Coculture of primary human colon monolayer with human gut bacteria

Jianbo Zhang, Victor Hernandez-Gordillo, Martin Trapecar, Charles Wright, Mao Taketani, Kirsten Schneider, Wen Li Kelly Chen, Eric Stas, David T. Breault, Rebecca L. Carrier, Christopher A. Voigt, Linda G. Griffith

Research output: Contribution to journalReview articlepeer-review


The presence of microbes in the colon impacts host physiology. Therefore, microbes are being evaluated as potential treatments for colorectal diseases. Humanized model systems that enable robust culture of primary human intestinal cells with bacteria facilitate evaluation of potential treatments. Here, we describe a protocol that can be used to coculture a primary human colon monolayer with aerotolerant bacteria. Primary human colon cells maintained as organoids are dispersed into single-cell suspensions and then seeded on collagen-coated Transwell inserts, where they attach and proliferate to form confluent monolayers within days of seeding. The confluent monolayers are differentiated for an additional 4 d and then cocultured with bacteria. As an example application, we describe how to coculture differentiated colon cells for 8 h with four strains of Bacteroides thetaiotaomicron, each engineered to detect different colonic microenvironments via genetically embedded logic circuits incorporating deoxycholic acid and anhydrotetracycline sensors. Characterization of this coculture system reveals that barrier function remains intact in the presence of engineered B. thetaiotaomicron. The bacteria stay close to the mucus layer and respond in a microenvironment-specific manner to the inducers (deoxycholic acid and anhydrotetracycline) of the genetic circuits. This protocol thus provides a useful mucosal barrier system to assess the effects of bacterial cells that respond to the colonic microenvironment, and may also be useful in other contexts to model human intestinal barrier properties and microbiota–host interactions.

Original languageEnglish (US)
Pages (from-to)3874-3900
Number of pages27
JournalNature protocols
Issue number8
StatePublished - Aug 2021
Externally publishedYes

ASJC Scopus subject areas

  • General Biochemistry, Genetics and Molecular Biology


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