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Culture and analysis of kidney tubuloids and perfused tubuloid cells-on-a-chip

Nature Protocols volume 16, pages 2023–2050 (2021)Cite this article

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Abstract

Advanced in vitro kidney models are of great importance to the study of renal physiology and disease. Kidney tubuloids can be established from primary cells derived from adult kidney tissue or urine. Tubuloids are three-dimensional multicellular structures that recapitulate tubular function and have been used to study infectious, malignant, metabolic, and genetic diseases. For tubuloids to more closely represent the in vivo kidney, they can be integrated into an organ-on-a-chip system that has a more physiological tubular architecture and allows flow and interaction with vasculature or epithelial and mesenchymal cells from other organs. Here, we describe a detailed protocol for establishing tubuloid cultures from tissue and urine (1–3 weeks), as well as for generating and characterizing tubuloid cell–derived three-dimensional tubular structures in a perfused microfluidic multi-chip platform (7 d). The combination of the two systems yields a powerful in vitro tool that better recapitulates the complexity of the kidney tubule with donor-specific properties.

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Fig. 1: Barrier integrity assay in the OrganoPlate using fluorescent probes.
Fig. 2: Passaging tubuloids.
Fig. 3: Overview of the method for modeling perfused 3D tubular structures made of primary kidney tubuloids in the OrganoPlate.
Fig. 4: Overview of correct patterning of an ECM gel and seeding tubuloids in the OrganoPlate 3-Lane.
Fig. 5: Overview of the barrier integrity assay with a fluorescent dye in the OrganoPlate 3-Lane.
Fig. 6: Overview of correct installation of the OrganoTEER for TEER measurements of cultures in the OrganoPlate.
Fig. 7: Analysis of transepithelial transport images.
Fig. 8: Tubuloid-derived tubular structures in the OrganoPlate 3-Lane characterized by immunofluorescence staining.

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Data availability

The main data discussed in this protocol are available in the article figures and in the supporting primary research paper (https://www.nature.com/articles/s41587-019-0048-8). The raw datasets are available for research purposes from the corresponding author upon request.

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Acknowledgements

F.A.Y.Y., F.S., C.M.E.A., M.B.R., M.C.V. and H.C. acknowledge the support of the partners of ‘Regenerative Medicine Crossing Borders’ (RegMed XB), Powered by Health~Holland, Top Sector Life Sciences & Health, and support by the Dutch Ministry of Education, Culture and Science for the Gravitation Program 024.003.103 ‘Materials Driven Regeneration’.

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Author notes

  1. These author contributed equally: Linda Gijzen, Fjodor A. Yousef Yengej.

Authors and Affiliations

  1. Mimetas BV, Leiden, the Netherlands

    Linda Gijzen, Marianne K. Vormann, Arnaud Nicolas, Dorota Kurek, Paul Vulto & Henriette L. Lanz

  2. Hubrecht Institute–Royal Netherlands Academy of Arts and Sciences and University Medical Center Utrecht, Utrecht, the Netherlands

    Fjodor A. Yousef Yengej, Frans Schutgens, Carola M. E. Ammerlaan & Hans Clevers

  3. Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, the Netherlands

    Fjodor A. Yousef Yengej, Frans Schutgens, Carola M. E. Ammerlaan, Maarten B. Rookmaaker & Marianne C. Verhaar

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Contributions

D.K., H.L.L., P.V. and H.C. conceived the strategy for this study. L.G., F.A.Y.Y., F.S. and C.M.E.A. designed and performed the experiments and data analysis. M.K.V. designed and performed the transepithelial transport experiments. A.N. designed the transepithelial electrical resistance experiments. L.G. and F.A.Y.Y. wrote the manuscript with comments from all authors. P.V., M.B.R., H.L.L, M.C.V. and H.C. oversaw the research.

Corresponding author

Correspondence to Hans Clevers.

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Competing interests

L.G., M.K.V., A.N., D.K., H.L.L. and P.V. are employees of Mimetas BV, the Netherlands. P.V. is shareholder of the same company. OrganoPlate, OrganoFlow, and OrganoTEER are registered trademarks of Mimetas BV. H.C. is an inventor on several patents related to organoids. A full disclosure can be found at https://www.uu.nl/staff/JCClevers/Additional%20functions.

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Peer review information Nature Protocols thanks Joseph Vincent Bonventre, Ryuji Morizane and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Schutgens, F. et al. Nat. Biotechnol. 37, 303–313 (2019): https://doi.org/10.1038/s41587-019-0048-8

Extended data

Extended Data Fig. 1 Expression levels of markers of the different nephron segments are stable in tubuloids over multiple passages, except for a decrease in the loop of Henle–specific transporter SLC12A1/NKCC2.

Normalized expression of SLC4A4/NBCe1 and SLC5A2/SGLT2 (proximal tubule), SLC12A1/NKCC2 (loop of Henle), SLC12A3/NCC (distal tubule) and SCNN1A/ENaCα (collecting duct principal cells) in tubuloids in passages 4, 7 and 10. Tubuloids from two different donors were analyzed for all genes except SLC4A4/NBCe1 and SLC5A2/SGLT2 (both one donor). Expression was normalized to the housekeeping gene RPLP0. Mean and SEM are shown.

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Gijzen, L., Yousef Yengej, F.A., Schutgens, F. et al. Culture and analysis of kidney tubuloids and perfused tubuloid cells-on-a-chip. Nat Protoc 16, 2023–2050 (2021). https://doi.org/10.1038/s41596-020-00479-w

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