Articular chondrocyte spheroids (Chondrosphere®) (co.don AG)

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.