Chondrocytes are isolated from articular cartilage, obtained from undamaged articular condyles of patients suffering from knee cartilage defects. Chondrocytes are expanded in vitro and condensed into spheroids (chondrospheres).
Articular cartilage was obtained from macroscopically undamaged human articular condyles. Cartilage (60-100 mg) was digested in a 50 ml Falcon tube using 20-25 U/mg collagenase type II at 37˚C for 8 hours in a gyratory shaker (110 rpm). Isolated cells were washed and resuspended in culture medium (DMEM and Ham's 1:1) with the addition of human serum. No growth factors, cytokines or other supplements were added. Chondrocytes were seeded in 75 cm2 culture flasks and maintained in a humidified 37˚C and 5% CO2 incubator. Medium was changed twice a week. After reaching confluence, the cells were trypsinized with trypsin-EDTA, and cultured in 225 cm2 flasks. For generation of spheroids, 2x105 cells of third passage chondrocytes were seeded in hydrogel-coated 96-well-plates. After 1 week, four to twelve single spheroids were transferred into a single well, allowing coalescence of spheroids.