Epiblast Stem Cell line 7 (NINDS - National Institute of Health)

E5.5  mouse embryos were dissected out from their decidua layer, into HEPES-buffered medium. In order to isolate the epiblast layer, an incision was made at the boundary between the extra-embryonic ectoderm and the epiblast layers. The embryonic fragment consisting of the epiblast and the overlying visceral endoderm layer, was digested in a solution of  trypsin and  pancreatin in PBS and subsequently returned to the initial HEPES-buffered dissection medium. The isolated epiblast fragments were then cultured on a feeder cell layer in DMEM/F12, supplemented with  knockout serum replacement (20%),  FGF2 (5 ng/ml), 2-mercaptoethanol (0.1 mM ),  L-glutamine (2 mM), and  non-essential amino acids (X1).