Juvenile neocartilage (RevaFlex™) (ISTO technologies)

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.