Optimization of bovine embryonic fibroblast feeder layer prepared by Mitomycin C

文献类型： 外文期刊

第一作者： Zhao, Fang

作者： Zhao, Fang;Yu, Jianning;Ding, Qiang;Chen, Kunlin;Xia, Shuwen;Qian, Yong;Wang, Huili;Zhong, Jifeng;Gao, Yundong;Lin, Zhiping

作者机构：

关键词： Bovine embryonic fibroblasts; Mitomycin C; Cell proliferation; Feeder cells; Bovine pluripotent stem cell

期刊名称：CELL AND TISSUE BANKING （ 影响因子：1.752； 五年影响因子：1.978 ）

ISSN： 1389-9333

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收录情况： SCI

摘要： 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 mu g/mL Mitomycin C for 3 h or 16 mu g/mL Mitomycin C for 3 h were significantly higher than that in bEF-feeder treated with 8 mu g/mL Mitomycin C for 8 h or bEFs treated with 6 mu g/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 mu g/mL Mitomycin C for 3 h or 16 mu g/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.

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