Optimization of bovine embryonic fibroblast feeder layer prepared by Mitomycin C

Feeder cells play important roles in In-vitro culture of stem cells. However, the preparation protocol of feeder cells produced by bovine embryonic fibroblast cells (bEFs) is still lack. In this study, the preparation of bEF-feeder by Mitomycin C was optimized with different concentrations and treatment time. The cell viability of bEFs was detected by CCK8 and 5-Ethynyl-2'-deoxyuridine. The growth of bESCs in each bEFs-feeder group was assessed by alkaline phosphatase staining and CCK8. Quantitative real time PCR was used to detect the mRNA expression of pluripotency-related genes of bESCs. Results showed that the proliferation of bEFs was significantly repressed while bEFs were treated with 14 ug/mL or 16 ug/mL Mitomycin C for 3 h, and the cell viability within 2-4 days after treatment was consistent with the 1st day. The numbers of bESCs clones in bEF-feeder treated with 14 μg/mL Mitomycin C for 3 h or 16 μg/mL Mitomycin C for 3 h were significantly higher than that in bEF-feeder treated with 8 μg/mL Mitomycin C for 8 h or bEFs treated with 6 μg/mL Mitomycin C for 9 h. The mRNA expression of pluripotency-related genes in bESCs cultured by bEF-feeder were higher than the MEF-feeder, the clone morphology of bESCs cultured in bEF-feeder was rounder and sharper than the MEF-feeder. In conclusion, the bEF-feeder prepared with 14 μg/mL Mitomycin C for 3 h or 16 μg/mL Mitomycin C for 3 h could effectively maintains the growth of bESCs, and bEF-feeder is more suitable for bESCs culture than the MEF-feeder.