Tgf β1 240 b 002

For chondrogenic differentiation, cells were cultured in a three-dimensional pellet culture: 200,000 cells were suspended in 0.2 mL of chondrogenic-inducing differentiation medium, consisting of DMEM high glucose, 1% penicillin/streptomycin, 1% ITS+ premix (354352; BD), 0.04 mg/mL proline (Sigma), 0.1 mM ascorbic acid, 0.1 μM dexamethasone, and 10 ng/mL TGF-β1 (240-B-002; R&D Systems). The pellets of the control group were suspended at the same density in 0.2 mL of chondrogenic differentiation medium without TGF-β1. Initially, L-MSC chondrogenesis was not induced; therefore, in a follow-up experiment, we cultured L-MSC pellets in the presence of TGF-β1 (10 ng/mL) and BMP-2 (250 ng/mL; R&D). The suspensions were placed in a 96-well round-bottom polystyrene plate (Corning Costar 7007) resulting in ultralow attachment of the cells, which was centrifuged for 5 min at 1,500 rpm at RT. Medium was changed every day for 2 weeks and thereafter every other day for another week. After 21 days, three pellets were collected from every condition for RNA isolation and qPCR analysis and two pellets for histological evaluation. We stained 5 μm thick sections with 0.125% Safranin O (Sigma) for proteoglycans in cartilage and counterstained with 0.4% Fast Green (Sigma); differentiation status was assessed by staining and morphology of the cell pellet.