Abstract:

BACKGROUND: Mouse embryonic fibroblast (MEF) as feeder layer can be effective to promote embryonic stem cells and to induce pluripotent stem cell proliferation and to maintain their undifferentiated and pluripotent characteristics, which are better than feeder-free or adding single cytokines in medium, furthermore, the feeder layer can provide embryonic development environment for the above two kinds of cells.
OBJECTIVE: To establish a stable and effective system of ICR MEF feeder layers to support embryonic stem cells and induce pluripotent stem cells in vitro.
METHODS: MEF of ICR mouse was isolated using the method of trypsin digestion to observe effects of different mass concentrations of trypsin and different digestion time on the amount and the proliferation activity of MEF. Various concentrations of mitomycin C were utilized to treat MEF for 1, 1.5, 2, 2.5, 3, 3.5 hours to prepare feeder layer cells. MTT assay was employed to determine proliferation activity of MEF, and to explore optimal condition for preparing feeder layer.
RESULTS AND CONCLUSION: The optimal mass concentration of trypsin for isolating MEF of ICR mouse was 0.05%; digestion time was 12-15 minutes; the optimal action mass concentration and time was 10 mg/L for 2.5 hours. Fibroblast feeder layer maintained for 8-12 days. The effects of 0.05% trypsin digestion for 15-20 minutes were superior to high mass concentration.  10 mg/L mitomycin C treated MEF for 2.5 hours can effectively inhibit the proliferation of the prepared feeder cells. Feeder layer cells prepared under this condition can support embryonic stem cells and induce pluripotent stem cells growth