Chondrocytes Cultured in Stirred Suspension with Serum-Free Medium Containing Pluronic-68 Aggregate and Proliferate While Maintaining Their Differentiated Phenotype

The study of chondrocyte biology requires culture conditions that maintain cell phenotype. Phenotype is rapidly lost in monolayer but is maintained in 3-dimensional scaffolds, which however, experience limited cell proliferation and limited mass transport. In this study, we cultured chondrocytes in aggregates in stirred spinner flask suspension cultures to control aggregate size and promote mass transport. A previously optimized serum-free medium, containing the following growth factors (GFs), epidermal growth factor, platelet-derived growth factor-BB, and basic fibroblast growth factor, all at 2 ng/mL, was used as a control medium. In addition, two modified media were tested: one containing Pluronic F-68 (PF-68) and the other containing PF-68 with 10 times greater GF concentration (20 ng/mL, medium PF-68= 10 x GF). Chondrocytes formed limited-size aggregates within 24 h and exhibited high viability (>95%), and cell concentration doubled in 7 days in the presence of PF-68. Low or no collagen I expression was found for any of the three media, whereas collagen II accumulated between cells, as revealed by a dense immunostaining. Integrin alpha 10, a marker of differentiated chondrocytes and chondrogenic cells, was also found to be highly expressed. Aggregates resulting from spinner culture were found to be relevant in vitro models and their use for cartilage repair to be also conceivable.