A new method for establishing ES cell lines from 129/ter、C57BL/6J mice was set up which was characterized by the murine embryonic fibroblast cell(MEF) feeder, the medium of rat heart cell-conditioned medium(RH-CM)for ES cells, and the consecutive digestion by the digestion liquid containing 1% serum. Every group of improved experiments was done with a control of routine method. The results showed that, compared with routine method, the improved way increased the ratio of ES cell lines of 129/ter mice from 11.8% to 33.3%, and of C57BL/6J from 3.7% to 13.3%. The difference is distinct. The passage culture of ES cells showed that, compared with medium added LIF, RH-CM not only inhibited the differentiation of murine ES cells, maintained its dipoild karyotype, but also promote its adherence growth. This kind of culture condition not only maintained the ES cells in an undifferentiated state and their normal dipoild karyotype, but also a series of other characteristics of totipotent embryonic stem cells during extended culture period.
