Briefly, total BM aspirate was directly seeded at a concentration of 50,000 total nucleated cells/cm2 in T75 plastic flask in Minimum Essential Medium Alpha (α-MEM) supplemented with 1% GlutagroTM, 10% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin (Pen/Strep) and incubated at 37 °C in an atmosphere of 5% CO2 and 95% relative humidity [33 (link)]. After 72 h, nonadherent cells were removed by medium change and the remaining adherent cells were then fed twice weekly thereafter with new medium. On day 14, colonies of adherent hBM-MSCs were detached and reseeded at 4000 cells/cm2 in the same culture conditions. Once cell cultures reached 70–80% confluence (7–8 days from the previous passage), cells were detached using 0.05% trypsin-0.53 mM Ethylenediaminetetraacetic acid (EDTA) and washed with 1× of phosphate buffered saline (PBS) (Corning Cellgro, Manassas, VA, USA), counted using Trypan Blue (Sigma-Aldrich, Milan, Italy) and subcultured at a concentration of 4 × 103 cells/cm2. At passage 2, cells were used experimentally.
Flow cytometry analysis was performed on hBM-MSCs obtained at passage 2; cells were positive for CD90, CD105, CD73 and negative for CD14, CD34, CD45, HLA-DR expression (Beckman Coulter, Brea, CA, USA) (data not shown) [34 (link)].
Flow cytometry analysis was performed on hBM-MSCs obtained at passage 2; cells were positive for CD90, CD105, CD73 and negative for CD14, CD34, CD45, HLA-DR expression (Beckman Coulter, Brea, CA, USA) (data not shown) [34 (link)].