Mesenchymal stem cells were isolated from femur bone marrow aspirates of male adult mice.
Bone marrow aspirates were diluted 1:1 in DMEM low-glucose and then filtered through a 70-μm nylon mesh. The resulting cell suspension was layered over 15 ml Ficoll-Paque Plus and centrifuged for 30 min at 800×g at room temperature. The supernatant and interface were combined, diluted to about 50 ml with PBS (0.1 M) and centrifuged for 10 min at 800×g. After discarding the supernatant, the pellet was suspended in 1 ml medium. The nucleated cells were counted, suspended at a concentration of 107/ml in growth medium (DMEM supplemented with L-glutamine (2 mg/ml), streptomycin (50 μg/ml) and non-heat inactivated fetal calf serum (10% (v/v)) and plated at 3×106/cm2 in 100-mm culture dishes. The cells were incubated for 3 days, and non-adherent cells were removed by replacing the medium in three washing steps. After the cultures reached confluence, the cells were lifted by incubation with Accutase at 37°C for 3–4 min. They were then diluted and plated at a density of 2,000 cells/cm2 in 100-mm culture dishes.