Isolation and culture of primary chondrocytes

A total of 30 C57BL/6 mice (age, 2-3 days) were purchased from the Animal Center of Chinese Academy of Sciences and were decapitated before chondrocytes were isolated from their articular cartilage. Briefly, the articular cartilages of each mouse were carefully extracted under aseptic conditions and cut into ~1-2 mm² slices, followed by washing with PBS three times at room temperature. The pieces were then digested using with DMEM/F12 medium supplemented with 0.1% collagenase II at 37˚C in a humidified atmosphere containing 5% CO₂ for 8 h. The cells were then collected via centrifugation at 1,000 x g for 3 min at 25˚C, washed with PBS three times, plated into cell culture flasks in DMEM/F12 supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin and incubated at 37˚C in an atmosphere of 95% air and 5% CO₂. The medium was changed after 24 h and the cells were harvested when 80-90% confluence was reached. Only chondrocytes from passages 1-2 were used in the present study to avoid the loss of phenotype. Light microscopy (upper panel original magnification, x100; lower panel original magnification, x200) was performed to observe the cell morphology of chondrocytes. Cells at passages 1-2 had a rounded or polygonal structure.