Abstract: The cumulus cells were obtained by digesting cumulusoocyte complexes with trypsin solution, and were cultured in DMEM/F12 media (control group) or McCoy′s5a media (four treatment groups) during primary culturing and subculturing. The McCoy′s5a were utilized for media in treatment 1. The media for treatment 2 were McCoy′s5a media containing 29 ng·mL-1 testosterone. Testosterone (29 ng·mL-1) and green tea polyphenols (15 μmol·L-1) were supplemented together into McCoy′s5a media in treatment 3. In treatment 4, optimized McCoy′s5a media were utilized for culturing cumulus cells. Optimized McCoy′s5a media were supplemented with transferrin, selenium and insulin based on media in treatment 3. The results show that the cumulus cells cultured with optimized McCoy′s5a media are normal in morphological characteristics, growth speed and karyotype. The contents of SOD and GSH and survival rate following thawing in cumulus cells cultured with optimized McCoy′s5a media are significant higher than that in cumulus cells of control. The optimized McCoy′s5a media with appropriate concentration of transferrin, selenium and insulin can instead of DMEM/F12 media containing serum to culture in vitro cumulus cells of cow.