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A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone

Nature Protocols volume 5, pages 550–560 (2010)Cite this article

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Abstract

Unlike humans, mouse bone marrow-derived mesenchymal stem cells (MSCs) cannot be easily harvested by adherence to plastic owing to the contamination of cultures by hematopoietic cells. The design of the protocol described here is based on the phenomenon that compact bones abound in MSCs and hematopoietic cells exist in the marrow cavities and the inner interfaces of the bones. The procedure includes flushing bone marrow out of the long bones, digesting the bone chips with collagenase type II, deprivation of the released cells and culturing the digested bone fragments, out of which fibroblast-like cells migrate and grow in the defined medium. The entire technique requires 5 d before the adherent cells are readily passaged. Further identification assays confirm that these cells are MSCs. We provide an easy and reproducible method to harvest mouse MSCs that does not require depletion of hematopoietic cells by sorting or immunomagnetic techniques.

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Figure 1: Dissection of mouse compact bones and the preparation of bone chips.
Figure 2: Morphological features of compact bone-derived mouse mesenchymal stem cells.
Figure 3: Immunophenotypic characterization and differentiation assays of mouse mesenchymal stem cells.
Figure 4: Comparison of cell yields of cultivation of the compact bones with the culturing of bone marrow from one female mouse aged 2–3 weeks.

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Acknowledgements

We thank Lindsey Berry (Keck School of Medicine, University of Southern California) for her critical reading of the paper and Dr. Hong-Mei Ning (Department of Cell Biology, Institute of Basic Medical Sciences, Beijing, China) for her helpful discussion. This study was supported by the National Key Basic Research Program of China (2005CB522705), the National Natural Science Foundation (30600309 and 30730043) and High-tech Research and Development Program of China (2007AA021109, 2007AA02Z454).

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  1. Department of Cell Biology, Institute of Basic Medical Sciences, Beijing, China

    Heng Zhu, Xiao-Xia Jiang, Hong Li, Xiao-Yan Wang, Hui-Yu Yao, Yi Zhang & Ning Mao

  2. Department of Experimental Hematology, Institute of Radiation Medicine, Beijing, China

    Zi-Kuan Guo

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Contributions

H.Z. and Z.-K.G. were involved in the conception, design and performance of the experiments, data analysis and interpretation and writing of the paper; X.-X.J. carried out the experiments and analyzed and interpreted data; H.L., X.-Y.W. and H.-Y.Y. collected and assembled the data; Y.Z. was involved in the conception and design of the experiments and the writing of the paper; and N.M. was involved in the conception and design of the experiments, provided financial and administrative support and was involved in the writing and final approval of the paper.

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Correspondence to Ning Mao.

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Zhu, H., Guo, ZK., Jiang, XX. et al. A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone. Nat Protoc 5, 550–560 (2010). https://doi.org/10.1038/nprot.2009.238

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