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Coculture of primary human colon monolayer with human gut bacteria

Nature Protocols volume 16, pages 3874–3900 (2021)Cite this article

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Abstract

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.

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Fig. 1: Schematic workflow to generate differentiated colon epithelial monolayers and coculture with engineered B. thetaiotaomicron.
Fig. 2: The success rate of generating usable monolayers and the cell viability of single cells from dissociated organoids used for monolayer seeding.
Fig. 3: Morphology of primary human colon organoids.
Fig. 4: Example of organoid expansion after each passaging.
Fig. 5: Representative images of failed monolayers that show regions with holes or the presence of excessive dead cells after exposure to bacterial medium.
Fig. 6: Representative images of organoids, monolayers and proliferative cells in those monolayers.
Fig. 7: Evaluation on the integrity of monolayers and response of B. thetaiotaomicron to inducers after coculture.

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Data availability

Source data are provided with this paper. Additional imaging data are in the Supplementary Tables or are available from the corresponding author upon reasonable request.

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Acknowledgements

This study was supported by the National Institute of Health R01EB021908, the Boehringer Ingelheim SHINE Program, the NIH P50 grant (P50-GM098792), Office of Naval Research Multidisciplinary University Research Initiatives Program (N00014-13-1-0074), Defense Agency Research Projects Agency Synergistic Discovery and Design (SD2; FA8750-17-C-0229) and National Science Foundation Semiconductor Synthetic Biology for Information Processing and Storage Technologies (SemiSynBio; CCF-1807575) program. We thank O. Yilmaz for providing the colon organoid of donor HC2978. We are grateful for the lab management support from H. Lee (MIT).

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Authors and Affiliations

  1. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Jianbo Zhang, Victor Hernandez-Gordillo, Martin Trapecar, Charles Wright, Mao Taketani, Kirsten Schneider, Wen Li Kelly Chen, Christopher A. Voigt & Linda G. Griffith

  2. Division of Endocrinology, Boston Children’s Hospital, Boston, MA, USA

    Eric Stas & David T. Breault

  3. Harvard Stem Cell Institute, Harvard University, Boston, MA, USA

    David T. Breault

  4. Department of Pediatrics, Harvard Medical School, Boston, MA, USA

    David T. Breault

  5. Department of Chemical Engineering, Northeastern University, Boston, MA, USA

    Rebecca L. Carrier

  6. Center for Gynepathology Research, Massachusetts Institute of Technology, Cambridge, MA, USA

    Linda G. Griffith

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Contributions

J.Z., C.W. and M.T. made the figures and tables. V.H.G, M.T., J.Z., K.S., C.W., M.T. and E.S. developed/validated the protocols. D.T.B., C.A.V. and L.G.G. supervised the experiments. J.Z., L.G.G., C.W. and C.A.V. wrote the manuscript with input from V.H.G, M.T. K.S., M.T. W.L.K.C., E.S., D.T.B. and R.L.C.

Corresponding author

Correspondence to Linda G. Griffith.

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Competing interests

C.A.V. and M.T. have filed a provisional patent based on this work. All other authors have no competing interests.

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Peer review information Nature Protocols thanks Nicole Royand the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Taketani, M. et al. Nat. Biotechnol. 38, 962–969 (2020): https://doi.org/10.1038/s41587-020-0468-5

Zhang, J. et al. Med 2, 74–98 (2021): https://doi.org/10.1016/j.medj.2020.07.001

Supplementary information

Supplementary Information

Supplementary Fig. 1.

Supplementary Table 1

Growth curve of B. thetaiotaomicron MT768

Supplementary Table 2

Calculation of RPUL from OD600 and luminescence data. Related to Fig. 7e

Supplementary Table 3

The OD600 and luminescence signal for bacterial cells collected from top and bottom of the apical compartment in the transwell inserts

Source data

Source Data Fig. 2

Statistical source data.

Source Data Fig. 6

Statistical source data.

Source Data Fig. 7

Statistical source data.

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Zhang, J., Hernandez-Gordillo, V., Trapecar, M. et al. Coculture of primary human colon monolayer with human gut bacteria. Nat Protoc 16, 3874–3900 (2021). https://doi.org/10.1038/s41596-021-00562-w

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