Intestinal organoid cocultures with microbes

Jens Puschhof, Cayetano Pleguezuelos-Manzano, Adriana Martinez-Silgado, Ninouk Akkerman, Aurelia Saftien, Charelle Boot, Amy de Waal, Joep Beumer, Devanjali Dutta, Inha Heo, Hans Clevers

Research output: Contribution to journal/periodicalBook/Film/Article reviewScientific

61 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)4633-4649
Number of pages17
JournalNature Protocols
Issue number10
Publication statusPublished - Oct 2021


  • Coculture Techniques
  • Intestines
  • Organoids


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