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Bone marrow-derived mesenchymal stem cells (Tulane University Health Sciences Center)

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.

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Adult Stem / Progenitor Cell
Homo sapiens
Bone marrow-derived mesenchymal stem cells