Articular cartilage-derived cells (ChondroCelect®) (TiGenix NV)

Normal articular cartilage was obtained from femoral condyles during arthroscopy. Up to 3 cartilage slices, 7-8 mm long and a few millimeters wide, were obtain during biopsy and placed in HBSS solution supplemented with 200 units/ml penicillin, 200 mg/ml streptomycin and 0.5 mg/ml amphotericin B (antibiotic–antimycotic solution). Cartilage slices were minced and washed twice in the supplemented HBSS for 5 minutes at 37°C. Chondrocytes were isolated from the cartilage biopsy in a GMP–approved facility, by overnight digestion at 37°C, in 0.2% crude type IV collagenase in high-glucose DMEM containing FBS (10%) and antibiotic–antimycotic solution. Cells were washed twice in DMEM containing FBS (10%) and antibiotic–antimycotic solution and then counted with Trypan blue staining to determine the number of viable cells. Generally, >95% of the cells were viable. Chondrocytes were plated at a density of 5x10⁴ cells/cm² in growth medium (DMEM containing FBS (10%) and 100 units/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml amphotericin B) and cultured in a humidified 5% (vol/vol) CO₂ atmosphere. Cells were detached 10 days after confluence by trypsin–EDTA treatment. Chondrocyte phenotype was then characterized. Each batch of ChondroCelect® is quantitatively graded using a ChondroCelect® score (CC score) based on the quantitative gene expression profile developed to predict the cells' ability to form stable hyaline cartilage in vivo.