Preliminary Study Of Embryonic Stem Cells In Vitro Culture System

Objective: Based on the pre-study of our research group, optimizing the feeder layer system and stabilizing the culture conditions to explore the suitable conditions of our laboratory for in vitro culturing embryonic stem cells(ESCs); sum up experimental cells data and standardize the description of experimental cells.Methods: (1).In vitro isolate and culture mouse embryonic fibroblasts (MEFs) by the way of tissue digestion. Measure MEFs viability and proliferation forms with MTT. MEFs were detected Vimentin by immunohistochemical staining. Treat MEFs with Mitomycin C(10μg/ml) to make feeder layers. (2).Obtain 3.5dpc(day post coitum, dpc) mouse embryos by nature copulation and superovulation, in vitro culture embryo to develop into expanded blastocyst and hatch out from the zona pellucida(ZP) on the MEFs feeder layer. Remove inner cell mass(ICM) clump from the surrounding trophoblast cells and disaggregat it by use of trypsin. Transfer the cell suspension into one fresh MEFs feeder layer and observe the cultures every day. (3).Collect and culture the discarded human embryos after IVF-ET to explore the in vitro culture conditions. (4).Basing on the authority of book to standardize the description and formulate table form of experimental cells.Results: (1).MEFs were isolated and cultured in vitro from 10~18dpc mouse embryos, the MEFs from 13~15dpc mouse embryos were better. The cells were aging after 5 passages. Growth curve showed that the cells were all in increased logarithmic phase in the third day. Immunohistochemical staining showed that about 90% cells expressed Vimentin. MEFs were made into feeder layer by Mitomycin C(10μg/ml). (2).We could obtain 8~12 embryos from each mouse by nature copulation. The feeder layer which we made could support the embryos to hatch out from the ZP and form ICM clump. (3).We had standardized the description and formulated table form of the experimental cells.Conclusions: (1).We have optimized the feeder layer system and detected the cells. (2).We have explored the condition of embryos culture and the way to form ICM clump.