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Intestinal organoid cocultures with microbes

Nature Protocols volume 16, pages 4633–4649 (2021)Cite this article

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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.

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Fig. 1: Overview of the protocol steps.
Fig. 2: Organoid microinjections.
Fig. 3: Visualizing organoid cocultures.

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Data availability

All previously unpublished data are included in the figures. Raw image files are available from the corresponding author upon request.

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Acknowledgements

We thank E. Allen-Vercoe and A. Robinson for provision of bacterial strains and discussions on bacterial culturing conditions. The development of the methods was supported by CRUK grant OPTIMISTICC (C10674/A27140) (J.P., C.P.-M. and H.C.), the Gravitation projects CancerGenomiCs.nl, and the Netherlands Organ-on-Chip Initiative (024.003.001) from the Netherlands Organisation for Scientific Research (NWO) funded by the Ministry of Education, Culture and Science of the government of the Netherlands (J.P., C.P.-M. and H.C.), the Oncode Institute (partly financed by the Dutch Cancer Society), the European Research Council under ERC Advanced Grant Agreement no. 67013 (J.P., D.D., I.H. and H.C.) and NETRF/Petersen Accelerator (J.B.).

Author information

Author notes

  1. Devanjali Dutta

    Present address: Laboratory of Stem Cell Bioengineering, Institute of Bioengineering, School of Life Sciences (SV), École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

  2. Inha Heo

    Present address: Janssen Pharmaceutica N.V., Beerse, Belgium

  3. These authors contributed equally: Jens Puschhof, Cayetano Pleguezuelos-Manzano.

Authors and Affiliations

  1. Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, Utrecht, the Netherlands

    Jens Puschhof, Cayetano Pleguezuelos-Manzano, Adriana Martinez-Silgado, Ninouk Akkerman, Aurelia Saftien, Charelle Boot, Amy de Waal, Joep Beumer, Devanjali Dutta, Inha Heo & Hans Clevers

  2. Oncode Institute, Hubrecht Institute, Utrecht, the Netherlands

    Jens Puschhof, Cayetano Pleguezuelos-Manzano, Adriana Martinez-Silgado, Ninouk Akkerman, Aurelia Saftien, Charelle Boot, Amy de Waal, Joep Beumer, Devanjali Dutta, Inha Heo & Hans Clevers

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Contributions

All authors contributed to the development of the organoid co-culture methods described in this protocol. J.P., C.P.M. and H.C. wrote the manuscript with input and corrections from all authors. A.M.S., C.P.M. and J.P. prepared figures.

Corresponding author

Correspondence to Hans Clevers.

Ethics declarations

Competing interests

H.C. is inventor on multiple patents held by the Dutch Royal Netherlands Academy of Arts and Sciences that cover organoid technology: PCT/NL2008/050543, WO2009/022907; PCT/NL2010/000017, WO2010/090513; PCT/IB2011/002167, WO2012/014076; PCT/IB2012/052950, WO2012/168930; PCT/EP2015/060815, WO2015/173425; PCT/EP2015/077990, WO2016/083613; PCT/EP2015/077988, WO2016/083612; PCT/EP2017/054797,WO2017/149025; PCT/EP2017/065101, WO2017/220586; PCT/EP2018/086716; and GB1819224.5. H.C.’s full disclosure is given at https://www.uu.nl/staff/JCClevers/.

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Peer review information Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.

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Key references using this protocol

Pleguezuelos-Manzano, C. et al. Nature 580, 269–273 (2020): https://doi.org/10.1038/s41586-020-2080-8

Heo, I. et al. Nat. Microbiol. 3, 814–823 (2018): https://doi.org/10.1038/s41564-018-0177-8

Lamers, M. M. et al. Science 369, 50–54 (2020): https://doi.org/10.1126/science.abc1669

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Puschhof, J., Pleguezuelos-Manzano, C., Martinez-Silgado, A. et al. Intestinal organoid cocultures with microbes. Nat Protoc 16, 4633–4649 (2021). https://doi.org/10.1038/s41596-021-00589-z

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