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High-throughput automated organoid culture via stem-cell aggregation in microcavity arrays

Abstract

Stem-cell-derived epithelial organoids are routinely used for the biological and biomedical modelling of tissues. However, the complexity, lack of standardization and quality control of stem cell culture in solid extracellular matrices hampers the routine use of the organoids at the industrial scale. Here, we report the fabrication of microengineered cell culture devices and scalable and automated methods for suspension culture and real-time analysis of thousands of individual gastrointestinal organoids trapped in microcavity arrays within a polymer-hydrogel substrate. The absence of a solid matrix substantially reduces organoid heterogeneity, which we show for mouse and human gastrointestinal organoids. We use the devices to screen for anticancer drug candidates with patient-derived colorectal cancer organoids, and apply high-content image-based phenotypic analyses to reveal insights into mechanisms of drug action. The scalable organoid-culture technology should facilitate the use of organoids in drug development and diagnostics.

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Fig. 1: Organoid array technology.
Fig. 2: GI organoid array cultures.
Fig. 3: Organoid arrays robustness.
Fig. 4: Analysis of organoid homogeneity and reproducibility.
Fig. 5: Automated high-content phenotypic drug screening.
Fig. 6: Organoid phenotypic drug screening performances and hit map.
Fig. 7: Phenotypic hit analysis.
Fig. 8: RNA-seq analysis.

Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are too large to be publicly shared, yet they are available for research purposes from the corresponding author on reasonable request. The RNA-seq data have been deposited at the NCBI Gene Expression Omnibus, and are accessible under the GEO Series accession number GSE148347.

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Acknowledgements

We thank F. Ringnalda for the initial establishment of colon organoid cultures in the Schwank laboratory; G. Rogler, B. Morell and M. Scharl for providing intestinal rest material; S. Allazetta for providing PEG hydrogel microbeads; N. Landwehr for help with video production; and M. Leleu for the bioinformatics analysis. Intestinal tissue samples were obtained from the Department of Gastroenterology and Hepatology, University Hospital Zürich under ethical approval from the Cantonal Ethics Committee of the Canton Zürich, Switzerland (EK-1755). Colorectal tumour samples were obtained from the Department of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois under ethical approval from the Cantonal Ethics Committee of the Canton Vaud, Switzerland (CER-VD: 2017-00359). This research was funded by the project ‘OPERON’ from the Personalized Health and Related Technologies Initiative from the ETH Board, as well as École Polytechnique Fédérale de Lausanne (EPFL).

Author information

Affiliations

Authors

Contributions

N.B., S.H. and M.P.L. conceived the study, designed experiments, analysed data and wrote the manuscript. F.K. and G.T. helped to design the screening experiments and analysed the data. K.H. designed the drug panel and gave valuable feedback on experimental designs. C.C. helped conduct mouse intestinal organoid experiments. N.G., T.R. and G.S. gave feedback on experimental designs. T.R. and G.S. helped to design experiments and analysed data with human cells. F.K., K.H., N.G., T.R., G.S. and G.C. provided inputs on the manuscript.

Corresponding author

Correspondence to Matthias P. Lutolf.

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Competing interests

The Ecole Polytechnique Fédérale de Lausanne has filed for patent protection on the technology described herein (PCT/IB2014/067242, published as CA2972057A1, CN107257850A, EP3237597A1, WO2016103002A1 JP2018504103A and US2018264465A1; and PCT/EP2017/073357, published as EP3296018A1, EP3515600A1, WO2018050862A1 and US2020010797A1), and S.H., N.B., N.G. and M.P.L. are named as inventors on those patents; S.H., N.B. and M.P.L. are shareholders in SUN bioscience SA, which is commercializing those patents.

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Supplementary information

Supplementary Information

Supplementary figures, tables and video captions.

Reporting Summary

Supplementary Video 1

Time-lapse video of aggregating mouse ISCs from 100 single sorted LGR5–GFP mouse ISCs.

Supplementary Video 2

Time-lapse video of growing and differentiating mouse intestinal organoids from 100 single sorted LGR5–GFP mouse ISCs.

Supplementary Video 3

Three-day time-lapse video of CRC organoids exposed to gambogic acid (10 μM), the positive-control condition.

Supplementary Video 4

Three-day time-lapse video of CRC organoids exposed to the vehicle (DMSO, diluted 1:1,000), the negative-control condition.

Supplementary Video 5

Three-day time-lapse video of CRC organoids exposed to 1 μM afuresertib.

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Brandenberg, N., Hoehnel, S., Kuttler, F. et al. High-throughput automated organoid culture via stem-cell aggregation in microcavity arrays. Nat Biomed Eng 4, 863–874 (2020). https://doi.org/10.1038/s41551-020-0565-2

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