Comparison of human juvenile and adult donor-derived chondrocyte sheets for scalable allogeneic regenerative therapy

Background: Regeneration of damaged cartilage is challenging, and no reproducible regenerative therapies using mass-producible cell products have been established. This study evaluated the cartilage regeneration capability and therapeutic scalability using cell sheets derived from routinely available surgical waste cartilage tissues of young and adult patients, while also investigating the mechanisms that define the characteristics of each cell type. Methods:We compared the viability, proliferation, and cell sheet characteristics of juvenile cartilage-derived chondrocytes (JCCs) from patients with polydactyly (2.2 ± 1.6 years) and adult cartilage-derived chondrocytes (ACCs) from patients with femoroacetabular impingement (FAI) (34.1 ± 10.6 years)in vitro. Thein vivocartilage regeneration capability of each cell sheet was validated in a nude rat knee cartilage defect model using histological O’Driscoll score evaluation on Safranin-O-stained tissues and immunohistochemistry. JCC sheets (n = 13) and ACC sheets (n = 8) were analyzed using established bulk RNA sequencing pipelines for gene ontology (GO) analysis, gene set enrichment analysis (GSEA), and ingenuity pathway analysis (IPA). Interferon gamma (IFN-γ) was applied to JCC sheet culture for confirmation of the interferon signaling involvement in cell proliferation, cell sheet characteristics, and chondrogenic differentiation.Results:JCC demonstrated higher colony-forming ability and stable high proliferation compared to ACC. Both JCC and ACC sheets formed positively stained hyaline cartilage for Safranin-O, type II collagen, aggrecan, and human vimentin, while being negative for type I collagen, four weeks after rat transplantation. However, the regenerated cartilage from ACC sheet transplantation was found to be thinner compared to that from JCC sheet transplantation. Comprehensive gene analysis revealed significant activation of IFN signaling in the ACC sheets. Furthermore, the addition of exogenous IFN dramatically reduced the proliferation and cartilage formation capability of JCC.Conclusions:JCC sheets exhibit high therapeutic scalability due to their proliferation and cartilage regeneration capabilities presumably derived from sustained low IFN-γ activity. Consideration of the donor age and tissue inflammatory status is essential for the cell source in allogeneic cell therapies. Given their sustainable sourcing from routine surgical discards, JCCs present a commercially viable and scalable option for allogeneic regenerative therapy in cartilage repair.