High-resolution 3D imaging of fixed and cleared organoids (original) (raw)

Nature Protocols volume 14, pages 1756–1771 (2019)Cite this article

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Abstract

In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids’ phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose–glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.

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All data generated or analyzed during this study are included in this published article (and its supplementary information files).

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Acknowledgements

We are very grateful for the technical support from the Princess Máxima Center for Pediatric Oncology and to the Hubrecht Institute and Zeiss for imaging support and collaborations. All the imaging was performed at the Princess Máxima imaging center. This work was financially supported by the Princess Máxima Center for Pediatric Oncology. J.F.D. was supported by a Marie Curie Global Fellowship and a VENI grant from the Netherlands Organisation for Scientific Research (NWO). J.E.V. was supported by the Australian National Health and Medical Research Council (NHMRC).

Author information

Author notes

  1. These authors contributed equally: Maria Alieva, Lianne M. Wellens.
  2. These authors jointly supervised this work: Jane E. Visvader, Hans Clevers, Anne C. Rios.

Authors and Affiliations

  1. Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands
    Johanna F. Dekkers, Maria Alieva, Lianne M. Wellens, Hendrikus C. R. Ariese, Ellen J. Wehrens, Hans Clevers & Anne C. Rios
  2. Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Center (UMC) Utrecht, Utrecht, the Netherlands
    Johanna F. Dekkers, Huili Hu & Hans Clevers
  3. Department of Cancer Research, Oncode Institute, Hubrecht Institute–KNAW Utrecht, Utrecht, the Netherlands
    Johanna F. Dekkers, Maria Alieva, Lianne M. Wellens, Hendrikus C. R. Ariese, Huili Hu, Koen C. Oost, Hugo J. G. Snippert, Ellen J. Wehrens, Hans Clevers & Anne C. Rios
  4. Cancer Genomics Center (CGC), Utrecht, the Netherlands
    Johanna F. Dekkers, Maria Alieva, Lianne M. Wellens, Hendrikus C. R. Ariese, Huili Hu, Ellen J. Wehrens, Hans Clevers & Anne C. Rios
  5. Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia
    Paul R. Jamieson & Jane E. Visvader
  6. Regenerative Medicine Center Utrecht, University Medical Center, Utrecht University, Utrecht, the Netherlands
    Annelotte M. Vonk, Gimano D. Amatngalim & Jeffrey M. Beekman
  7. Department of Pediatric Pulmonology, Wilhelmina Children’s Hospital, University Medical Center, Utrecht University, Utrecht, the Netherlands
    Annelotte M. Vonk, Gimano D. Amatngalim & Jeffrey M. Beekman
  8. Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands
    Koen C. Oost & Hugo J. G. Snippert
  9. Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia
    Jane E. Visvader

Authors

  1. Johanna F. Dekkers
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  2. Maria Alieva
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  3. Lianne M. Wellens
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  4. Hendrikus C. R. Ariese
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  5. Paul R. Jamieson
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  6. Annelotte M. Vonk
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  7. Gimano D. Amatngalim
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  8. Huili Hu
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  9. Koen C. Oost
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  10. Hugo J. G. Snippert
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  11. Jeffrey M. Beekman
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  12. Ellen J. Wehrens
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  13. Jane E. Visvader
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  14. Hans Clevers
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  15. Anne C. Rios
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Contributions

J.F.D. designed the study, performed experiments and interpreted data; J.F.D., M.A., L.M.W., H.C.R.A. and P.R.J. performed experiments and analyzed data; J.F.D., P.R.J., A.M.V., G.D.A., H.H. and J.M.B. performed the organoid culturing. K.C.O. and H.J.G.S. provided fluorescent constructs; A.C.R. helped design the study and carried out data interpretation; J.F.D. and A.C.R. cowrote the manuscript. J.E.V., H.C. and E.J.W. helped with data interpretation, manuscript writing and corrections.

Corresponding author

Correspondence toAnne C. Rios.

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Competing interests

H.C. is named as inventor on several patents related to organoid technology. J.F.D. is named as inventor on one patent related to the organoid technology.

Additional information

Journal peer review information: Nature Protocols thanks Xavier Gidrol, Melissa Skala and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Hu, H. et al. Cell 175, 1591–1606.e19 (2018): https://doi.org/10.1016/j.cell.2018.11.013

Sachs, N. et al. EMBO J. 38, e100300 (2019): https://doi.org/10.15252/embj.2018100300

Supplementary information

Supplementary Video 1

This video highlights the intricate 3D features of delicate organoid structures that can be imaged at cellular or even subcellular resolution with this sample-preparation protocol.

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Dekkers, J.F., Alieva, M., Wellens, L.M. et al. High-resolution 3D imaging of fixed and cleared organoids.Nat Protoc 14, 1756–1771 (2019). https://doi.org/10.1038/s41596-019-0160-8

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