Bioengineering 3D airway tubes to study epithelial morphogenesis

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My long-term research program aims to elucidate the fundamental cellular and molecular mechanisms driving human lung development. Human fetal lung development remains largely unknown especially the pressures of air flow in the lungs on epithelial cell behaviour, reminiscent of the early period of post-natal life. Model organisms and current in-vitro cell models cannot be used to study fundamental mechanisms driving human lung cell fate decisions as there are genetic, cellular and/or ultrastructural complexities that are not recapitulated in any of the current systems. Importantly, generating tubular structures to mimic the airways with cellular, extracellular matrix, and biomechanical forces has not been achieved. Previous attempts at developing `airway tubes' have used cells incapable of self-organization and/or multicellular differentiation. Therefore, the mechanisms driving cellular behaviour as they form in 3D airway conduits during development remain unclear. In this proposal, we aim to develop a novel 3D airway mimetic with proper extracellular matrices (ECM) and physiological airflow to study epithelial cell fate decisions and patterning under physiologically relevant stresses such as air flow. We hypothesize that the 3D airway mimetic will induce epithelial morphogenesis with spatial distribution, cellular composition and function similar to native tissues. Aim 1. To optimize 3D tube bioprinting using defined biological extracellular matrices. We will first determine the appropriate substratum topography and ECM that is needed to support epithelial cell viability, expansion and polarization. Aim 2. To optimize bidirectional air flow perfusion in a microfluidic device that will sustain integrity of the epithelium. We will determine the viability of human bronchial epithelial cells in the tubes exposed to defined bidirectional lumenal air flow in our prefabricated microfluidic device. The influence of airflow on epithelial morphogenesis (differentiation and spatial organization) will be tested for cellular viability, epithelial integrity and assessed for morphological and ion transport function similar to native tissue. We will also compare to static fluid-filled tubes reminiscent of lungs in-utero. Aim 3. To investigate epithelial morphogenesis under physiological airflow. Here, we will utilize pluripotent stem cell-derived basal stem cells capable of multicellular differentiation. We will monitor epithelial morphogenesis by live imaging and determine the cellular distribution and cell fate of the derivatives. Cilia beating, a measure of functional activity and ciliated cell differentiation, a key hallmark of the proximal airways, will be assessed in the 3D tubes using fluorescent bead tracking. Significance. We will create the first humanized perfusable 3D airway mimetic using state-of-the art bioprinting technology and stem cell engineering to study fundamental mechanisms driving human airway development.