Formation of functionally robust human neocartilage from multiple donors using highly expanded costochondral cells

Successful translation of allogeneic tissue-engineered neocartilage requires abundant functional cells. Human costal cartilage is a promising cell source, yet the effects of donor variability and extensive passaging remain unexplored. Therefore, this study investigated the functional (i.e. morphological, histological, mechanical, and biochemical) properties of hyaline-like neocartilage generated from high-passage human costochondral cells. A cell banking system was applied to seven donors to create master cell banks and subsequently working cell banks to fabricate P5 and P9 constructs using the processes of conservative chondrogenic passaging, aggregate rejuvenation, and self-assembly. Cell morphology or gene expression levels of these cells were correlated with mechanical and biochemical properties to identify predictive markers of neocartilage functional properties. Cells from younger donors (⩽3 months) expanded 8.7-fold more than cells from older donors (9-14 years). Cumulative expansion factors from P0 reached 3124-17397 at P5 and 1.4-36.2 million at P9. Aggregate rejuvenation was as effective at P9 as at P5 in restoring a hyaline cartilage-like phenotype, evidenced by increased cell circularity, upregulation of chondrogenic genes (e.g. >310-fold forACAN; >200 000-fold forCOL2A1; >2500-fold forChm-1), and robust neocartilage functional properties. At P5, one young donor exhibited the highest functional properties (e.g. aggregate modulus = 310 kPa, Young's modulus = 2.9 MPa, GAG/ww = 9.5%, COL/ww = 3.1%). At P9, a different young donor had the highest functional properties (e.g. aggregate modulus = 220 kPa, Young's modulus = 2.3 MPa, GAG/ww = 8.6%, COL/ww = 3.4%). Gene expression levels after aggregate rejuvenation were identified to be predictive of neocartilage functional properties. For example,ACAN, Chm-1, andMIApositively correlated with Young's modulus, ultimate tensile strength (UTS), and GAG/dw, withChm-1also correlating with shear modulus.SOX9correlated with Young's modulus and GAG/dw, whileMMP13inversely correlated with UTS and GAG/dw. These findings support the translational potential of extensively passaged human costochondral cells, the need for donor screening, and the utility of gene expression markers to predict neocartilage functional properties.