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Generating mouse multipotent stromal cells (MSC) from bone-marrow cells is usefull for a wide range of applications. Effectively, these MSC can differentiate into adipocytes, osteocytes [See “Binding to Secreted Bone Matrix in vitro” (Tormo et al., 2014)] or chondrocytes upon culture in specific differentiation medium.

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Isolation of Multipotent Stromal Cells from Mouse Bone Marrow

Immunology > Immune cell isolation > Maintenance and differentiation
Authors: Aurélie Jeanne Tormo
Aurélie Jeanne TormoAffiliation: Département de Pharmacologie, Université de Montréal, Montreal, Quebec, Canada
For correspondence: jf.gauchat@umontreal.ca
Bio-protocol author page: a1121
Moutih Rafei
Moutih RafeiAffiliation: Département de Pharmacologie, Université de Montréal, Montreal, Quebec, Canada
Bio-protocol author page: a1124
 and Jean-François Gauchat
Jean-François GauchatAffiliation: Département de Pharmacologie, Université de Montréal, Montreal, Quebec, Canada
For correspondence: jf.gauchat@umontreal.ca
Bio-protocol author page: a1123
Vol 4, Iss 4, 2/20/2014, 2285 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.1031

[Abstract] Generating mouse multipotent stromal cells (MSC) from bone-marrow cells is usefull for a wide range of applications. Effectively, these MSC can differentiate into adipocytes, osteocytes [See “Binding to Secreted Bone Matrix in vitro” (Tormo et al., 2014)] or chondrocytes upon culture in specific differentiation medium.

Materials and Reagents

  1. 6-8 weeks old mouse
  2. PBS without Ca2+ and Mg2+ (Wisent, catalog number: 311-01-CL)
  3. Dulbecco's Modified Eagle's Medium High glucose with stable L-glutamine (DMEM) (Wisent, catalog number: 319-015-CL)
  4. Fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 12483)
  5. Penicillin/Streptomycin solution (Wisent, catalog number: 450-201-EL)
  6. Trypan blue (Life Technologies, Gibco®, catalog number: 15250-061)
  7. Trypsin 0.05%/EDTA 0.53 mM (Wisent, catalog number: 325-042-EL)
  8. APC-conjugated anti-CD31 antibody (clone MEC13.3) (BD Biosciences, catalog number: 551262)
  9. FITC-conjugated anti-CD45 antibody (clone 30-F11) (BD Biosciences, catalog number: 553080)
  10. APC-conjugated anti-CD44 antibody (clone IM7) (BD Biosciences, catalog number: 559250)
  11. PE-conjugated anti-CD105 antibody (clone MJ7/18) (BD Biosciences, catalog number: 562759)
  12. FITC-conjugated anti-CD90 antibody (clone 5E10) (BD Biosciences, catalog number: 555595)

Equipment

  1. Scissors and forceps
  2. Syringe 1cc with 27 Gauge x 1-½ needle (BD, catalog numbers: BD-309659 and BD-305109)
  3. Petri dishes 100 x 20 mm (BD, catalog number: DL-353003)
  4. 50 ml conical tubes (Progene®, catalog number: 71-5000-B)
  5. Table top centrifuge
  6. Culture hood
  7. Hemocytometer
  8. T-25 flask (BD, catalog number: 353108)
  9. 37 °C, 5% CO2 Cell culture incubator
  10. Flow cytometer (e.g. BD LSRFortessa)

Procedure

  1. Under culture hood, flush tibia and femora from one 6-8 weeks old mouse with FBS-free DMEM in a dish with DMEM previously warm at 37 °C (see Video 1).

    Video 1. Mouse-bone marrow collection

    To play the video, you need to install a newer version of Adobe Flash Player.

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  2. With a syringe and needle, aspirate and eject 2-3 times the bonne marrow to disrupt it.
  3. Transfer in a 50 ml conical tube.
  4. Centrifuge 10 min at 430 x g at 4 °C.
  5. Resuspend the pellet with DMEM supplemented with 15% FBS and Penicillin/Streptomycin at a density of 106 cells/ml.
  6. Plate 1 x 107 cells (10 ml) in a T25 flask.
  7. Incubate at 37 °C-5 % CO2 for 7-10 days without changing medium.
  8. Remove the supernatant in order to keep only adherent cells and wash the flask 2 times with 5 ml of PBS.
  9. Trypsinize cells for expansion. For cells trypsinization: add 0.5 ml of trypsin on the rinsed cells. Wait 3 to 5 min until cells peel off the plastic surface. Transfer cells in a 15 ml tube and add quickly 10 ml of DMEM containing 10% FBS in order to inhibit trypsin action.
  10. After 7 - 10 days, assess the phenotype of the growing adherent cells by flow cytometry.
    1. Stain cells with fluorescein isothiocyanate (FITC)-labeled anti-CD45 (clone 30-F11) and with allophycocyanine (APC)-labeled anti-CD31 (clone MEC133). These antibody should be diluted 1/200. Cells should be negative for these two markers as CD45 is a hematopoietic cells marker, and CD31 is an endothelial cells marker.
    2. Stain cells with APC-labeled anti-CD44 (clone IM7), phycoerythrin (PE)-labeled anti-CD105 (clone MJ7/18) and with FITC-labeled anti-CD90 (clone 5E10), all used at a 1/200 dilution. Cells should be positive for these three markers.

Acknowledgments

This protocol is adapted from Tormo et al. (2013).

References

  1. Tormo, A. J., Beaupre, L. A., Elson, G., Crabe, S. and Gauchat, J. F. (2013). A polyglutamic acid motif confers IL-27 hydroxyapatite and bone-binding properties. J Immunol 190(6): 2931-2937.
  2. Tormo, A. J., Beauséjour, C. and Gauchat, J. F. (2014). Binding to secreted bone matrix in vitro. Bio-protocol 4(4): e1030.



How to cite this protocol: Tormo, A. J., Rafei, M. and Gauchat, J. (2014). Isolation of Multipotent Stromal Cells from Mouse Bone Marrow. Bio-protocol 4(4): e1031. DOI: 10.21769/BioProtoc.1031; Full Text



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