Poster V6 - Uncovering the effects of fibrotic stiffening on airway epithelial function

Introduction: The epithelial cells that line the airways of the lung are exposed to air on their apical surface and a pliable mesenchymal tissue on their basal surface. Air-liquid interface (ALI) cultures mimic the boundary condition on the apical surface of the epithelium, but the traditional membranes that are used as substrata are orders of magnitude stiffer than the mesenchymal tissues of the lung. Here, we generated ALI culture models that more accurately mimic the physical boundary conditions of the lung epithelium by grafting polyacrylamide (PA) gels onto the surfaces of transwell membranes. We used this novel experimental platform to test the effects of fibrotic stiffening on the phenotype of lung epithelial cells.

Materials and

Methods: We tuned the stiffness of PA gels to cover the physiological range of the healthy and fibrotic lung. We found that using a photoinitiator, benzophenone, enhances the grafting of PA gels onto the transwell membranes without affecting the intended elastic moduli of the gels. We cultured human bronchial epithelial (16HBE14o) cells on transwells grafted with PA gels of different stiffness and characterized cellular morphology and phenotype by measuring the projected cell area and the nucleocytoplasmic intensity ratio of the mechanosensor YAP. We also measured transepithelial electrical resistance (TEER), conducted RT-PCR analysis for markers of differentiation, and performed immunostaining analysis for markers of tight junctions.

Results, Conclusions, and Discussions: Using our ALI culture model, we found that human airway epithelial cells exhibit different morphologies and functional phenotypes when cultured at ALI on PA gels of different stiffness. On stiffer substrata, these cells show increased projected cell area and nuclear localization of YAP. Additionally, we found that the permeability of the epithelium is sensitive to substratum stiffness, which has implications for barrier function of the lung under conditions of fibrosis. We are currently using this engineered culture model to investigate the effects of fibrotic stiffening on viral transmission and recovery after airway injury.

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