Human bronchial organoids represent a highly advanced 3D cell culture model system that reflects complex features of the airway epithelium, including structure, developmental aspects, tissue-specific functions and heterogeneous cell-cell interactions. Thus, they serve as an ideal model to study physiological differentiation and activation processes of the bronchial epithelial cells, as well as pathophysiological mechanisms of lower airway diseases. We have established, refined and validated a series of protocols for the generation, perpetuation and characterization of human bronchial lung organoids based on somatic cells derived from surgical lung tissue samples as well as from primary bronchial epithelial cells which may be obtained from healthy and diseased donors. Such organoids are cultured in an extracellular matrix gel with a serum-free medium containing a precisely adjusted growth factor cocktail. This maintains the balance between the self-renewal and differentiation capacities of the local progenitor cells, allowing the long-term culture of organoids if specific splitting and dilution as well as freezing/thawing procedures are carried out regularly and appropriately. Here we provide these detailed protocols to enable researchers to apply the organoid technology and to generate highly comparable and complementary data. Furthermore, we present several examples for the detailed characterization and analysis of human bronchial organoids, such as gene-level expression analysis, covering single cell RNA sequencing, as well as imaging and metabolic activity-based assays.
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