Human MSCs were isolated from bone marrow aspirates of the iliac crest of normal adult donors, after informed consent.
Nucleated cells were isolated with a density gradient (Ficoll-Paque) and resuspended in complete culture medium: alpha minimal essential medium (αMEM), supplemented with fetal bovine serum (FBS, 20%), lot-selected for rapid growth of MSCs, penicillin (100 U/ml), streptomycin (100 µg/ml), and L-glutamine (2 mM). All nucleated cells (25-71 million) were plated in 20 ml medium on a 180-cm2 culture dish and incubated at 37°C in 5% CO2. After 24 hours, nonadherent cells were discarded, and adherent cells were thoroughly washed twice with phosphate-buffered saline (PBS). The cells were incubated for 4-11 days, harvested with trypsin (0.25%) and EDTA (1 mM) for 5 minutes at 37°C, and replated at 3-50 cells/cm2 in an intercommunicating system of culture flasks (6,300 cm2 ). After 7-12 days, the cells were harvested with trypsin/EDTA, suspended at 1 × 10^6 cells/ml in dimethylsulfoxide (5%) and FBS (30%), and frozen in 1-ml aliquots in liquid nitrogen (passage 1 cells). To expand a culture, a frozen vial of MSCs was thawed, plated in a 60 cm2 culture dish, and incubated for 4 days (passage 2 cells). MSCs were cultured at 10 cells/cm2 , 50 cells/cm2, 100 cells/cm2, and 1,000 cells/cm2 on 60-cm2 dishes.