Long-term expandable auricular chondrocytes reveal a conserved nine-gene decline module underlying senescence-independent dedifferentiation.

Introduction: Primary chondrocytes often lose matrix-forming capacity during in vitro expansion, limiting scalable cartilage engineering. Here, we examined aging-independent regulation of chondrogenic function during long-term expansion of primary auricular chondrocytes and sought molecular features associated with late-passage functional decline. Methods: Mouse auricular chondrocytes (mACs) were serially expanded in 2D culture up to passage 40 or 50 (P50) under high- or low-seeding conditions. Proliferation, morphology, and senescence status were assessed by growth curves, phase-contrast imaging, and SA-β-gal staining. Chondrogenic capacity was evaluated using 3D pellet culture and toluidine blue staining. Bulk RNA sequencing of matched 2D and 3D samples was followed by PCA, differential expression, and pathway enrichment analyses. Key transcriptional signatures were validated by RT-qPCR in mouse auricular chondrocytes. Results: Both seeding conditions supported sustained proliferation across extended passaging. Although not immortalized, mACs maintained stable proliferation for more than P 50 with minimal senescence-associated features, exhibiting cell line-like proliferative behavior. Despite preserved growth, proteoglycan-rich matrix production declined at P50. Transcriptomic analyses identified culture dimensionality as the dominant source of variance, with 2D cultures showing passage-dependent drift and 3D pellets retaining a more stable chondrogenic profile. Cross-comparisons identified a conserved nine-gene decline module ( Conclusion: Auricular chondrocytes exhibit cell line-like proliferative capacity with limited senescence activation while progressively losing matrix-forming competence at ultra-late passages. A conserved nine-gene decline module provides molecular benchmarks for passage-resolved assessment and may inform optimization of scalable auricular cartilage manufacturing.