Abstract:

BACKGROUND: Mouse embryonic fibroblasts (MEFs) feeder is more frequently used to culture embryonic stem cells (ESCs) and can retain ESCs proliferative potential in an undifferentiated state. But the preparation of feeder cells is elaborate and multiplicity.
OBJECTIVE: To develop a simple and efficient method to establish MEFs feeder layers in vitro.
METHODS: Mouse fetuses of 13.5-day gestational age were chosen to isolate MEFs as feeder layer. MEFs were isolated using two simply methods in vitro. The change of cells about appearance and amount was observed by inverted microscope; frozen MEFs were thawed through different methods, and the survival rate of MEFs was contrasted. MEFs were inactivated with different concentration mitomycin C and different time treatment to prepare MEFs feeder layer.
RESULTS AND CONCLUSION: The primary MEFs isolated using the two simple methods were in the best state and adequate. The two methods were better to culture primary mouse embryonic fibroblast cells, and complex manipulate protocol could be avoided. After thawed by the simple method, MEFs were treated with 10 mg/L mitomycin C for 1.5-2.0 hours or 1 mg/L mitomycin C overnight based on different confluence conditions.