![]() ![]() Besides the heterogenic cell populations within the airways, the subepithelial extracellular matrix (ECM) has been shown to play a key role in airway structural integrity as well as in cell regulatory functions such as cell activation, proliferation and differentiation 8. Each of these cells found in the interstitial layer of the airways play key roles in the cell–cell communication that influence normal function and disease 5, 6, 7. ![]() Additionally, there is significant interplay with the pseudostratified epithelium and its basal three-dimensional (3D) microenvironment that includes subepithelial fibroblasts, immune cells, endothelial cells, and smooth muscle cells depending on airway size. For many diseases there is dysregulation in these epithelial cell subtypes that directly corresponds with the disease including asthma, chronic obstructive pulmonary disorder, and cystic fibrosis 3, 4. ![]() The airway epithelium is heterogeneous with distinct specialized cells including ciliated cells, goblet cells, club cells, basal cells, ionocytes, and neuroendocrine cells 2. The airways are at the interface of the internal and external environment of the human body facilitating a variety of functions including mucociliary clearance, airway humidification, pathogen/particulate sensing and defense, and signaling to the underlying mesenchyme and immune system 1, 2. The human bronchial tree is a complicated heterogeneous system that has important functions beyond being a simple barrier and conduit for air exchange. This study highlights the feasibility and versatility of a 3D airway OTE model to model the multiple components of the human airway 3D microenvironment. Modulation of hydrogel stiffness did not negatively impact HBE differentiation and could be a valuable variable to alter epithelial phenotype. 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 mature epithelial markers relating to goblet, club, and ciliated cells. Variations of this model were analyzed during 28 days of ALI culture by evaluating epithelial confluence, trans-epithelial electrical resistance, and epithelial phenotype via multispectral immuno-histochemistry and next-generation sequencing. We demonstrate the versatility of the OTE model by evaluating the impact of these features on human bronchial epithelial (HBE) cell phenotype. Here, we report the development and characterization of a physiologically relevant air–liquid interface (ALI) 3D airway ‘organ tissue equivalent’ (OTE) model with three novel features: native pulmonary fibroblasts, solubilized lung ECM, and hydrogel substrate with tunable stiffness and porosity. 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. The human airways are complex structures with important interactions between cells, extracellular matrix (ECM) proteins and the biomechanical microenvironment. ![]()
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