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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
$99.00 per year
only $8.25 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
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.
References
Lancaster, M. A. & Knoblich, J. A. Organogenesis in a dish: modeling development and disease using organoid technologies. Science 345, 1247125 (2014).
Dutta, D., Heo, I. & Clevers, H. Disease modeling in stem cell-derived 3D organoid systems. Trends Mol. Med. 23, 393–410 (2017).
Fatehullah, A., Tan, S. H. & Barker, N. Organoids as an in vitro model of human development and disease. Nat. Cell Biol. 18, 246–254 (2016).
Ranga, A., Gjorevski, N. & Lutolf, M. P. Drug discovery through stem cell-based organoid models. Adv. Drug Deliv. Rev. 69–70, 19–28 (2014).
Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).
Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 1762–1772 (2011).
Akkerman, N. & Defize, L. H. K. Dawn of the organoid era: 3D tissue and organ cultures revolutionize the study of development, disease, and regeneration. Bioessays https://doi.org/10.1002/bies.201600244 (2017).
Schneeberger, K. et al. Converging biofabrication and organoid technologies: the next frontier in hepatic and intestinal tissue engineering? Biofabrication 9, 013001 (2017).
Gracz, A. D. et al. A high-throughput platform for stem cell niche co-cultures and downstream gene expression analysis. Nat. Cell Biol. 17, 340–349 (2015).
Gunasekara, D. B. et al. Development of arrayed colonic organoids for screening of secretagogues associated with enterotoxins. Anal. Chem. 90, 1941–1950 (2018).
Francies, H. E., Barthorpe, A., McLaren-Douglas, A., Barendt, W. J. & Garnett, M. J. Drug sensitivity assays of human cancer organoid cultures. Methods Mol. Biol. 1576, 339–351 (2019).
van de Wetering, M. et al. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell 161, 933–945 (2015).
Vlachogiannis, G. et al. Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science 359, 920–926 (2018).
Wang, Y. et al. A microengineered collagen scaffold for generating a polarized crypt-villus architecture of human small intestinal epithelium. Biomaterials 128, 44–55 (2017).
Gobaa, S. et al. Artificial niche microarrays for probing single stem cell fate in high throughput. Nat. Methods 8, 949–955 (2011).
Yin, X. et al. Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny. Nat. Methods 11, 106–112 (2014).
Sato, T. et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469, 415–418 (2011).
Zhang, Y., Lukacova, V., Reindl, K. & Balaz, S. Quantitative characterization of binding of small molecules to extracellular matrix. J. Biochem. Biophys. Methods 67, 107–122 (2006).
Gjorevski, N. et al. Designer matrices for intestinal stem cell and organoid culture. Nature 539, 560–564 (2016).
Pastuła, A. et al. Three-dimensional gastrointestinal organoid culture in combination with nerves or fibroblasts: a method to characterize the gastrointestinal stem cell niche. Stem Cells Int. 2016, 3710836 (2016).
Horvay, K. et al. Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium. EMBO J. 34, 1319–1335 (2015).
Yang, T.-L. B. et al. Mutual reinforcement between telomere capping and canonical Wnt signalling in the intestinal stem cell niche. Nat. Commun. 8, 14766 (2017).
Sato, T. & Clevers, H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340, 1190–1194 (2013).
Dekkers, J. F. et al. A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat. Med 19, 939–945 (2013).
Ley, S. et al. Screening of intestinal crypt organoids: a simple readout for complex biology. SLAS Discov. 22, 571–582 (2017).
Hannan, N. R. F. et al. Generation of multipotent foregut stem cells from human pluripotent stem cells. Stem Cell Rep. 1, 293–306 (2013).
Fujii, M. et al. A colorectal tumor organoid library demonstrates progressive loss of niche factor requirements during tumorigenesis. Cell Stem Cell 18, 827–838 (2016).
Carpenter, A. E. et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7, R100 (2006).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Anders, S., Pyl, P. T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).
McCarthy, D. J., Chen, Y. & Smyth, G. K. Differential expression analysis of multifactor RNA-seq experiments with respect to biological variation. Nucleic Acids Res. 40, 4288–4297 (2012).
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
Authors and Affiliations
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
Ethics declarations
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.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Supplementary Information
Supplementary figures, tables and video captions.
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.
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41551-020-0565-2
This article is cited by
-
Harnessing 3D in vitro systems to model immune responses to solid tumours: a step towards improving and creating personalized immunotherapies
Nature Reviews Immunology (2024)
-
Microfluidic high-throughput 3D cell culture
Nature Reviews Bioengineering (2024)
-
Chemically-defined and scalable culture system for intestinal stem cells derived from human intestinal organoids
Nature Communications (2024)
-
Application of organoids in otolaryngology: head and neck surgery
European Archives of Oto-Rhino-Laryngology (2024)
-
Applications of lung cancer organoids in precision medicine: from bench to bedside
Cell Communication and Signaling (2023)