The human airways are complex structures with important interactions between cells, extracellular matrix (ECM) 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 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. We demonstrate the versatility of the OTE model by evaluating the impact of these features on human bronchial epithelial (HBE) cell phenotype. 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. 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. Modulation of hydrogel stiffness did not negatively impact HBE differentiation and could be a valuable variable to alter epithelial phenotype. This study highlights the feasibility and versatility of a 3D airway OTE model to model the multiple components of the human airway 3D microenvironment.

人体气道是复杂的结构，细胞、细胞外基质 (ECM) 蛋白和生物力学微环境之间存在重要的相互作用。一个强大的、分化良好的体外培养系统可以准确地模拟这些相互作用，将为研究正常和病理性气道生物学提供有用的工具。在这里，我们报告了生理相关的气液界面（ALI）3D气道“器官组织等效物”（OTE）模型的开发和表征，该模型具有三个新特征：天然肺成纤维细胞、溶解的肺ECM和具有可调节刚度和弹性的水凝胶基质。孔隙率。我们通过评估这些特征对人支气管上皮 (HBE) 细胞表型的影响来证明 OTE 模型的多功能性。通过多光谱免疫组织化学和下一代测序评估上皮汇合、跨上皮电阻和上皮表型，分析了 ALI 培养 28 天期间该模型的变化。水凝胶基质中包含溶解的肺ECM和天然肺成纤维细胞的培养物形成分化良好的ALI培养物，其维持屏障功能并表达与杯状细胞、棒状细胞和纤毛细胞相关的成熟上皮标记物。水凝胶硬度的调节不会对 HBE 分化产生负面影响，并且可能是改变上皮表型的一个有价值的变量。这项研究强调了 3D 气道 OTE 模型对人体气道 3D 微环境的多个组成部分进行建模的可行性和多功能性。