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.