Prolonged In Vitro Expansion Shapes the Neuro-Supportive Potential of Jaw Periosteum Secretomes: Implications for Secretome Product Quality.

Nerve injuries are frequent complications of complex oral and maxillofacial surgical procedures, particularly following extensive tumor resections. Secretome-based, cell-free therapies derived from mesenchymal stromal cells have emerged as promising regenerative approaches; however, robust manufacturing requires the identification of critical quality attributes (CQAs) that ensure product potency and consistency. The influence of replicative senescence during in vitro expansion on the quality of jaw periosteum-derived mesenchymal stromal cell (JPC) secretomes has not yet been established. This study investigated whether the expansion state of JPCs affects the composition and neuro-supportive potency of their secretomes. Secretomes from four independent JPC donors were collected separately at early and late passages, pooled within each passage-specific preparation, and applied to human induced pluripotent stem cell-derived neurons. Neuronal survival, neurite outgrowth, and neuronal marker expression were assessed as functional readouts. Secretome composition was characterized by quantitative proteomics and enzyme-linked immunosorbent assay (ELISA) of selected senescence-associated secretory phenotype (SASP) factors. Secretomes derived from early-passage JPCs significantly enhanced neuronal survival and neurite outgrowth, whereas late-passage secretomes displayed reduced neuro-supportive activity. Proteomic profiling identified a pronounced shift toward inflammatory and stress-associated signaling, whereas performed ELISAs confirmed senescence-associated remodeling of the secretome, including increased abundance of SASP-associated factors in late-passage preparations. These findings demonstrate that prolonged in vitro expansion profoundly influences both the composition and biological potency of JPC-derived secretomes. Collectively, this study identifies the passage-associated senescence-like phenotype of JPCs as a key determinant of secretome quality and supports its consideration as a critical quality attribute for the manufacturing and standardization of JPC-derived secretome products. Monitoring and controlling the expansion state of JPCs may therefore be essential to ensure the consistency, potency, and clinical translation of secretome-based regenerative therapies.