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Chondrocytes are isolated from the patient and cultured on a type I/III collagen porcine membrane.
Cartilage tissue (50–150 mg) is obtained from the nonweight-bearing supracondylar region around the femoral condyles and placed in serum-free nutrient medium. Cartilage specimens are minced and washed three times in culture medium containing Ham's F12 medium supplemented with HEPES buffer (10 mmol/l), gentamicin sulfate (50 μg/ml [approximately 70 μmol/l]), amphotericin B (2 μg/ml [2.2 μmol/l]), and l-ascorbic acid (50 μg/ml [300 μmol/l]). The minced cartilage is digested for 16 hours, in a spinner bottle, in 10 ml culture medium containing clostridial collagenase (1 mg/ml [150 U/l]) and deoxyribonuclease I (0.1 mg/ml [25,000 U/l]). The cells are then filtered through nylon mesh with a pore diameter of 25 micrometers, washed three times, counted (range, 180,000 to 455,000 cells), resuspended in culture medium supplemented with 15% of the patient's serum (autologous serum), and seeded at a cell density of 5000 to 10,000 cells per square centimeter in 25-cm 2 or 75-cm2 culture flasks. After acceptable cell density is ascertained in vitro, cells are seeded onto the collagen membrane.