Objective:To establish a stable and effective system of mouse embryonic fibroblasts(MEFs) feeder layer in order to separate and culture mouse embryonic stem cells(ESC) in vitro.Methods:the MEFs were isolated from the mouse embryos at 13.5~14.5dpc(days post-coitus) through primary tissue digestion and tissue piece culture.The optional planting density of cell was determined according to the cellular growth curve detected by MTT and the morphology in the culture dish under inverted microscope.The optional concentration of mitomycin C and the time treating with mitomycin on MEFs were determined by MTT assay.Murine blastcysts(3.5dpc) were cultured on the feeder layer treated by mitomycin C and ESC growth was observed.Result:The method of tissue digestion and tissue piece culture is an efficient way to obtain primary MEFs;1×105~2×105/ml cells is an optional planting density;MEFs proliferation could be effeciently repressed after being treated with mitomycin C 10μg/ml for 3.5 hours or 20μg/ml for 2.5 hours

and the quantity of MEFs could maintain at least for 7 days.ES clone could be obtained from the mouse blastcysts(3.5dpc) after being cultured on the feeder layer.Conclusion:MEFs feeder layer could effectively support mouse embryonic stem cells to grow.