Characterization of human airway-derived decellularized extracellulr matrix hydrogels and their effects on airway epithelial cell growth, organization and differentiation.

Three-dimensional human airway models are useful tools to study respiratory development, disease and regeneration. However, commonly used substrates such as Matrigel™ differ substantially from native airway ECM, and effects on cell behavior remain incompletely characterized. Human airway-derived decellularized ECM (AW-dECM) hydrogels represent a potentially more physiological alternative, but their properties relative to conventional matrices are undefined. We performed a comprehensive characterization of AW-dECM and Matrigel™ hydrogels by assessing ECM composition, stiffness and viscoelasticity and evaluated effects on primary human bronchial epithelial cell behavior at air-liquid interface. AW-dECM (15 mg/mL; 30 mg/mL) exhibited lower stiffness and greater similarity to native airway tissue than standard concentration (SC; 8 mg/mL) or high concentration (HC; 19 mg/mL) Matrigel™. All hydrogels displayed typical viscoelastic behavior. Proteomic analysis revealed collagen I and VI enrichment in AW-dECM that was absent in Matrigel™. These differences lead to distinct cellular organization: SC Matrigel™ promoted organoid formation, HC Matrigel™ supported tubule-like structures/monolayers, and AW-dECM favored confluent monolayers. Gene expression varied by substrate; stiffer matrices promoted greater secretory differentiation and AW-dECM increased expression of MMP9, MMP7, and TJP1. Inhibition of actomyosin contractility enhanced cell adhesion/spreading on softer matrices, further supporting the influence of matrix properties on airway epithelial cell behavior. AW-dECM and Matrigel™ exhibit distinct mechanical and biochemical properties that differentially influence airway epithelial cell growth, organization, and differentiation. This work establishes a detailed characterization of human airway-derived ECM hydrogels and provides a framework linking matrix properties with airway epithelial responses, thereby informing the development of more physiological airway models.