Intestinal organoid cocultures with microbes.

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.