Development of a Bronchial Wall Model: Triple Culture on a Decellularized Porcine Trachea

In vitro coculture models mimicking the bronchial barrier are a significant step forward in investigating the behavior and function of the upper respiratory tract mucosa. To date, mostly synthetic materials have been used as substrates to culture the cells. However, decellularized tissues provide a more in vivo-like environment based on the native extracellular matrix. In this study, an in vitro, bronchial wall coculture model has been established using a decellularized, porcine luminal trachea membrane and employing three relevant human cell types. The tissue was decellularized and placed in plastic transwell supports. The human bronchial epithelial cell line, 16HBE14o-, was seeded on the apical side of the membrane with the human lung fibroblast cell line, Wi-38, and/or the microvascular endothelial cell line, ISO-HAS-1, seeded on the basolateral side. Transepithelial electrical resistance (TER) was measured over 10 days and tight/adherens junctions (ZO-1, occludin/β-catenin) were studied through immunofluorescence. Scanning electron microscopy (SEM) was performed to evaluate microvilli and cilia formation. All cultures grew successfully on the membrane. TER values of 555 Ω·cm² (±21, SEM) were achieved in the monoculture. Cocultures with fibroblasts reached 565 Ω·cm² (±41, SEM), with endothelial cells at 638 Ω·cm² (±37, SEM), and the triple culture achieved 552 Ω·cm² (±38, SEM). ZO-1, occludin, and β-catenin were expressed in 16HBE14o- under all culture conditions. Using SEM, a dense microvilli population was found. Prominent cell-cell contacts and clusters of emerging cilia could be identified. Fibroblasts and endothelial cells strengthened the formation of a tight barrier by the 16HBE14o-. Thus, the coculture of three relevant cell types in combination with native decellularized scaffolds as a substrate approaches more closely the in vivo situation and could be used to study mechanisms of upper respiratory damage and regeneration.