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A reliable and economical method for gaining mouse embryonic fibroblasts capable of preparing feeder layers

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Abstract

Mouse embryonic fibroblasts (MEFs) are widely used to prepare feeder layers for culturing embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in vitro. Transportation lesions and exorbitant prices make the commercially obtained MEFs unsuitable for long term research. The aim of present study is to establish a method, which enables researchers to gain MEFs from mice and establish feeder layers by themselves in ordinary laboratories. MEFs were isolated from ICR mouse embryos at 12.5–17.5 day post-coitum (DPC) and cultured in vitro. At P2–P7, the cells were inactivated with mitomycin C or by X-ray irradiation. Then they were used to prepare feeder layers. The key factors of the whole protocol were analyzed to determine the optimal conditions for the method. The results revealed MEFs isolated at 12.5–13.5 DPC, and cultured to P3 were the best choice for feeder preparation, those P2 and P4–P5 MEFs were also suitable for the purpose. The P3–P5 MEFs treated with 10 μg/ml of mitomycin C for 3 h, or irradiated with X-ray at 1.5 Gy/min for 25 Gy were the most suitable feeder cells. Treating MEFs with 10 μg/ml of mitomycin C for 2.5 h, 15 μg/ml for 2.0 h, or irradiating the cells with 20 Gy of X-ray at 2.0 Gy/min could all serve as alternative methods for P3–P4 cells. Our study provides a reliable and economical way to obtain large amount of qualified MEFs for long term research of ESCs or iPSCs.

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Abbreviations

MEFs:

Mouse embryonic fibroblasts

ESCs:

Embryonic stem cells

hESCs:

Human embryonic stem cells

iPSCs:

Induced pluripotent stem cells

DPC:

Day post-coitum

FGF:

Fibroblast growth factor

FBS:

Fetal bovine serum

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Acknowledgments

The authors thank Drs. Aidong Wu and Bing Yan at Radiotherapy Department, Affiliated Anhui Provincial Hospital, Anhui Medical University for their generous help in irradiation process; and Dr. Xiao Lei at Zhejiang University for presenting hESCs.

Conflict of interest

The authors have no conflict of interests to declare.

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Authors and Affiliations

  1. Blood Bank, Affiliated Anhui Provincial Hospital, Anhui Medical University, Hefei, 230001, China

    Guangming Jiang, Xiaoju Wan, Ming Wang, Jianhua Zhou & Jian Pan

  2. Laboratory Department, Affiliated Anhui Provincial Hospital, Anhui Medical University, Hefei, 230001, China

    Baolong Wang

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  1. Guangming Jiang
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  2. Xiaoju Wan
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  3. Ming Wang
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  4. Jianhua Zhou
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  5. Jian Pan
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  6. Baolong Wang
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Correspondence to Baolong Wang.

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Jiang, G., Wan, X., Wang, M. et al. A reliable and economical method for gaining mouse embryonic fibroblasts capable of preparing feeder layers. Cytotechnology 68, 1603–1614 (2016). https://doi.org/10.1007/s10616-014-9815-z

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