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Preparing ductal epithelial organoids for high-spatial-resolution molecular profiling using mass spectrometry imaging

Nature Protocols volume 17, pages 962–979 (2022)Cite this article

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Abstract

Organoid culture systems are self-renewing, three-dimensional (3D) models derived from pluripotent stem cells, adult derived stem cells or cancer cells that recapitulate key molecular and structural characteristics of their tissue of origin. They generally form into hollow structures with apical–basolateral polarization. Mass spectrometry imaging (MSI) is a powerful analytical method for detecting a wide variety of molecules in a single experiment while retaining their spatiotemporal distribution. Here we describe a protocol for preparing organoids for MSI that (1) preserves the 3D morphological structure of hollow organoids, (2) retains the spatiotemporal distribution of a vast array of molecules (3) and enables accurate molecular identification based on tandem mass spectrometry. The protocol specifically focuses on the collection and embedding of the organoids in gelatin, and gives recommendations for MSI-specific sample preparation, data acquisition and molecular identification by tandem mass spectrometry. This method is applicable to a wide range of organoids from different origins, and takes 1 d from organoid collection to MSI data acquisition.

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Fig. 1: Organoid collection and preparation for MSI and, in parallel, molecular identification (MS2).
Fig. 2: Cutoff pipette tips.
Fig. 3: Freezing of gelatin-embedded organoids.
Fig. 4: Appearance of the organoids at various stages of the protocol.
Fig. 5: PDAC organoid morphology.
Fig. 6: High-spatial-resolution MALDI-MSI.
Fig. 7: High-mass-resolution MALDI-MSI.
Fig. 8: Number of m/z values that could be assigned to a distinct molecular species based on exact mass (mass error <2 ppm).
Fig. 9: MALDI-MS2 on spotted organoid.
Fig. 10: Number of m/z values that could be assigned to a distinct molecular species on the basis of exact mass and MS2 data.

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Data availability

The example data are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank Molecular Horizon Srl, Italy, for the development of LipostarMSI and their support in analyzing organoids with their software. This work was supported by NWO-NWA Startimpuls (grant no. 400.17.604). The authors thank the NUTRIM graduate program for the personal grants supporting R.D.W.V. and M.R.A. This work was financially supported by the Dutch province of Limburg through the LINK program.

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Author notes

  1. These authors contributed equally: Rianne D. W. Vaes, Merel R. Aberle.

Authors and Affiliations

  1. Maastricht MultiModal Molecular Imaging institute (M4I), Maastricht University, Maastricht, the Netherlands

    Brenda Bakker & Ron M. A. Heeren

  2. Department of Surgery, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University, Maastricht, the Netherlands

    Rianne D. W. Vaes, Merel R. Aberle, Tessa Welbers, Sander S. Rensen & Steven W. M. Olde Damink

  3. Leiden Academic Center for Drug Research, Division of System Biomedicine and Pharmacology, Leiden University, Leiden, the Netherlands

    Thomas Hankemeier

  4. Department of General, Visceral and Transplant Surgery, University Hospital Aachen, Aachen, Germany

    Steven W. M. Olde Damink

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  1. Brenda Bakker
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Contributions

B.B.: writing, protocol development, data acquisition, data processing. R.D.W.V.: writing, organoid culture, protocol development. M.R.A.: writing, organoid culture, protocol development. T.W.: writing, protocol development, organoid culture. T.H.: supervision. S.S.R.: writing, supervision, funding, project conception. S.W.M.O.D.: supervision, funding, project conception. R.M.A.H.: supervision, funding, project conception.

Corresponding author

Correspondence to Ron M. A. Heeren.

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The authors declare no competing interests.

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Nature Protocols thanks Aleksandra Aizenshtadt, Choi-Fong Cho, Amanda Hummon, Hanne Røberg-Larsen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Ščupáková, K. et al. Angew Chem. Int. Ed. Engl. 59, 17447–17450 (2020): https://doi.org/10.1002/anie.202007315

Ellis, S. R. et al. Nat. Methods 15, 515–518 (2018): https://doi.org/10.1038/s41592-018-0010-6

Supplementary information

Supplementary Information

Supplementary Methods 1–6, Supplementary Figs. 1–4 and Supplementary Table 1.

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Bakker, B., Vaes, R.D.W., Aberle, M.R. et al. Preparing ductal epithelial organoids for high-spatial-resolution molecular profiling using mass spectrometry imaging. Nat Protoc 17, 962–979 (2022). https://doi.org/10.1038/s41596-021-00661-8

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