**2.1 Materials**

1.Growth medium (pH 7.1–7.5): Dulbecco's modified Eagle's medium (DMEM)-low glucose (Sigma-Aldrich, St Louis, MO), 10% fetal bovine serum (FBS,

*Isolation and Expansion of Mesenchymal Stem/Stromal Cells, Functional Assays and Long-Term… DOI: http://dx.doi.org/10.5772/intechopen.100286*

heat-inactivated) (Atlanta Biologicals, Lawrenceville, GA), 0.37 g% sodium bicarbonate, and 1% penicillin–streptomycin. Adjust growth medium to pH 7.1–7.5.


#### **2.2 Isolation and culture expansion of human BM-MSCs**

1.Make sure BM sample, 1 × PBS + 2% FBS + 1 mM EDTA, Ficoll-Paque and centrifuge are all at room temperature.


*Isolation and Expansion of Mesenchymal Stem/Stromal Cells, Functional Assays and Long-Term… DOI: http://dx.doi.org/10.5772/intechopen.100286*


35.Harvest cells. This culture is passage 1. MSCs at passage 1 can be frozen in liquid nitrogen (see the next) or continue to serially passage.
