Type ia crude bacterial collagenase

To obtain conditioned medium, C-PCs and BM-MSCs were seeded (1.0 ×10⁵ cells per well) into 6-well plates and cultured in DMEM++ (2 mL per well). Cells were cultured for 4 days in a 37°C cell incubator. Medium was collected, centrifuged (1700 RPM, for 5 minutes) and supernatant was used as conditioned media. Meniscocytes were isolated from rat menisci from three month old Lewis rats. Menisci were diced into small fragments, digested with Pronase (Roche, Indianapolis, IN, USA) (2.0 mg/ml in 1xHBSS) for 30 minutes at 37°C in a shaking water bath, washed in 1xHBSS and further digested using Type IA Crude Bacterial Collagenase (Sigma-Aldrich, St. Louis, MO, USA) (1.0 mg/ml) for 8 hrs at 37°C in a shaking water bath. Cells were passed through a nylon cell strainer (100 μm pore size) to remove undigested tissue. Meniscocytes were expanded in DMEM++ in a 37°C cell incubator prior to experiments. Meniscocytes were seeded (5.0 ×10⁴ cells per well) into a 12-well plate. After 24 hours, media was changed to 600 μL of conditioned media from BM-MSC, CPCLA1, CPCL3, or unconditioned DMEM++ media (control). Cells were cultured for 6 days and cell numbers were quantified using a hemocytometer. Images were taken using a Nikon Eclipse 90i microscope.

Primary human OAC and OA-MSC were isolated from discarded articular cartilage tissues as previously described.³ In brief, cartilage collected from non-lesion area of knee was washed with PBS three times and cut into small pieces. The cartilage fragments were treated with 2.0 mg/ml Pronase (Roche, Indianapolis, IN, USA) for 30 minutes at 37°C in shaking water bath. Cartilage fragments were then washed with DMEM twice and digested with 1.0 mg/mL Type IA Crude Bacterial Collagenase (Sigma-Aldrich, St. Louis, MO, USA) for 8 hours at 37°C in shaking water bath. Hundred micrometers of nylon cell strainer was utilized to remove the undigested cartilage clumps. Cells were then washed twice with DMEM supplemented with 10% FBS and counted by a hemocytometer. OA-MSC was enriched using differential cell adhesion method with 10 μg/mL fibronectin at 37°C. After 20 minutes, adherent cells were OA-MSC, which were further used for cell culture or cell line generation, while the non-adherent cells were OA chondrocyte.³ OA-MSC were confirmed to be CD166 positive while OAC were confirmed to be CD166 negative by flow cytometry analysis, as described previously.^{3 ,22 (link)} All the cells were mycoplasma negative. Human BMSCs were purchased from ATCC (ATCC PCS500012, VA, USA), and OA-MSC cell lines were established as described previously.³