Passage 4 BMSCs at a density of 5 x [10.sup.5] cells/mL in 0.2 mL L-DMEM without FBS were added into the upper chamber of a 6.5 mm diameter transwell insert with a pore size of 8 [micro]m.
Passage 4 BMSCs at a density of 1 x [10.sup.5] cells/mL were seeded in 96-well plates with 0.1 mL L-DMEM (10% FBS) and incubated for 24 hours after application of CCK-8 kit, and values of OD450 for each group were read by the microplate reader according to manufacturer's protocol.
Cells were expanded in 35 mm culture plates until passage 3 or 4 and then were induced for 14 days in an adipogenic induction medium (L-DMEM supplemented with 10% FBS, 0.5 mM isobutylmethylxanthine (IBMX) (Sigma-Aldrich), 1mM dexamethasone, 10 mM insulin, 200 mM indomethacin, and 1% antibiotic/antimycotic solution).
The induction medium consisting of L-DMEM, 10% FBS, 0.1 [micro]M dexamethasone, 10 mM [beta]-glycerophosphate, 0.05 mM ascorbate-2- phosphate, and 1% antibiotic/antimycotic solution was used to induce osteogenic differentiation in vitro.
The pellets were cultured with complete medium (L-DMEM, 10% FBS, 100 U/mL penicillin, and streptomycin) at 37[degrees]C in a 5% C[O.sub.2] atmosphere.
The cells were then digested with pancreatic enzymes and diluted with L-DMEM at [10.sup.5] cells per milliliter.