Isolation of Bone Marrow Mesenchymal Stem Cells:
Initially, 10-15 ml of bone marrow samples were prepared. The bone marrow sample was diluted 1: 1 with PBS, then it was slowly poured onto the ficoll, and the cells were centrifuged at room temperature for 20 min at room temperature with minimal acceleration and braking. After re-centrifugation, the mononuclear cells were separated and diluted in PBS (2: 1) and centrifuged again at room temperature at 400 xg for 10 min. The cell pellets were counted and centrifuged again at room temperature for 4 min at 400 xg. At this stage, the cell pellets were suspended in DMEM medium containing 10% FBS at a concentration of 107 cells / ml. Approximately 30 ml of culture medium containing FBS was added to each sample and cells were cultured at approximately 50,000 cells/cm in T-175 flask (1 ml of mononuclear cell suspension to 30 ml of flask culture medium). The cells were slowly mixed in the culture medium and the flasks were transferred to a 37 ° C incubator with 50% CO2. After 4 to 7 days, non-adherent cells were removed by supernatant removal and 30 ml of fresh medium was added to the flask. Culture medium was replaced twice a week. After 5 to 10 days of plating (in some cases more) MSCs cells reaching 80-90% density, were moved into other flasks.