A Chondrosphere-Based Scaffold Free Approach to Manufacture an In Vitro Articular Cartilage Model

In vitro engineering of human articular cartilage (AC) is a regenerative medicine challenge. The main objective of this study was the development of a repeatable scaffold-free in vitro model of chondrocyte spheroid-based treatments of cartilage defects, to allow for systematic study and further optimization of this type of treatment. Human articular chondrocytes (HC) and immortalized mesenchymal cells differentiated in chondrocytes (Y201-Cs) were cultured in round-bottom 96-well plates to produce multicellular spheroids and their growth kinetics, and viability was evaluated over 7 days of culture. Then, the spheroids were assembled and cultured for 21 days on a gelatin-coated poly(lactic-co-glycolic acid) electrospun membrane (10 spheroids/cm²), following a protocol in line with the clinically approved Chondrosphere^® (CO.DON AG) technique. Both HC and Y201-C cells formed compact and viable spheroids after 7 days of culture with a reduction of diameter over the 7 days from 1300 ± 150 μm to 600 ± 90 μm and from 1250 ± 60 μm to 800 ± 20 μm for HC and Y201-C, respectively. When the spheroids were transferred onto the support membrane, these adhered on the membrane itself and fused themselves, producing collagen type II (COL2A1) and aggrecan (ACAN), according to gene expression and glycosaminoglycans quantification analyses. We detected higher expression of COL2A1 in HC cells, while the Y201-C constructs were characterized by an increased ACAN expression. The approach we presented allows a standardizable production of spheroids with predictable geometry and the creation of a reproducible scaffold-free in vitro AC-like construct showing high expression of chondrogenic markers, using both HC and Y201-C. In addition, the bankable Y201-C cells provide an effective base model for experimentation with the spheroid approach to further enhance the process. Impact statement This is first work on the development of a repeatable scaffold-free in vitro model based on an optimized protocol in line with a recent clinically approved Chondrosphere^® (CO.DON AG) technique. In addition, we demonstrated that a bankable cell type (Y201-C) could produce an engineered cartilage-like construct, giving a repeatable model as a key tool for experimentation of therapeutic treatment ahead of studies with heterogeneous cell populations.