Abstract:

In order to isolate the ES cells from Kunming mouse efficiently, the feeder cells, developmental stage of embryos and culture conditions were optimized in the study. Mouse embryonic fibroblast (MEF) were inoculated at concentrations of 1×10⁴, 1×10⁵ and 1×10⁶·mL^-1 as the feeder layers of ES cells after they were treated with Mitomycin C within 3 passages, and cultured in H-DMEM+15%KSR+LIF, the growth of the ES cells were observed. The effects of developmental stages of embryos and added SCF, SCF+insulin in culture medium on growth of Kunming mouse ES cells were investigated. The results showed that the formation rates of the 1^st and 2^nd passage ES cells in 1×10⁵·mL^-1 MEF were higher than those cultured on the other 2 groups(P<0.05). The 2^nd passage ES cells colonies formation rate of blastocysts were higher than that of compacted morula(P<0.05). It had improved the attachment rate was significantly enhanced(P<0.05) when the culture medium added with SCF, and the highest attachment rate, 1^st and 2^nd passage ES cells colonies formation rate were got when the culture medium added with SCF and insulin (P<0.05). The isolated ES cells, which were positive for AKP staining and immunofluorescence against antigens of Oct-4 and SSEA-1, had a series of characters of mice ES cells. These results suggest that the blastocysts on the MEF with a density of 1×10⁵·mL^-1 and addition with SCF and insulin in the culture medium is more suitable for the isolation and passage of Kunming mouse ES cells.