A Behavior-Integrated Potency Assay for Quantitative Evaluation of Extracellular Matrix Remodeling by Mesenchymal...

Reliable potency assays are critical for ensuring the therapeutic consistency and manufacturing quality of mesenchymal stem/stromal cells (MSCs). Conventional assays based on soluble factor quantification often fail to capture extracellular matrix (ECM) remodeling capacity and exhibit high variability. We developed a biologically relevant, image-based potency assay that directly quantifies MSCs-mediated ECM remodeling under defined biophysical conditions. A collagen-coated (CL) surface that provides a stable, proliferation-suppressive microenvironment, enabling single-cell quantification of remodeling activity using collagen hybridizing peptide staining. The ECM remodeling index (I_CHP+) defined an optimal working range (3.0 × 10³ ≤ cells/cm² ≤ 4.5 × 10³), where spatially separated MSCs achieved high analytical precision (CV < 10%). The CL-based assay demonstrated robust repeatability across MSCs sources and biologically relevant quantitation limits. Integration of I_CHP+ with γH2AX, enabled simultaneous assessment of functional potency and cell health. Response surface methodology modeling of culture duration, seeding density, and passage number revealed a dome-shaped relationship, with maximal remodeling at early passages (N_p1-N_p3) and 3-5 days of culture, whereas extended culture (> 6 days) decreased potency alongside elevated γH2AX expression. The model exhibited strong predictive performance (R² > 0.95), supporting its utility for process optimization. Collectively, this mechanism-based potency assay enables standardized platform for potency evaluation and manufacturing control for consistent, high-quality MSCs therapeutics.