Development of a Novel Air-Liquid Interface Airway Tissue Equivalent Model for In Vitro Respiratory Modeling Studies

The human airways are complex structures with important interactions between cells, extracellular matrix proteins and the biomechanical microenvironment. A robust, well-differentiated in vitro culture system that accurately models these interactions would provide a useful tool for studying normal and pathological airway biology. Here, we report the analysis of a physiologically relevant air-liquid interface (ALI) 3D airway ‘organ tissue equivalent’ (OTE) model with three novel features: native pulmonary fibroblasts, solubilized lung extracellular matrix (ECM), and hydrogel substrate with tunable stiffness and porosity. We demonstrate the versatility of the OTE model by evaluating the impact of these features on HBE cell phenotype. Variations of this model were analyzed during 28 days of ALI culture by evaluating epithelial confluence, trans-epithelial resistance (TEER), and epithelial phenotype via multispectral immuno-histochemistry and next-generation sequencing (NGS). Cultures that included both solubilized lung ECM and native pulmonary fibroblasts within the hydrogel substrate formed well differentiated ALI cultures that maintained a barrier function and expressed similar levels of goblet, club and ciliated markers to native airway tissue. Modulation of hydrogel stiffness did not negatively impact HBE differentiation and could be a valuable variable to alter epithelial phenotype. This study highlights the capability and versatility of a 3D airway OTE model to model the multiple components of the human airway 3D microenvironment. Gene expression profiling analysis of RNA-Seq data for 3D airway OTE models with varying stiffness and 2D airway epithelial cultures. The 3D OTE cultures were fabricated with hydrogels of 3 stiffnesses (2.6kPa, 5.1kPa, 9.8kPa) each containing solubilized lung ECM and native human lung fibroblasts.