Optimal conditions of collagenase treatment for isolation of articular chondrocytes from aged human tissues

INTRODUCTION: There are various types of cartilage, including the auricular and articular cartilages. These cartilages have different functions, and their matrix volume and density of chondrocytes may differ. Thus, different protocols may be required to digest different types of cartilage. METHODS: In this study, we examined protocols for the digestion of articular and auricular cartilages and determined the optimal conditions for articular cartilage digestion. RESULTS: Our histological findings showed that the articular cartilage has a larger matrix area and fewer cells than the auricular cartilage. In 1-mm(2) areas of articular and auricular cartilages, the average numbers of cells were 44 and 380, respectively, and the average matrix areas were 0.94 and 0.77 mm(2), respectively. The maximum numbers of viable cells (approximately 1 × 10(5) cells/0.1 g of tissue) were obtained after digestion in 0.15, 0.3, or 0.6% collagenase for 24 h, in 1.2% collagenase for 6 h, or in 2.4% collagenase for 4 h. In tissues incubated in 0.15 or 0.3% collagenase, the cell numbers were lower than 1 × 10(5), even at 24 h, possibly reflecting incomplete digestion of cartilage. No significant differences were observed in the results of apoptosis assays for all collagenase exposure times and concentrations. However, cell damage appeared to be greater when collagenase concentrations were high. When cells obtained after digestion with different concentrations of collagenase were seeded at a density of 3000 cells/cm(2), they yielded the maximum cell numbers after 1 week. CONCLUSIONS: We recommend a 24-h incubation in 0.6% collagenase as the optimal condition for chondrocyte isolation from articular cartilage. Moreover, we found that the optimum cell-seeding density is approximately 3000 cells/cm(2). Conditions determined in this study would maximize the yield of isolated articular chondrocytes and enable the generation of a large quantity of cultured cells.