To optimize the chondrocyte numbers obtained after collagenase digestion for cartilage tissue engineering, we examined the enzyme concentration and incubation time for collagenase digestion. The appropriate cell density in the chondrocyte primary culture was also verified. The collagenase digestion conditions that maximized the viable cell numbers were 24 h in 0.15% and 0.3% collagenase, 6 h in 0.6%, and 4 h in 1.2%, leading to similar to 5 x 10(5) cells from 0.05 g. When seeded at 10,000 cells/cm(2), all of these cells became almost confluent within 1 week. Cells digested in 0.3% for 24 h or 0.6% for 6 h and seeded at 3000 cells/cm(2) may also be acceptable, and similarly reached confluence within 1 week. Results regarding expression of the p53, tumor necrosis factor-a, and interleukin-1 beta genes, as well as apoptosis enzyme-linked immunosorbent assay results, show that excessive collagenase exposure may decrease chondrocyte viability or activity. We recommend a 24-h incubation in 0.3% collagenase or 6 h in 0.6% collagenase, and a cell-seeding density of 3000-10,000 cells/cm(2). These conditions maximize the harvest of isolated chondrocytes from a small amount of biopsied tissue and significantly aid in obtaining a large quantity of cultured cells in a short period.