Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

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

Nature Protocols volume 4, pages 102–106 (2009)Cite this article

Abstract

We explain a protocol for straightforward isolation and culture of mesenchymal stem cells (MSCs) from mouse bone marrow (BM) to supply researchers with a method that can be applied in cell biology and tissue engineering with minimal requirements. Our protocol is mainly on the basis of the frequent medium change in primary culture and diminishing the trypsinization time. Mouse mesenchymal stem cells are generally isolated from an aspirate of BM harvested from the tibia and femoral marrow compartments, then cultured in a medium with Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) for 3 h in a 37 °C–5% CO2 incubator. Nonadherent cells are removed carefully after 3 h and fresh medium is replaced. When primary cultures become almost confluent, the culture is treated with 0.5 ml of 0.25% trypsin containing 0.02% ethylenediaminetetraacetic acid for 2 min at room temperature (25 °C). A purified population of MSCs can be obtained 3 weeks after the initiation of culture.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Morphological features of mMSCs.
Figure 2: Cell surface markers and differentiation capacity of mouse mesenchymal stem cells (mMSCs).

Similar content being viewed by others

References

  1. Fuchs, E. & Segre, J.A. Stem cells: a new lease on life. Cell 100, 143–155 (2000).

    Article  CAS  Google Scholar 

  2. Pittenger, M.F. et al. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147 (1999).

    Article  CAS  Google Scholar 

  3. Friedenstein, A.J., Gorskaja, J.F. & Kulagina, N.N. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp. Hematol. 4, 267–274 (1976).

    CAS  PubMed  Google Scholar 

  4. Bianco, P., Riminucci, M., Gronthos, S. & Robey, P.G. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 19, 180–192 (2001).

    Article  CAS  Google Scholar 

  5. Sun, S. et al. Isolation of mouse marrow mesenchymal progenitors by a novel and reliable method. Stem Cells 21, 527–535 (2003).

    Article  CAS  Google Scholar 

  6. Prockop, D.J. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276, 71–74 (1997).

    Article  CAS  Google Scholar 

  7. Baksh, D., Song, L. & Tuan, R.S. Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J. Cell Mol. Med. 8, 301–316 (2004).

    Article  CAS  Google Scholar 

  8. Horwitz, E.M. et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proc. Natl. Acad. Sci. USA 99, 8932–8937 (2002).

    Article  CAS  Google Scholar 

  9. Koc, O.N. et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J. Clin. Oncol. 18, 307–316 (2000).

    Article  CAS  Google Scholar 

  10. Petite, H. et al. Tissue-engineered bone regeneration. Nat. Biotechnol. 18, 959–963 (2000).

    Article  CAS  Google Scholar 

  11. Quarto, R. et al. Repair of large bone defects with the use of autologous bone marrow stromal cells. N. Engl. J. Med. 344, 385–386 (2001).

    Article  CAS  Google Scholar 

  12. Grinnemo, K.H. et al. Xenoreactivity and engraftment of human mesenchymal stem cells transplanted into infarcted rat myocardium. J. Thorac. Cardiovasc. Surg. 127, 1293–1300 (2004).

    Article  CAS  Google Scholar 

  13. Barry, F.P. Mesenchymal stem cell therapy in joint disease. Novartis. Found Symp. 249, 86–102, 170–4, 239–41 (2003).

    PubMed  Google Scholar 

  14. Peister, A. et al. Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood 103, 1662–1668 (2004).

    Article  CAS  Google Scholar 

  15. Phinney, D.G., Kopen, G., Isaacson, R.L. & Prockop, D.J. Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation. J. Cell Biochem. 72, 570–585 (1999).

    Article  CAS  Google Scholar 

  16. Baddoo, M. et al. Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection. J. Cell Biochem. 89, 1235–1249 (2003).

    Article  CAS  Google Scholar 

  17. Tropel, P. et al. Isolation and characterisation of mesenchymal stem cells from adult mouse bone marrow. Exp. Cell Res. 295, 395–406 (2004).

    Article  CAS  Google Scholar 

  18. Eslaminejad, M.B., Nikmahzar, A., Taghiyar, L., Nadri, S. & Massumi, M. Murine mesenchymal stem cells isolated by low density primary culture system. Dev. Growth Differ. 48, 361–370 (2006).

    Article  CAS  Google Scholar 

  19. Nadri, S. & Soleimani, M. Isolation murine mesenchymal stem cells by positive selection. In Vitro Cell Dev. Biol. Anim. 43, 276–282 (2007).

    Article  CAS  Google Scholar 

  20. Nadri, S. et al. An efficient method for isolation of murine bone marrow mesenchymal stem cells. Int. J. Dev. Biol. 51, 723–729 (2007).

    Article  CAS  Google Scholar 

  21. Modderman, W.E., Vrijheid-Lammers, T., Lowik, C.W. & Nijweide, P.J. Removal of hematopoietic cells and macrophages from mouse bone marrow cultures: isolation of fibroblastlike stromal cells. Exp. Hematol. 22, 194–201 (1994).

    CAS  PubMed  Google Scholar 

  22. Falla, N. et al. Characterization of a 5-fluorouracil-enriched osteoprogenitor population of the murine bone marrow. Blood 82, 3580–3591 (1993).

    CAS  PubMed  Google Scholar 

  23. Van Vlasselaer, P., Falla, N., Snoeck, H. & Mathieu, E. Characterization and purification of osteogenic cells from murine bone marrow by two-color cell sorting using anti-Sca-1 monoclonal antibody and wheat germ agglutinin. Blood 84, 753–763 (1994).

    CAS  PubMed  Google Scholar 

  24. Wang, Q.R. & Wolf, N.S. Dissecting the hematopoietic microenvironment. VIII. Clonal isolation and identification of cell types in murine CFU-F colonies by limiting dilution. Exp. Hematol. 18, 355–359 (1990).

    CAS  PubMed  Google Scholar 

  25. Kitano, Y., Radu, A., Shaaban, A. & Flake, A.W. Selection, enrichment, and culture expansion of murine mesenchymal progenitor cells by retroviral transduction of cycling adherent bone marrow cells. Exp. Hematol. 28, 1460–1469 (2000).

    Article  CAS  Google Scholar 

  26. Muraglia, A., Cancedda, R. & Quarto, R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J. Cell Sci. 113 (Part 7): 1161–1166 (2000).

    CAS  PubMed  Google Scholar 

  27. Digirolamo, C.M. et al. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br. J. Haematol. 107, 275–281 (1999).

    Article  CAS  Google Scholar 

  28. Keating, A., Horsfall, W., Hawley, R.G. & Toneguzzo, F. Effect of different promoters on expression of genes introduced into hematopoietic and marrow stromal cells by electroporation. Exp. Hematol. 18, 99–102 (1990).

    CAS  PubMed  Google Scholar 

  29. Deans, R.J. & Moseley, A.B. Mesenchymal stem cells: biology and potential clinical uses. Exp. Hematol. 28, 875–884 (2000).

    Article  CAS  Google Scholar 

  30. Otsuka, T. et al. Expression of the c-kit ligand and interleukin 6 genes in mouse bone marrow stromal cell lines. Stem Cells 12, 409–415 (1994).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by Iran Stem Cell Technology Institute.

Author information

Author notes

  1. Masoud Soleimani and Samad Nadri: Both the authors contributed equally to this work.

Authors and Affiliations

  1. Department of Hematology, Faculty of Medical Science, Tarbiat Modares University, P.O. Box, 14115-111, Tehran, Iran

    Masoud Soleimani

  2. Department of Stem Cells and Tissue Engineering, Stem Cell Technology Institute, P.O. Box 14155-3174, Tehran, Iran

    Samad Nadri

  3. Department of Biomedical Engineering and Medical Physics, Faculty of Medicine, Shahid Beheshti University (MC), Tehran, 19395-4719, Iran

    Samad Nadri

Authors
  1. Masoud Soleimani
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Samad Nadri
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Masoud Soleimani or Samad Nadri.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Soleimani, M., Nadri, S. A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. Nat Protoc 4, 102–106 (2009). https://doi.org/10.1038/nprot.2008.221

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2008.221

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing