Tissue Engineering Architectural Cues for in vitro Models of Respiratory Epithelium

Diseases affecting the respiratory system are one of the major leading causes of death worldwide. Epithelial dysfunction is associated with many lung diseases and therefore tools for primary cell culture are important for generating models of human lung epithelium. Respiratory epithelium is comprised of many diverse cell types that inhabit structurally distinct regions in the lung and airways. In vitro approaches to generate or repair epithelium are inadequate as they do not incorporate the specific architecture that lung and airway tissues exhibit in vivo, resulting in disorganized and dysfunctional epithelial cells. We hypothesized micropatterned biophysical cues, in the form of stiffness and topography, will induce alignment of airway epithelial cells along a defined tissue axis. We also hypothesized that culture of distal lung epithelial cells in biomimetic architecture will extend the maintenance of viability and phenotype. We employed three scaffolding approaches with microengineered biophysical cues to control epithelial behaviour. In the first two approaches, we applied micropatterning techniques to create hydrogel systems that were compatible with air-liquid interface (ALI) culture of airway epithelium. Although our stiffness-patterning platform did not support primary culture, we found that primary epithelial cells can be cultured on collagen vitrigel membranes and groove topography induces morphological alignment for up to 14 days of ALI culture. In the third approach, we generated a poly-dimethylsiloxane culture substrate with alveolar-mimetic curvature for culture of lung epithelial cells. Specifically, primary mouse cells grown in cavity culture conditions remain viable (96 ± 4% vs. 2 ± 1% on flat controls) and maintained expression of phenotypic markers (surfactant protein C, aquaporin-5) over one week. These findings demonstrate the profound influence of biophysical cues on epithelial behaviours including spreading, polarization and phenotype. Our rationally-designed biomaterial substrates are able to mimic numerous aspects of the extracellular matrix and direct the behaviour of adult primary cells derived from mammalian trachea and lung epithelial progenitors. Biomaterial scaffolds with defined architectural cues advance the capabilities of lung and airway epithelial models to instruct tissue function and provide platforms for understanding mechanisms of lung disease and repair.Ph.D