Gibco collagenase type 2

Synovium was harvested from the femoropatellar joints of 5 systemically healthy horses (ages 2–14 years) euthanized for reasons other than this study and free of femoropatellar joint disease. The isolated synovium was weighed and digested for 2 h at 37°C under constant rotation with synoviocyte media [high glucose (4.5 g/L) DMEM medium with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 1 mM sodium pyruvate, 25 mM HEPES, penicillin (100 units/mL), and streptomycin (100 μg/ml)] added at 10 mL/g tissue and containing 1.5 mg/mL Gibco® collagenase type II (ThermoFisher Scientific, Waltham, MA, USA) (50 (link), 51 (link)). The resulting digest was passed through a 100 μm filter and centrifuged at 800 g for 10 min. The cell pellet was then washed twice with fresh synoviocyte media and live synoviocyte count was determined using a Cellometer® Auto 2000 and ViaStain^TM AOPI Staining Solution (Nexcelom Bioscience LLC, Lawrence, MA, USA). Synoviocytes were frozen in aliquots of 10 × 10⁶ cells/mL in liquid nitrogen until use.

Cartilage was harvested from the femoral trochlear ridges of a 2-year-old Thoroughbred gelding free of orthopedic disease and euthanized for reasons other than this study. The isolated cartilage was weighed and digested overnight (16–18 h) at 37°C under constant rotation with chondrocyte media [Ham's F12 medium with 10% FBS, 25 mM HEPES, ascorbic acid (50 μg/mL), α-ketoglutarate (30 μg/mL), L-glutamine (300 μg/mL), penicillin (100 units/mL), and streptomycin (100 μg/ml)] containing 0.75 mg/mL of Gibco® collagenase type II (ThermoFisher Scientific, Waltham, MA, USA) (52 (link), 53 (link)). The resulting digest was passed through a 100 μm filter and centrifuged at 800 g for 10 min. The cell pellet was then washed twice with fresh chondrocyte media. Cells were resuspended in chondrocyte media and live chondrocyte count was determined using a Cellometer® Auto 2000 and ViaStain^TM AOPI Staining Solution. Chondrocytes were frozen in aliquots of 10 × 10⁶ cells/mL in liquid nitrogen until use.