Objective To reduce the animal component contamination for human embryonic stem cells ( hESCs ) and to simplify hESCs culture process , we develop a new coating substrate which can support the hESCs growth without dif -ferentiation, and is easy to store and use. Methods Mouse embryonic fibroblasts(MEF)were fixed on the surface of plate by methanol.hESCs were cultured on this new substrate and were passaged every 5 to 6 days.After 10 passages, we checked the cell morphology , alkaline phosphatase expression , embryonic specific markers and the differentiation ability in vitro.Results After 10 passages , the hESCs grew well on this new substrate and maintained the typical hESCs morpholo -gy.Alkaline phosphatase staining was positive .Immunofluorescence staining showed that the expressions of Oct 4, SSEA4, Tra-1-60 were positive .The cells formed embryoid body in vitro .Conclusions This methanol-fixed MEF substrate can support the growth of undifferentiated hESCs .The coating material can be produced in large scale and stored for a long time.It provides a new and relatively easy way to amplify hESCs .