Clostridial collagenase ia

Full-depth articular cartilage was excised and immersed in Dulbecco’s Modified Eagle Medium (DMEM) (with phenol red and 4.5 g/L glucose) supplemented with N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES) 10 mM, penicillin (100 U/ml) and streptomycin (0.1 mg/ml) (all from Lonza, Verviers, Belgium). After three washings, chondrocytes were released from cartilage by sequential enzymatic digestions with 0.5 mg/ml hyaluronidase type IV S (Sigma-Aldrich, Bornem, Belgium) for 30 min at 37°C, 1 mg/ml pronase E (Merck, Leuven, Belgium) for 1 h at 37°C and 0.5 mg/ml clostridial collagenase IA (Sigma-Aldrich, Bornem, Belgium) for 16 to 20 h at 37°C. The enzymatically isolated cells were then filtered through a nylon mesh (70 μm), washed three times, counted and filled to the density of 0.1 x 10⁶ cells/ml of DMEM (with phenol red and 4.5 g/L glucose) supplemented with 10% fetal bovine serum, 10 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine (all from Lonza, Verviers, Belgium), 20 μg/ml proline and 50 μg/ml vitamin C (Sigma-Aldrich, Bornem, Belgium) [12 (link)].

Full-depth articular cartilage was excised and immersed in Dulbecco’s Modified Eagle Medium (DMEM) (with phenol red and 4.5 g/L glucose) supplemented with N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES) 10 mM, penicillin (100 U/ml) and streptomycin (0.1 mg/ml) (all from Lonza, Verviers, Belgium). After three washings, chondrocytes were released from cartilage by sequential enzymatic digestions with 0.5 mg/ml hyaluronidase type IV S (Sigma-Aldrich, Bornem, Belgium) for 30 min at 37°C, 1 mg/ml pronase E (Merck, Leuven, Belgium) for 1 h at 37°C and 0.5 mg/ml clostridial collagenase IA (Sigma-Aldrich, Bornem, Belgium) for 16 to 20 h at 37°C. The enzymatically isolated cells were then filtered through a nylon mesh (70 μm), washed three times, counted and filled to the density of 0.25 x 10⁶ cells/ml (bovine chondrocytes) or 0.1 x 10⁶ cells/ml (human chondrocytes) of DMEM (with phenol red and 4.5 g/L glucose) supplemented with 10% fetal bovine serum, 10 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM glutamine (all from Lonza, Verviers, Belgium) and 20 μg/ml proline (Sigma-Aldrich, Bornem, Belgium)

Full-depth articular cartilage was excised and immersed in Dulbecco’s Modified Eagle Medium (DMEM) (with phenol red and 4.5 g/L glucose) supplemented with N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES) (10 mM), penicillin (100 U/mL) and streptomycin (0.1 mg/mL) (all from Lonza, Belgium). After three washes, chondrocytes were released from cartilage by sequential enzymatic digestions with 0.5 mg/mL hyaluronidase type IV S (Sigma-Aldrich, Belgium) for 30 min at 37°C, 1 mg/mL pronase E (Merck, Belgium) for 1 h at 37°C and 0.5 mg/mL clostridial collagenase IA (Sigma-Aldrich, Belgium) for 16–20 h at 37°C. The enzymatically isolated cells were then filtered through a nylon mesh (70 µm), washed three times and counted.