Bone marrow-derived stem cells (Tehran University of Medical Sciences)

Cells were obtained from patients (for autologous treatment). A bone marrow aspiration of 20 ml was performed from the iliac crest. Mononuclear cells were separated using Ficoll hypaque and cultured in DMEM supplemented with fetal bovine serum (10%). After reaching confluence, cells were detached with trypsin/EDTA, and subcultured. Repeated passages were performed until the required cell number was obtained. Confluent cells of last passage were washed with tyrode solution and incubated in M199 for 60 minutes. Cells were separated using trypsin/EDTA and washed 3 times with M199 and 1% HSA. Precipitates were diluted with heparinized M199 to obtain 6×10⁶ cells/ml.