Isolation of Multipotent Stromal Cells from Mouse Bone Marrow    

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
   
   
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.