2. Academic Validation
  3. Optimization of bovine embryonic fibroblast feeder layer prepared by Mitomycin C

Optimization of bovine embryonic fibroblast feeder layer prepared by Mitomycin C

  • Cell Tissue Bank. 2022 Jul 28. doi: 10.1007/s10561-022-10027-3.
Fang Zhao 1,  Jianning Yu 1,  Qiang Ding 1,  Kunlin Chen 1,  Shuwen Xia 1,  Yong Qian 1,  Yundong Gao 2,  Zhiping Lin 3,  Huili Wang 4,  Jifeng Zhong 5
Affiliations

Affiliations

  • 1 Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base, Ministry of Science and Technology / Key Laboratory of Crop and Animal Integrated Farming, Ministry of Agriculture / Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, Jiangsu, China.
  • 2 Shandong OX Livestock Breeding Co.,Ltd, Jinan, 250100, Shandong, China.
  • 3 Jiangsu Youyuan Dairy Research Institute, Nanjing, 211100, Jiangsu, China.
  • 4 Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base, Ministry of Science and Technology / Key Laboratory of Crop and Animal Integrated Farming, Ministry of Agriculture / Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, Jiangsu, China. wanghuili318@163.com.
  • 5 Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base, Ministry of Science and Technology / Key Laboratory of Crop and Animal Integrated Farming, Ministry of Agriculture / Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, Jiangsu, China. zhongjifeng64@sina.cn.
PMID: 35896934 DOI: 10.1007/s10561-022-10027-3
Abstract

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.

Keywords

Bovine embryonic fibroblasts; Bovine pluripotent stem cell; Cell proliferation; Feeder cells; Mitomycin C.

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