Organoid and Cell-Surface Engineering for Investigation of Human Respiratory Pathophysiology

The human conducting airway is composed of polarized pseudostratified epithelium, with cilia movement and mucus secretion taking place on the apical side, which faces the external environment and directly interacts with respiratory pathogens. However, conventional airway organoids engineered from airway basal cells in extracellular matrix-embedded culture have an apical-in conformation making the apical surface difficult to access and precluding effective investigation of airway pathology in a physiologically relevant manner. In addition, due to cilia’s nanoscale dimension and high beating frequency, quantitative assessment of their motility remains a sophisticated task. In this thesis, these limitations were addressed by the development of a simple yet robust technique for reproducible engineering of airway organoids with an apical-out conformation, mimicking the epithelial polarity of the native airway. Propelled by exterior-facing cilia beating, the mature apical-out airway organoids exhibited stable rotational motion when being surrounded by Matrigel. We developed a computational framework leveraging computer vision algorithms to quantify organoid rotation and validated its correlation with direct measurement of cilia motility. We further established the feasibility of using organoid rotation to recapitulate and measure defective cilia motility generated by chemotherapy-induced respiratory toxicity and by CCDC39 mutations in cells from primary ciliary dyskinesia patient. Building on the innovative features of the apical-out airway model allowing non-invasive administration of respiratory threats and non-invasive sampling and analysis of respiratory secretions, we sensitively detected inflammation-induced goblet cell hyperplasia and mucus hypersecretion following stimulation with cytokines and particulate matter-induced respiratory injuries to the mature and developing airway epithelium. 

From a cell-surface engineering perspective, it is of particular interest to understand how cell-surface accessibility regulates the adhesion and the assembly between cells for multicellular assembly to achieve complex tissue. However, cell-surface glycocalyx, composed of delicate biopolymers that can extend hundreds of nanometers from the external surface of the phospholipid bilayer, serves as a physiological barrier regulating cellular accessibility to other cells and to macromolecules. Thus, this thesis also explored the cell-surface glycocalyx further, validating the feasibility of using DNA origami nanostructures as a functional measure of cell-surface glycocalyx barrier integrity. Altogether, we expect the organoid and cell-surface engineering techniques presented in this thesis to be adopted to develop high-throughput assays for personalized and expedited modeling of genetic and environmental respiratory diseases allowing better investigation of respiratory pathophysiology and effective therapeutic development.