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Controlling the polarity of human gastrointestinal organoids to investigate epithelial biology and infectious diseases

Nature Protocols volume 16, pages 5171–5192 (2021)Cite this article

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Abstract

Human epithelial organoids—3D spheroids derived from adult tissue stem cells—enable investigation of epithelial physiology and disease and host interactions with microorganisms, viruses and bioactive molecules. One challenge in using organoids is the difficulty in accessing the apical, or luminal, surface of the epithelium, which is enclosed within the organoid interior. This protocol describes a method we previously developed to control human and mouse organoid polarity in suspension culture such that the apical surface faces outward to the medium (apical-out organoids). Our protocol establishes apical-out polarity rapidly (24–48 h), preserves epithelial integrity, maintains secretory and absorptive functions and allows regulation of differentiation. Here, we provide a detailed description of the organoid polarity reversal method, compatible characterization assays and an example of an application of the technology—specifically the impact of host–microbe interactions on epithelial function. Control of organoid polarity expands the possibilities of organoid use in gastrointestinal and respiratory health and disease research.

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Fig. 1: Overview of protocol to generate GI apical-out human organoids.
Fig. 2: Apical-out human GI organoids preserve tight junctions, multi-lineage differentiation and polarized secretion.
Fig. 3: Kinetics of polarity reversal in suspension and preservation of basolateral-out organoid polarity with the addition of soluble BME.
Fig. 4: S. Typhimurium infection of human ileal enteroids.
Fig. 5: Dextran diffusion barrier integrity assay.
Fig. 6: Fatty acid uptake assay.
Fig. 7: Solutions to two common problems with the polarity-reversal protocol.

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Data availability

All data generated or analyzed during this study are included in this published article and in our primary research article21. No datasets were generated or analyzed during the current study. Source data are provided with this paper. Additional source data underlying the figures are available from the corresponding author upon request.

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Acknowledgements

We thank R. Cooper for reagent preparation and the laboratory of C. Kuo for support in deriving GI organoids. This study was supported by the Stanford Child Health Research Institute Postdoctoral Award (to J.Y.C.), NIH T32AI007328-29 (support to J.Y.C.), Novo Nordisk Foundation Challenge Programme (to M.R.A and M.M.-C.), NIH U19AI116484-01 (to M.R.A. and D.M.M.), Bill and Melinda Gates Foundation/Stanford CHSI Pilot Grant (to M.R.A. and D.M.M) and OPP1113682 (to M.R.A).

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Author notes

  1. These authors contributed equally: Julia Y. Co, Mar Margalef-Català.

Authors and Affiliations

  1. Division of Infectious Diseases, Department of Pediatrics, Stanford University, Stanford, CA, USA

    Julia Y. Co, Mar Margalef-Català & Manuel R. Amieva

  2. Department of Microbiology and Immunology, Stanford University, Stanford, CA, USA

    Denise M. Monack & Manuel R. Amieva

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  1. Julia Y. Co
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  2. Mar Margalef-Català
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Contributions

J.Y.C. and M.M.-C developed the protocol for the apical-out GI organoids and the referenced apical-out lung organoids, respectively, and performed the experiments. J.Y.C., M.M.-C., D.M.M. and M.R.A. conceived the study and prepared the manuscript.

Corresponding author

Correspondence to Manuel R. Amieva.

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The authors declare no competing interests.

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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

Co, J. Y. et al. Cell Rep. 26, 2509–2520 (2019): https://doi.org/10.1016/j.celrep.2019.01.108

Salahudeen, A. A. et al. Nature 588, 670–675 (2020): https://doi.org/10.1038/s41586-020-3014-1

Supplementary information

41596_2021_607_MOESM1_ESM.mov

Supplementary Video 1 Human gastroid polarity reversal. Time-lapse DIC microscopy movie of a gastroid immediately after removal from BME shows that the process of polarity reversal occurs by morphogenetic eversion. The same gastroid was fixed, stained and imaged by 3D confocal microscopy to confirm apical-out polarity (F-actin–rich microvilli (arrow) face outward). Nuclei are stained with DAPI (blue; ThermoFisher Scientific, cat. no. D3571; RRID: AB_2307445), and the actin cytoskeleton is stained with AlexaFluor 660 phalloidin (F-actin, white; ThermoFisher, cat. no. A22285), both diluted 1:500. Scale bar, 10 µm.

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Co, J.Y., Margalef-Català, M., Monack, D.M. et al. Controlling the polarity of human gastrointestinal organoids to investigate epithelial biology and infectious diseases. Nat Protoc 16, 5171–5192 (2021). https://doi.org/10.1038/s41596-021-00607-0

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