Epithelial organoid culture systems are currently attracting considerable interest due to their potential application in several fields, including disease modelling and drug discovery. However, their structural organisation renders the apical surface of the epithelium inaccessible, hindering their use in the study of host-pathogen interactions. This thesis explores innovative methods for developing intestinal, bronchial, and nasal organoid culture systems that expose the apical surface of epithelial cells, facilitating their use in disease modeling and drug screening. We first provide a workflow for the establishment of intestinal epithelial organoid-derived monolayers, as well as an optimised a protocol for the generation of apical-out intestinal organoids. This protocol relies on the removal of the extracellular matrix (ECM) hydrogel from established ECM-embedded organoid cultures that induces the switch in the orientation of the epithelium. We then present a novel method to generate apical-out airway organoids in a scalable, standardised and chemically-defined manner by differentiating aggregates of 2D-expanded bronchial epithelial cells in suspension in the absence of any ECM hydrogel. These organoids show size homogeneity, robust ciliogenesis and susceptibility to respiratory virus infections, making them suitable for antiviral drug screenings. We further describe a technique to induce secretory cell differentiation in apical-out airway organoids, increasing their cellular complexity and physiological relevance. Finally, we outline the successful generation of highly-homogeneous apical-out nasal organoids, and explore workflow modifications to facilitate efficient differentiation and survival of these organoids. These advancements in organoid culture systems provide valuable tools for studying host-pathogen interactions in a more accessible and high-throughput manner
