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Updated: Oct 25, 2025
An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions
Published on: June 30, 2021
Intestinal organoid cocultures with microbes
Jens Puschhof1,2, Cayetano Pleguezuelos-Manzano1,2, Adriana Martinez-Silgado1,2
1Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, Utrecht, the Netherlands.
Abstract:
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.
Insights
This protocol details culturing human organoids with bacteria to study host-microbe interactions. It covers methods for assessing bacterial and organoid health, spatial relationships, and genetic changes.
Area of Science:
- Microbiology
- Cell Biology
- Gastroenterology
Background:
- Adult stem cell-derived organoids serve as powerful ex vivo models for human epithelial tissues.
- Studying host-microbe interactions is crucial for understanding gut health and disease.
- Existing methods lack precise control for investigating specific bacterial interactions with human tissues.
Purpose of the Study:
- To provide a detailed protocol for the co-culture of human organoids with microbes.
- To enable controlled investigation of host-microbe interactions in the human small intestine and colon.
- To establish methods for assessing the impact of bacterial colonization on organoid physiology and genetics.
Main Methods:
- Co-culture of human small intestinal and colon organoids with individual bacterial species.
- Microinjection techniques for introducing bacteria into 3D organoids (lumen and periphery) and 2D culture systems.
- Characterization of co-cultures using assays for bacterial and organoid cell viability, growth kinetics, fluorescence live microscopy, and scanning electron microscopy.
Main Results:
- Detailed protocols for successful co-culture of organoids and bacteria are presented.
- Methods for assessing bacterial and organoid viability and growth kinetics are established.
- Techniques for analyzing spatial relationships and genetic alterations (gene expression, mutations) are discussed.
Conclusions:
- This protocol offers a robust framework for studying host-microbe interactions using human organoids.
- It facilitates precise investigation into how specific bacteria influence human epithelial tissue models.
- The methods support comprehensive analysis from cellular responses to genetic-level changes.
