Chondrocytes (ChondroArt™) (Educell)

Human articular chondrocytes are isolated from articular cartilage. Cartilage tissue is rinsed with phosphate-buffered saline (PBS), diced into small fragments and digested with collagenase II (0.05% (w/v)) for 18 h at 37⁰C. The digestion suspension is washed by centrifugation (5 min at 300g) in DMEM/F-12 medium containing FBS (10%), fungizone (2 mg/ml) and gentamycin (1 ml/ml).The pellet is resuspended in 1 ml culture medium. Cells are seeded in 25 cm2 culture flasks in monolayers at a concentration of 3,000 cells/cm2 and cultivated at 37⁰C under a 5% CO₂ atmosphere. Culture medium is exchanged twice per week. After reaching 80%–90% confluence, cells are harvested using trypsin/EDTA solution (0.02%). 

Cells were seeded on a fibrinogen-collagen scaffold or on an alginate-agarose hydrogel scaffold:

A two-component fibrin glue (fibrinogen and thrombin solution) is prepared using a standard technique, first adding the fibrinogen solution to the cell suspension and thrombin only after cell seeding. Trypsinized chondrocytes taken from monolayers (2nd passages) are resuspended in an equal volumes of culture medium and fibrinogen solution. Scaffolds are seeded with 400,000 cells resuspended in a final volume of 300 ml. Chondrocytes are seeded by dropping a cell suspension directly onto the sponge. After a 30-min incubation at room temperature, the thrombin solution, prepared with PBS (1:10), is added onto each side of the constructs for 2 h. Constructs are placed in six-well plates in culture medium containing FBS (10%) and cultivated for 3 weeks. Culture medium is changed twice per week.

Samples of alginate/agarose hydrogels with a final cell density of 10⁶ cells/ml, are prepared according to the manufacturer’s recommendations. Once gelled, all the constructs are removed from the mold, washed extensively with PBS, and cultured in DMEM/F-12 medium with human serum (15% (v/v)) and gentamycin (50 mg/ml), in a humidified incubator, at 37⁰C and 5% CO_2, for 14 days. Culture medium is replaced every 2 days.