Abstract:

BACKGROUND: Mouse embryonic stem cell line SF1-G is derived from morula of female C57BL/6 mice mating with male M. spretus. Usually SF1-G was cultured on the layer of STO feeders. STO cells were expensive, whereas mouse embryonic fibroblasts (MEFs) were easier to get, and had more advantage in supporting embryonic stem cells growth, such as the formation of embryonic stem cell clones and maintaining the normal karyoplast of embryonic stem cells. Thus, it was very useful to establish a culture system in which SF1-G cells could amplify in an undifferentiated status.

OBJECTIVE: To explore and establish an effective method of isolating and culturing MEFs and prepare the feeder cells for mouse embryonic stem cell (SF1-G cells) proliferation. 

METHODS: The mouse primary MEFs were isolated from ICR mouse fetus at 12.5 to 14.5 day gestational ages. The 3 to 5 passages MEFs were treated by mytomycin C to inhibit cell proliferation. The treated MEFs were used as feeder cells for culturing SF1-G cell. The karyotype of SF1-G cell was detected by chromosome G staining process. The expression of alkaline phosphatase (AKP) of SF1-G cells was tested. Oct4 and Nanog gene expressions were also tested by RT-PCR.

RESULTS AND CONCLUSION: MEFs were successfully isolated and cultured from mouse fetus. Feeder cells prepared MEFs of 3 to 5 passages could be used to support SF1-G cell growth with clear boundaries of clone. The SF1-G cells had normal karyotype, showing the positive results of AKP and expressing specific transcription factors. This experiment established an effective method of isolating and culturing MEFs and prepared feeder cells for propagating and culturing mouse embryonic stem cells, and SF1-G cells can be cultured in undifferentiated state in a laboratory.