed similar to protein expressions. The cell lineage marker expressions showed faint or none. The cell viability and proliferation activity were increased in time-dependent manner in our optimized HTS system. In conclusion, the novel HTS system using ES cells can by useful for developing models for drug discovery as well as toxicological screening in the near future.