Adult-stem-cell-derived organoids model human epithelial tissues ex vivo, which enables the study of host-microbe interactions with great experimental control. This protocol comprises methods to coculture organoids with microbes, particularly focusing on human small intestinal and colon organoids exposed to individual bacterial species. Microinjection into the lumen and periphery of 3D organoids is discussed, as well as exposure of organoids to microbes in a 2D layer. We provide detailed protocols for characterizing the coculture with regard to bacterial and organoid cell viability and growth kinetics. Spatial relationships can be studied by fluorescence live microscopy, as well as scanning electron microscopy. Finally, we discuss considerations for assessing the impact of bacteria on gene expression and mutations through RNA and DNA sequencing. This protocol requires equipment for standard mammalian tissue culture, or bacterial or viral culture, as well as a microinjection device.

The lung epithelium has long been overlooked as a key player in tuberculosis disease. In addition to acting as a direct barrier to Mycobacterium tuberculosis (Mtb), epithelial cells (EC) of the airways and alveoli act as first responders during Mtb infections; they directly sense and respond to Mtb by producing mediators such as cytokines, chemokines and antimicrobials. Interactions of EC with innate and adaptive immune cells further shape the immune response against Mtb. These three essential components, epithelium, immune cells and Mtb, are rarely studied in conjunction, owing in part to difficulties in coculturing them. Recent advances in cell culture technologies offer the opportunity to model the lung microenvironment more closely. Herein, we discuss the interplay between lung EC, immune cells and Mtb and argue that modelling these interactions is of key importance to unravel early events during Mtb infection.

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Tuberculosis (TB) is the world's most lethal infectious disease caused by a bacterial pathogen, Mycobacterium tuberculosis . This pathogen evades the immune defenses of its host and grows intracellularly in immune cells, particularly inside macrophages.