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.

成体干细胞衍生的类器官离体模拟人类上皮组织，这使得研究宿主-微生物相互作用成为可能，并具有很好的实验控制。该协议包括与微生物共培养类器官的方法，特别关注暴露于单个细菌物种的人类小肠和结肠类器官。讨论了微注射到 3D 类器官的内腔和外围，以及将类器官暴露于 2D 层中的微生物。我们提供了详细的协议，用于表征关于细菌和类器官细胞活力和生长动力学的共培养。空间关系可以通过荧光活显微镜以及扫描电子显微镜来研究。最后，我们讨论了通过 RNA 和 DNA 测序评估细菌对基因表达和突变影响的考虑因素。