Whole knees derived from deceased subjects spanning birth to 13 years of age (juvenile), are processed aseptically in accordance with current Good Tissue Practices to harvest articular cartilage from the proximal femur and distal tibia. The chondrocytes form cartilage discs, about the size of a quarter, which are nearly identical in toughness and composition to hyaline cartilage.
Minced cartilage is disaggregated by sequential enzymatic dissociation in pronase (0.2%), followed by overnight digestion, using a mixture of 0.07% collagenase and 0.04% hyaluronidase in HL-1 chemically defined, serum-free medium.
Neocartilage is produced by seeding 0.5-1.0 ×106 nonexpanded chondrocytes per cm2 in HL-1 medium or a proprietary chemically defined medium displaying growth characteristics similar to HL-1. After day 3 of culture, cultures are supplemented with ascorbate (50 µg/mL), with medium exchange occurring every 3-4 days. Cultures are maintained at 37°C in a humidified environment consisting of 5% CO2. Neocartilage is harvested and used on days 44-63 of culture.