Establishment and Culture of Human Intestinal Organoids Derived from Adult Stem Cells

Cayetano Pleguezuelos-Manzano, Jens Puschhof, Stieneke van den Brink, Veerle Geurts, Joep Beumer, Hans Clevers, Cayetano Pleguezuelos-Manzano, Jens Puschhof, Stieneke van den Brink, Veerle Geurts, Joep Beumer, Hans Clevers

Abstract

Human intestinal organoids derived from adult stem cells are miniature ex vivo versions of the human intestinal epithelium. Intestinal organoids are useful tools for the study of intestinal physiology as well as many disease conditions. These organoids present numerous advantages compared to immortalized cell lines, but working with them requires dedicated techniques. The protocols described in this article provide a basic guide to establishment and maintenance of human intestinal organoids derived from small intestine and colon biopsies. Additionally, this article provides an overview of several downstream applications of human intestinal organoids. © 2020 The Authors. Basic Protocol 1: Establishment of human small intestine and colon organoid cultures from fresh biopsies Basic Protocol 2: Mechanical splitting, passage, and expansion of human intestinal organoids Alternate Protocol: Differentiation of human intestinal organoids Basic Protocol 3: Cryopreservation and thawing of human intestinal organoids Basic Protocol 4: Immunofluorescence staining of human intestinal organoids Basic Protocol 5: Generation of single-cell clonal intestinal organoid cultures Support Protocol 1: Production of Wnt3A conditioned medium Support Protocol 2: Production of Rspo1 conditioned medium Support Protocol 3: Extraction of RNA from intestinal organoid cultures.

Keywords: adult stem cells; human intestinal organoids; organoid cryopreservation; organoid culture establishment; organoid differentiation; organoid immunofluorescence; organoid passage; single-cell clonal organoid culture; specialized organoid reagents.

Conflict of interest statement

H.C. is the inventor on several patents related to organoid technology; his full disclosure is given at https://www.uu.nl/staff/JCClevers/.

© 2020 The Authors.

Figures

Figure 1
Figure 1
Establishment of human colon organoid lines from fresh biopsy of normal tissue. Representative images from two organoid lines. Cells are shown after establishment of cultures (left), before the first split (middle), and after the first split (right). P indicates passage number; d indicates days after seeding or passage; scale bars, 0.5 mm.
Figure 2
Figure 2
Mechanical splitting and expansion of human intestinal organoids. (A) Schematic of culture procedure. (B) Glass Pasteur pipette connected to a 10‐μl tip used for mechanical disruption of organoids. (C) Representative bright‐field microscopy images of organoid cultures at days 0, 1, 2, and 4 after mechanical split. Scale bars, 2 mm. (D) Examples of a healthy organoid (top), composed mostly of stem/transit amplifying cells, and a suboptimal organoid (bottom), with signs of differentiation (thickening of wall). Scale bars, 0.4 mm.
Figure 3
Figure 3
Differentiation of human intestinal organoids. (A) Representative images of organoids differentiated towards the enteroendocrine cell lineage as in Beumer et al. (2020). Scale bars, 0.2 mm. (B) Representative images of organoids differentiated towards the enterocyte lineage. Scale bars, 0.4 mm. Inset: Detail of differentiated organoid with enterocytes (elongated cells, arrowhead). Scale bar, 0.1 mm.
Figure 4
Figure 4
Confocal immunofluorescence images of human intestinal organoids cultured under expansion conditions. DAPI, nuclei; KI67, proliferation marker; phalloidin, F‐actin staining. Scale bar, 0.1 mm.
Figure 5
Figure 5
Clonal expansion of human intestinal organoid cultures. (A) Schematic of process. (B) Gating strategy for sorting of living cells used for clonal organoid outgrowth. Cells in P3 gate were sorted and seeded. (C) Single‐cell organoid outgrowth after 10 days. Arrowhead indicates organoid of proper size for picking. Scale bar, 2 mm. (D) Organoid fragments after trypsinization of a single picked organoid. Scale bars, 1 mm (left) and 0.4 mm (inset at right).

References

    1. Bar‐Ephraim, Y. E. , Kretzschmar, K. , & Clevers, H. (2019). Organoids in immunological research. Nature Reviews Immunology, 20(5), 279–293. doi: 10.1038/s41577-019-0248-y.
    1. Beumer, J. , Artegiani, B. , Post, Y. , Reimann, F. , Gribble, F. , Nguyen, T. N. , … Clevers, H. (2018). Enteroendocrine cells switch hormone expression along the crypt‐to‐villus BMP signalling gradient. Nature Cell Biology, 20(8), 909–916. doi: 10.1038/s41556-018-0143-y.
    1. Beumer, J. , Puschhof, J. , Bauzá‐Martinez, J. , Martínez‐Silgado, A. , Elmentaite, R. , James, K. R. , … Clevers, H. (2020). High‐resolution mRNA and secretome atlas of human enteroendocrine cells. Cell, 181(6), 1291–1306.e19. doi: 10.1016/j.cell.2020.04.036.
    1. Bigorgne, A. E. , Farin, H. F. , Lemoine, R. , Mahlaoui, N. , Lambert, N. , Gil, M. , … de Saint Basile, G. (2014). TTC7A mutations disrupt intestinal epithelial apicobasal polarity. Journal of Clinical Investigation, 124(1), 328–337. doi: 10.1172/JCI71471.
    1. Blokzijl, F. , de Ligt, J. , Jager, M. , Sasselli, V. , Roerink, S. , Sasaki, N. , … van Boxtel, R. (2016). Tissue‐specific mutation accumulation in human adult stem cells during life. Nature, 538(7624), 260–264. doi: 10.1038/nature19768.
    1. Christensen, S. , Van der Roest, B. , Besselink, N. , Janssen, R. , Boymans, S. , Martens, J. W. M. , … Van Hoeck, A. (2019). 5‐Fluorouracil treatment induces characteristic T>G mutations in human cancer. Nature Communications, 10(1), 4571. doi: 10.1038/s41467-019-12594-8.
    1. Clevers, H. (2013). The intestinal crypt, a prototype stem cell compartment. Cell, 154(2), 274–284. doi: 10.1016/j.cell.2013.07.004.
    1. Clevers, H. (2016). Modeling development and disease with organoids. Cell, 165(7), 1586–1597. doi: 10.1016/j.cell.2016.05.082.
    1. Dekkers, J. F. , Wiegerinck, C. L. , de Jonge, H. R. , Bronsveld, I. , Janssens, H. M. , de Winter‐de Groot, K. M. , … Beekman, J. M. (2013). A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nature Medicine, 19(7), 939–945. doi: 10.1038/nm.3201.
    1. Dieterich, W. , Neurath, M. F. , & Zopf, Y. (2020). Intestinal ex vivo organoid culture reveals altered programmed crypt stem cells in patients with celiac disease. Scientific Reports, 10(1), 3535. doi: 10.1038/s41598-020-60521-5.
    1. Dijkstra, K. K. , Cattaneo, C. M. , Weeber, F. , Chalabi, M. , van de Haar, J. , Fanchi, L. F. , … Voest, E. E. (2018). Generation of tumor‐reactive T cells by co‐culture of peripheral blood lymphocytes and tumor organoids. Cell, 174(6), 1586–1598.e12. doi: 10.1016/j.cell.2018.07.009.
    1. Drost, J. , & Clevers, H. (2018). Organoids in cancer research. Nature Reviews Cancer, 18(7), 407–418. doi: 10.1038/s41568-018-0007-6.
    1. Drost, J. , van Jaarsveld, R. H. , Ponsioen, B. , Zimberlin, C. , van Boxtel, R. , Buijs, A. , … Clevers, H. (2015). Sequential cancer mutations in cultured human intestinal stem cells. Nature, 521(7550), 43–47. doi: 10.1038/nature14415.
    1. Farin, H. F. , Jordens, I. , Mosa, M. H. , Basak, O. , Korving, J. , Tauriello, D. V. F. , … Clevers, H. (2016). Visualization of a short‐range Wnt gradient in the intestinal stem‐cell niche. Nature, 530(7590), 340–343. doi: 10.1038/nature16937.
    1. Freire, R. , Ingano, L. , Serena, G. , Cetinbas, M. , Anselmo, A. , Sapone, A. , … Senger, S. (2019). Human gut derived‐organoids provide model to study gluten response and effects of microbiota‐derived molecules in celiac disease. Scientific Reports, 9(1), 7029. doi: 10.1038/s41598-019-43426-w.
    1. Fujii, M. , Clevers, H. , & Sato, T. (2019). Modeling human digestive diseases with CRISPR‐Cas9‐modified organoids. Gastroenterology, 156(3), 562–576. doi: 10.1053/j.gastro.2018.11.048.
    1. Fujii, M. , Matano, M. , Nanki, K. , & Sato, T. (2015). Efficient genetic engineering of human intestinal organoids using electroporation. Nature Protocols, 10(10), 1474–1485. doi: 10.1038/nprot.2015.088.
    1. Fujii, M. , Matano, M. , Toshimitsu, K. , Takano, A. , Mikami, Y. , Nishikori, S. , … Sato, T. (2018). Human intestinal organoids maintain self‐renewal capacity and cellular diversity in niche‐inspired culture condition. Cell Stem Cell, 23(6), 787–793.e6. doi: 10.1016/j.stem.2018.11.016.
    1. Geurts, M. H. , de Poel, E. , Amatngalim, G. D. , Oka, R. , Meijers, F. M. , Kruisselbrink, E. , … Clevers, H. (2020). CRISPR‐based adenine editors correct nonsense mutations in a cystic fibrosis organoid biobank. Cell Stem Cell, 26(4), 503–510.e7. doi: 10.1016/j.stem.2020.01.019.
    1. Janda, C. Y. , Dang, L. T. , You, C. , Chang, J. , de Lau, W. , Zhong, Z. A. , … Garcia, K. C. (2017). Surrogate Wnt agonists that phenocopy canonical Wnt and β‐catenin signalling. Nature, 545(7653), 234–237. doi: 10.1038/nature22306.
    1. Miao, Y. , Ha, A. , de Lau, W. , Yuki, K. , Santos, A. J. M. , You, C. , … Garcia, K. C. (2020). Next-generation surrogate Wnts support organoid growth and deconvolute Frizzled pleiotropy in vivo. Cell Stem Cell, 2020;S1934–5905(20), 30358–1. doi: .
    1. Nanki, K. , Fujii, M. , Shimokawa, M. , Matano, M. , Nishikori, S. , Date, S. , … Sato, T. (2020). Somatic inflammatory gene mutations in human ulcerative colitis epithelium. Nature, 577(7789), 254–259. doi: 10.1038/s41586-019-1844-5.
    1. Pleguezuelos‐Manzano, C. , Puschhof, J. , Rosendahl Huber, A. , van Hoeck, A. , Wood, H. M. , Nomburg, J. , … Clevers, H. (2020). Mutational signature in colorectal cancer caused by genotoxic pks + E. coli. Nature, 580(7802), 269–273. doi: 10.1038/s41586-020-2080-8.
    1. Sato, T. , Stange, D. E. , Ferrante, M. , Vries, R. G. J. , van Es, J. H. , van den Brink, S. , … Clevers, H. (2011). Long‐term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology, 141(5), 1762–1772. doi: 10.1053/j.gastro.2011.07.050.
    1. Sato, T. , Vries, R. G. , Snippert, H. J. , van de Wetering, M. , Barker, N. , Stange, D. E. , … Clevers, H. (2009). Single Lgr5 stem cells build crypt‐villus structures in vitro without a mesenchymal niche. Nature, 459(7244), 262–265. doi: 10.1038/nature07935.
    1. Schwank, G. , Koo, B.‐K. , Sasselli, V. , Dekkers, J. F. , Heo, I. , Demircan, T. , … Clevers, H. (2013). Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell, 13(6), 653–658. doi: 10.1016/j.stem.2013.11.002.
    1. van de Wetering, M. , Francies, H. E. , Francis, J. M. , Bounova, G. , Iorio, F. , Pronk, A. , … Clevers, H. (2015). Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell, 161(4), 933–945. doi: 10.1016/j.cell.2015.03.053.
    1. van Rijn, J. M. , Ardy, R. C. , Kuloğlu, Z. , Härter, B. , van Haaften‐Visser, D. Y. , van der Doef, H. P. J. , … Boztug, K. (2018). Intestinal failure and aberrant lipid metabolism in patients with DGAT1 deficiency. Gastroenterology, 155(1), 130–143.e15. doi: 10.1053/j.gastro.2018.03.040.
    1. Xu, Q. , Wang, Y. , Dabdoub, A. , Smallwood, P. M. , Williams, J. , Woods, C. , … Nathans, J. (2004). Vascular development in the retina and inner ear: Control by Norrin and Frizzled‐4, a high‐affinity ligand‐receptor pair. Cell, 116(6), 883–895. doi: .

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