Sw1353 human chondrosarcoma cells

The ATDC5 mouse chondrogenic cell line was obtained from Riken BioResource Center (Tsukuba, Japan) and was cultured in DMEM/F12 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Biological Industries, ISRAEL) at 37 °C under 5% CO₂. The SW1353 human chondrosarcoma cells were purchased from ATCC and cultured in DMEM medium (Gibco, USA) supplemented with 10% FBS at 37 °C under 5% CO₂. Primary articular chondrocytes were isolated from the articular cartilage of newborn mice (within 3 days). Rib cartilage from several mouse pups was dissected under a stereo light microscope, and the articular cartilage was incubated in 0.25% trypsin for 30 min. The cartilage was then digested in 0.1% collagenase type II (Worthington, USA) for 6 h, and chondrocytes were seeded in a T25 cell culture flask at a density of 2 × 10⁵ cells per flask and cultured at 37 °C with 5% CO₂ in DMEM/F12 medium supplemented with 1% penicillin-streptomycin (P/S) solution (Gibco) and 10% FBS. For the chondrogenic differentiation assay, cells were cultured in differentiation medium containing DMEM, streptomycin (100 µg/mL), penicillin (100 U/mL), 1% insulin-transferrin-selenous acid (ITS) Premix (Gibco), _L-proline (40 µg/mL), TGF-β1 (Novoprotein, 10 ng/mL), 0.1 µM dexamethasone and ascorbate 2-phosphate (50 µg/mL) in a 24-well plate. The media were refreshed every two days.