Generation of a human vascularized 3D airway model replicating native mucosal heterogeneity

<i>In vitro</i> models of the human airway are essential to study respiratory diseases and test potential therapeutics while reducing animal testing. Current models often use two-dimensional culture conditions rather than replicating the physiological 3D environment and do not allow direct cell-cell interactions between the diverse cell types found in the mucosa. Here, we provide a detailed step-by-step instruction for reproducibly generating a complex tri-culture model, which can be used to investigate the human airway environment in health and disease. The model is fabricated by preparing an epithelialized fibrin hydrogel with embedded endothelial and stromal cells. To assure complete differentiation into a mucociliary phenotype, samples are maintained at air-liquid interface (ALI) for 28 days. Afterwards, morphology and functionality can be validated using downstream analysis techniques such as immunohisto- and cytochemistry, electron microscopy, ciliary beating frequency analysis, measurement of mucociliary clearance and RNA isolation. After 4 weeks of maturation, a well-differentiated pseudostratified epithelium comprising basal, multiciliated and secretory cells is developed. We also observe a physiological ciliary beating frequency, mucus production and a functional particle clearance. Inside the hydrogel, endothelial cells form a three-dimensional network of vascular structures. These features make our model ideal for replicating human mucosal heterogeneity, especially compared to airway models using tumor-derived or immortalized cell lines, monocultures or rigid substrates. Hence, this protocol paves the way for fellow researchers to achieve robust airway <i>in vitro</i> modeling that can be performed in a standard cell culture lab without the need for extraordinary equipment or specialized expertise.