Event Abstract Back to Event Controlling the mechanical microenvironment of human airway epithelial cells for tracheal tissue engineering James Poon1, Thomas K. Waddell1, 2, 3 and Alison P. Mcguigan1, 4 1 University of Toronto, Institute of Biomaterials & Biomedical Engineering, Canada 2 University of Toronto, Institute of Medical Science, Canada 3 University Health Network, Division of Thoracic Surgery, Canada 4 University of Toronto, Department of Chemical Engineering & Applied Chemistry, Canada Many major organs are lined by sheets of epithelial tissue comprised of various cell types; therefore engineering epithelial constructs has emerged as an important field in tissue engineering. We are developing cell alignment tools to engineer functional epithelium for incorporation into engineered tracheas and other artificial organs. Airway epithelium rests on a basement membrane of aligned collagen fibres and significant local variation in mechanical stiffness exists at the scale of the individual cells [1]. However, most tissue engineering tools have been developed for endothelial, muscle, and nerve cells. These cell types are grown on solid culture substrates and do not polarize in a specialized apical-basal fashion. Currently, transwell membrane cultures are used to generate artificial adult airway epithelium in vitro. This air-liquid-interface (ALI) culture system allows for apical-basal polarization of the cells over a four-week maturation period; differentiation is indicated by the formation of motile cilia at the cell apical surface [2]. However, ALI culture does not create planar polarized epithelium. Planar polarity is required for correct alignment of motile cilia, which beat in coordinated waves in order to produce a transport function important for the clearance of mucus and foreign particles from the airway. The mechanical properties of the extracellular environment have been demonstrated to guide cell behaviour. Variations in substrate topography and stiffness influence focal adhesion formation and cytoskeleton organization in epithelial cells in vitro [3],[4]. We are particularly interested in using sub-cellular scale mechanical cues, in the form of groove topography and anisotropic substrate stiffness, to align motile cilia in primary human tracheal epithelial cells (HTECs). We hypothesize that primary HTECs will align and polarize in the direction of our mechanical signals, and produce more organized cilia beating than HTECs on unpatterned substrates. Preliminary studies indicate that HTECs grown on polydimethylsiloxane (PDMS) substrates with deep groove topography (3 µm depth) display a cell alignment response. We are currently fabricating, using photolithography and chemical etching techniques, groove arrays of various dimensions (1-50 µm spacing and 0.2-3 µm depth) to identify the optimal alignment response (whole cell, F-actin and nuclei) of primary cells on these features. These dimensions will be used to mould grooved gelatin inserts onto which HTECs will undergo ALI culture. In parallel, we are developing a simple spin-coating protocol to produce micron-scale anisotropic stiffness patterns, whereby a thin layer (<20μm) of 5% gelatin is spun onto a grooved 15% gelatin substrate to produce a flat surface with alternating regions of soft and stiff substrate. Mucociliary clearance, as assessed by video tracking of fluorescent bead movement on the surface of ALI cultures, is disorganized in control transwell cultures and expected to be organized or enhanced in patterned gel substrates. Engineering specific physical forces during tissue development and regeneration provides a novel strategy for recapitulating large airway epithelium. Understanding mechanical requirements will improve clinical impact by providing the basis for generating gel coatings with specific properties for lining the lumens of engineered airway replacements to guide epithelium organization and maturation.
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Nov 4, 2024
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