Abstract:

BACKGROUND: Studies have demonstrated that many factors may influence mouse embryonic fibroblasts (MEF) separation. Current researches on exploring these factors are based on semi-empirical results, which are lack of objective and quantitative data support.  
OBJECTIVE: To investigate the optimal conditions in establishing MEF as mouse embryonic stem cells’ feeder layer cells.
METHODS: Two different methods were conducted to isolate MEF from 13.5 days post coitum (dpc) Kunming mouse embryos. Trypan blue staining and cell counting of MEF were performed to study the relationship between cell livability and quantity. The MEF was digested by 0.05% trypsin at room temperature at various time points to analyze effects of dissociation adherent cell ability and cell viability at various digestion time points. Cells in feeder layer were inactivated using mitomycin for 2 and 2.5 hours. The cell growth curve was drawn. The optimal concentration and disposal time of mitomycin were explored. By various cryopreservation methods, the livability of MEF was compared after thawing. The optimal cryopreservation methods of MEF were screened.
RESULTS AND CONCLUSION: MEF was sensitive to trypsin. The shorter trypsinase digestion time and multiple times method obviously elevated cell viability and quantity. At room temperature, 4-6 minutes digestion time contributed to cell viability preservation. 10 mg/L 2.5 hours and 20 mg/L 2 hours mitomycin inactivation was suitable for establishing cells in feeder layer. Progressive temperature reduction made MEF adaptive to temperature changes, which significantly increased MEF livability and quantity after thawing.