Isolation and culture of mouse embryonic fibroblasts and preparation of feeder layers

doi:10.3969/j.issn.2095-4344.2014.45.017

Abstract

Abstract:

OBJECTIVE: To establish the optimal method for isolation and culture of Kunming mouse embryonic fibroblasts, and to evaluate the feasibility of preparing feeder layers for culture of human embryonic stem cells.

METHODS: Embryonic fibroblasts were isolated and cultured by different concentrations of trypsin from Kunming mouse fetuses in vitro. The biological characteristics and growth rule of mouse embryonic fibroblasts were investigated, and then the feeder layers for human embryonic stem cells culture were produced. The growth of human embryonic stem cells on the prepared feeder layer was tested.

RESULTS AND CONCLUSION: The optimal fetal age for preparing Kunming mouse embryonic fibroblast feeder layer was 13.5 days. Kunming mouse embryonic fibroblasts at different concentrations grew well with high purity and active proliferation by trypsin digestion method. There was no significant difference in the survival rate of cells after cryopreservation for 2 weeks, 1 month, 3 months and 6 months. The cells were proliferative from the second to fourth passage and declined sharply after the fifth passage. Human embryonic stem cells which grew on Kunming mouse embryonic fibroblasts feeder layers were still to remain the typical undifferentiated morphology and were strongly positive for alkaline phosphatase and periodic acid-Schiff after long-term subculture. The mouse embryonic fibroblasts can be used as the stable and high-quality feeder cells for human embryonic stem cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: embryonic stem cells, fibroblasts, trophoblasts

CLC Number:

R394.2

Hu San-qiang, Wang Yan-yan, Ma Yong-bin, Hu Jia-bo. Isolation and culture of mouse embryonic fibroblasts and preparation of feeder layers[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(45): 7306-7311.

Figures/Tables 5

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