Serum replacement with a growth factor-free synthetic substance in culture medium contributes to effective establishment of mouse embryonic stem cells of various origins

Objective: to evealuate whether serum replacement with growth factor-free synthetic substances contributed to the effective establishment of embryonic stem (ES) cells. Design: Randomized, prospective model study. Setting: Gamete and stem cell biotechnology laboratory at Seoul National University in Korea. Animal(s): F1 (C57BL6 x DBA2) mice. Intervention(s): Blastocysts of different origins were cultured in sperm-replaced media. Main Outcome Measure(s): Embryonic stem cell establishment. Result(s): Eight batches of ES cells were established from colony-forming inner cell mass cells after the replacement of fetal bovine serum (FBS) with synthetic knockout serum replacement (KSR) in mkDMEN. The established cells were positive for ES cell markers and formed both embryoid bodies in vitro and teratomas in vivo, but the established cell batches and control (transformed) ES cells responded differently to the culture media. Higher levels of cell viability were detected after the replacement with the 75:25 FBS-KSR mixture than with any other mixtures, and a gradual decrease in viability was detected as the KSR volume ratio was increased. The 75:25 FBS-KSR mixture-containing medium supported ES cell establishment of outbred ICR, F1 and F2 of C57/BL6/DBA2; F1 parthenogenetic and ES cell-completed tetraploid blastocysts; and single ES-cell cultures. Conclusion(s): A serum-replaced medium could be used for effective ES-cell establishment of various origins.