In vitro Isolation and Culturing of Mouse Primary Chondrocytes for Cartilage Biology.

Articular cartilage destruction leads to altered chondrocyte activity, which is a major causative factor in a variety of cartilage diseases, including osteoarthritis (OA) and rheumatoid arthritis (RA). As the most abundant cell type in cartilage, an in-depth study of the biological properties of chondrocytes is essential for the development of effective disease treatment strategies. Isolation and culturing of primary chondrocytes provide important experimental materials for the investigation of cartilage disease and contribute to unraveling the mechanisms of cartilage injury and repair. This protocol provides a detailed description of the in vitro isolation and culturing methods for primary chondrocytes derived from mice. By using the optimized collagenase II protocol, chondrocyte isolation can be completed within 8 h for neonatal mouse knee joint cartilage specimens. The cell yield is approximately 1-2 × 10³ cells/mg of cartilage tissue, varying with tissue freshness and initial cell density; the isolated cells exhibit >90% viability. This protocol uses mouse primary chondrocytes as an example, showing the morphological characteristics and adhesion status of cells cultured for 1, 2, and 5 days after isolation. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting showed that interleukin-1β (IL-1β) treatment reduced Collagen Type II Alpha 1 (Col2a1) and increased Matrix Metallopeptidase 13 (Mmp13) expression, confirming that isolated chondrocytes respond to inflammatory stimuli. Compared with conventional methods, this protocol employs a higher collagenase concentration to reduce the impact of prolonged isolation time on cell viability, while avoiding potential cell damage from trypsin treatment. The obtained primary chondrocytes are pure and viable, suitable for in-depth studies on cartilage injury-related mechanisms, which have important implications for in vitro research and the clinical treatment of cartilage disease.