Abstract: Objective To establish a serum-free and feeder-free culture system for human embryonic stem cells (HESc). Methods The HESX-01 cells were cultured in Knockout medium (the first group), self-made serum-free medium (the second group) and improved serum-free medium with bFGF and heparin (the third group), respectively. The morphology, clone number and clone size were observed after the twenty-fifth generation. And the surface labeling immunocytochemical analysis, Fluorescence-activated cell sorter analysis (FACS) and embryoid bodies formation were utilized to determine the character of HESc. Results After twenty-fifth passages the clones of the second group were gradually different- tiated, and the HESX-01 cells in the first and third group showed typical human embryonic stem cell morphology, they all expressed Nanog, Oct-4，Tra-1-60 and SSEA-4, but did not express SSEA-1, the same results were observed from FACS analysis. The HESc could form embryoid body in vivo. Besides, the significant differences were oberserved in the second group compared with the first and third group in clone nuber and size per view.Conclutions The self-made serum-free and feeder-free culture system for HESX-01 cells has been established, and the culture effect is similar with the publish recognized Knockout medium.

Key words: Key words: human embryonic stem cells, self-made serum-free, feeder-free, bFGF, heparin