Abstract
The intestinal epithelium is known as one of the most regenerative tissues in our body. The lining of the intestine is composed of a single layer of epithelial cells generated by rapidly renewing stem cells residing at the crypt bottoms, resulting in a flow of cells to the villus tips. The stereotypical crypt–villus architecture makes the intestine an ideal model for stem cell research. Based on recent advances in research of stem cell niche signals in vivo, we have established an intestinal epithelial stem cell culture method. Under this culture condition, single Lgr5+ intestinal stem cells (ISCs) or isolated whole crypts efficiently expand into three-dimensional spherical structures recapitulating the intestinal crypt–villus organization. These organoids can be passaged weekly and maintained for years in culture. Moreover, they can be cryopreserved. As intestinal organoids recapitulate many aspects of the epithelial biology and are amenable to most, if not all, current experimental manipulations, they are widely used to study stem cell biology, cell fate determination, gene function, and disease mechanism.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Leblond CP, Stevens CE (1948) The constant renewal of the intestinal epithelium in the albino rat. Anat Rec 100:357–377
Barker N, van Es JH, Kuipers J et al (2007) Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449:1003–1007
Sato T, Vries RG, Snippert HJ et al (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459:262–265
Sato T, Stange DE, Ferrante M et al (2011) Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141:1762–1772
Yui S, Nakamura T, Sato T et al (2012) Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell. Nat Med 18:618–623
Fukuda M, Mizutani T, Mochizuki W et al (2014) Small intestinal stem cell identity is maintained with functional Paneth cells in heterotopically grafted epithelium onto the colon. Genes Dev 28:1752–1757
Sato T, Clevers H (2013) Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340(6137):1190–1194
Koo B-K, Stange DE, Sato T, Karthaus W, Farin HF, Huch M, van Es JH, Clevers H (2011) Controlled gene expression in primary Lgr5 organoid cultures. Nat Methods 9(1):81–83
Schwank G, Koo B-K, Sasselli V et al (2013) Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13:653–658
Li X, Nadauld L, Ootani A et al (2014) Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture. Nat Med 20(7):769–777
Drost J, van Jaarsveld RH, Ponsioen B et al (2015) Sequential cancer mutations in cultured human intestinal stem cells. Nature 521:43–47
Matano M, Date S, Shimokawa M, Takano A, Fujii M, Ohta Y, Watanabe T, Kanai T, Sato T (2015) Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nat Med 21(3):256–262. https://doi.org/10.1038/nm.3802
Basak O, Beumer J, Wiebrands K, Seno H, van Oudenaarden A, Clevers H (2017) Induced quiescence of Lgr5+ stem cells in intestinal organoids enables differentiation of hormone-producing enteroendocrine cells. Cell Stem Cell 20:177–190.e4
Schuijers J, van der Flier LG, van Es J, Clevers H (2014) Robust cre-mediated recombination in small intestinal stem cells utilizing the olfm4 locus. Stem Cell Reports 3(2):234–241
Mustata RC, Vasile G, Fernandez-Vallone V, Strollo S, Lefort A, Libert F, Monteyne D, Pérez-Morga D, Vassart G, Garcia M-I (2013) Identification of Lgr5-independent spheroid-generating progenitors of the mouse fetal intestinal epithelium. Cell Reports 5:421–432
Grün D, Lyubimova A, Kester L, Wiebrands K, Basak O, Sasaki N, Clevers H, van Oudenaarden A (2015) Single-cell messenger RNA sequencing reveals rare intestinal cell types. Nature 525:251–255
Haber AL, Biton M, Rogel N et al (2017) A single-cell survey of the small intestinal epithelium. Nat Publ Group 551:333–339
Lindeboom RG, van Voorthuijsen L, Oost KC et al (2018) Integrative multi-omics analysis of intestinal organoid differentiation. Mol Syst Biol 14:e8227
Behjati S, Huch M, van Boxtel R et al (2014) Genome sequencing of normal cells reveals developmental lineages and mutational processes. Nat Publ Group 513:422–425
Gonneaud A, Jones C, Turgeon N, Lévesque D, Asselin C, Boudreau F, Boisvert F-M (2016) A SILAC-based method for quantitative proteomic analysis of intestinal organoids. Sci Rep 6:38195
Cristobal A, van den Toorn HWP, van de Wetering M, Clevers H, Heck AJR, Mohammed S (2017) Personalized proteome profiles of healthy and tumor human colon organoids reveal both individual diversity and basic features of colorectal cancer. Cell Reports 18:263–274
Beumer J, Artegiani B, Post Y, Reimann F, Gribble F, Nguyen TN, Zeng H, van den Born M, van Es JH, Clevers H (2018) Enteroendocrine cells switch hormone expression along the crypt-to-villus BMP signalling gradient. Nat Cell Biol 20:909–916
Gehart H, van Es JH, Hamer K, Beumer J, Kretzschmar K, Dekkers JF, Rios A, Clevers H (2019) Identification of enteroendocrine regulators by real-time single-cell differentiation mapping. Cell 176:1158–1173.e16
Nozaki K, Mochizuki W, Matsumoto Y, Matsumoto T, Fukuda M, Mizutani T, Watanabe M, Nakamura T (2016) Co-culture with intestinal epithelial organoids allows efficient expansion and motility analysis of intraepithelial lymphocytes. J Gastroenterol 51:206–213
Zhang Y-G, Wu S, Xia Y, Sun J (2014) Salmonella-infected crypt-derived intestinal organoid culture system for host-bacterial interactions. Physiol Rep 2:e12147–e12111
Heo I, Dutta D, Schaefer DA et al (2018) Modelling Cryptosporidium infection in human small intestinal and lung organoids. Nat Microbiol 3(7):814–823
Booth C, O’Shea JA, Freshney RI (2002) Isolation and culture of intestinal epithelial cells. Culture of epithelial cells, 2nd edn. Wiley-Liss, New York, pp 303–335
Farin HF, van Es JH, Clevers H (2012) Redundant sources of Wnt regulate intestinal stem cells and promote formation of Paneth cells. Gastroenterology 143(6):1518–1529.e7. https://doi.org/10.1053/j.gastro.2012.08.031
Kim KA (2005) Mitogenic influence of human R-spondin1 on the intestinal epithelium. Science 309:1256–1259
Barker N, Huch M, Kujala P et al (2010) Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6:25–36
Acknowledgments
The authors would like to thank Joep Beumer for advice on whole-mount imaging technique and critical reading of the manuscript, and Jeroen Korving and Harry Begthel for their advice on immunohistochemistry protocol.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Mizutani, T., Clevers, H. (2020). Primary Intestinal Epithelial Organoid Culture. In: Ordóñez-Morán, P. (eds) Intestinal Stem Cells. Methods in Molecular Biology, vol 2171. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0747-3_11
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0747-3_11
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0746-6
Online ISBN: 978-1-0716-0747-3
eBook Packages: Springer Protocols