Chondrocyte Isolation and Cell Culture
Full-thickness articular cartilage was aseptically harvested from the metacarpal-phalangeal joints of skeletally-mature bovine (obtained fresh from a local slaughterhouse; 20-24 months of age) to isolate primary chondrocytes by enzymatic digestion (Waldman et al. , 2002). Briefly, cartilage slices were incubated in protease (0.5% w/v) for 1 hour followed by 18 hours in collagenase A (0.15% w/v) in Ham’s F-12 media. For each isolation, tissue slices from at least two animals were pooled together to achieve adequate cell numbers. Viable cells (> 90%; by trypan blue dye exclusion) were seeded on collagen II-coated Millicell™ filters (10 mm dia.; Millipore) in 3D high density culture (2 x 106 cells/filter or 25,000 cells/mm2) (Kaupp & Waldman, 2008) and maintained in Ham’s F12 media containing 10 mM glucose and supplemented with 20% FBS, 100 µg/mL ascorbate, 20 mM HEPES, and 1% (v/v) penicillin/streptomycin. Cultures were grown under varying volumes of media (1 – 8 mL or 0.5 – 4 mL/106 cells) and maintained under normoxic conditions (5% CO2:95% atmospheric air; 95% relative humidity; 37°C) for 4 weeks with media exchanged every 2-3 days. The same approach was used for both rabbit chondrocytes (Charles River Laboratories) and human donor chondrocytes; approved by the University Animal Care Committee (Queen’s University) and Research Ethics Board (Queen’s University), respectively.