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Summary

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.

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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.