Establishment of Gastrointestinal Epithelial Organoids

Maxime M. Mahe1, Eitaro Aihara1, Michael A. Schumacher2, Yana Zavros2, Marshall H. Montrose2, Michael A. Helmrath3, Toshiro Sato4, Noah F. Shroyer5

1 These authors contributed equally to this work., 2 Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio, 3 Division of Pediatric Surgery, Cincinnati Children's Hospital Medical Research Center, Cincinnati, Ohio, 4 Department of Gastroenterology, School of Medicine, Keio University, Tokyo, 5 Division of Gastroenterology, Hepatology, and Nutrition, Cincinnati Children's Hospital Medical Research Center, Cincinnati, Ohio
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo130179
Online Posting Date:  December, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The intestinal epithelium constitutes a system of constant and rapid renewal triggered by proliferation of intestinal stem cells (ISCs), and is an ideal system for studying cell proliferation, migration, and differentiation. Primary cell cultures have proven to be promising for unraveling the mechanisms involved in epithelium homeostasis. In 2009, Sato et al. established a long‐term primary culture to generate epithelial organoids (enteroids) with crypt‐ and villus‐like epithelial domains representing the complete census of progenitors and differentiated cells. Similarly, isolated ISCs expressing Lgr5 (leucine‐rich repeat‐containing G protein–coupled receptor) can generate enteroids. Here, we describe methods to establish gastric, small intestinal, and colonic epithelial organoids and generate Lgr5+ve single cell–derived epithelial organoids. We also describe the imaging techniques used to characterize those organoids. This in vitro model constitutes a powerful tool for studying stem cell biology and intestinal epithelial cell physiology throughout the digestive tract. Curr. Protoc. Mouse Biol. 3:217‐240 © 2013 by John Wiley & Sons, Inc.

Keywords: gastrointestinal stem cells; 3‐dimensional cell culture; organoids; Lgr5 cell sorting; imaging

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Derivation of Enteroids from Small‐Intestinal Crypts
  • Alternate Protocol 1: Primary Gastric Epithelial Culture from the Fundus or Antrum
  • Alternate Protocol 2: Culture of Colonoids Derived from Colonic Crypts
  • Basic Protocol 2: Lgr5‐GFP+ve Gastrointestinal Stem Cell Sorting and Culture
  • Basic Protocol 3: Imaging of the Gastrointestingal Epithelial Organoids
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Derivation of Enteroids from Small‐Intestinal Crypts

  Materials
  • Mice: C57BL6/J strain (The Jackson laboratory) aged 6 to 8 weeks
  • 70% ethanol
  • Dulbecco's Phosphate‐Buffered Saline (DPBS) without Ca2+ and Mg2+ (DPBS: Thermo Fisher Scientific, cat. no. SH3002802)
  • Crypt chelating buffer (see recipe)
  • Dissociation buffer (see recipe), cold
  • Matrigel, growth factor reduced (GFR), phenol red free (R&D Systems)
  • Murine recombinant R‐spondin 1 (R&D Systems, 1000× stock; 1 mg/ml in sterile DPBS/0.1% BSA)
  • Murine recombinant Noggin (R&D Systems, 1000× stock; 100 µg/ml in sterile DPBS/0.1% BSA)
  • Human recombinant EGF (R&D Systems, 10,000× stock; 500 µg/ml in sterile DPBS/0.1% BSA)
  • Basal minigut medium (see recipe)
  • Complete minigut medium (see recipe)
  • Freezing medium (see recipe)
  • Isopropyl alcohol
  • Liquid N 2
  • Murine recombinant Wnt3a (R&D Systems, 1000× stock; 100 µg/ml in sterile DPBS/0.1% BSA)
  • Y27623 compound (Sigma‐Aldrich, 10 mM in ultrapure H 2O, filter sterilized with 0.22‐µm filter)
  • 24‐well plate
  • Tissue forceps
  • Surgical scissors
  • 10‐ml syringe with 18‐G needle
  • Razor blades
  • 15‐ and 50‐ml conical polypropylene tubes
  • Orbital shaker
  • 70‐µm cell strainer
  • 1‐ml syringe with 27½‐G needle (insulin syringe)
  • 5‐ml round‐bottom tubes
  • Refrigerated centrifuge
  • Inverted microscope
  • Cryovials
  • Freezing container (e.g., Mr. Frosty from Thermo Scientific Nalgene)
  • Liquid N 2 storage container
  • Additional reagents and equipment for rodent euthanasia (Donovan and Brown, )

Alternate Protocol 1: Primary Gastric Epithelial Culture from the Fundus or Antrum

  Additional Materials (also see protocol 1)
  • Mice: C57BL6/J strain (The Jackson laboratory) aged at least 6 weeks
  • Gastric gland chelating buffer: 5 mM EDTA in DPBS
  • Human recombinant FGF10 (PeproTech, 1000× stock; 100 µg/ml in sterile DPBS/0.1% BSA)
  • Human [Leu15]‐Gastrin I (Sigma‐Aldrich, 1000× stock; 10 µM in sterile DPBS/0.1% BSA)
  • N‐Acetylcysteine (Sigma‐Aldrich, 500× stock; 500 mM in ultrapure H 2O, filter sterilized with 0.22‐µm filter)
  • Silicone‐coated dish: silicone made in glass dish using SYLGARD 184 Silicone Elastomer kit according to manufacturer's instructions (Dow Corning, cat. no. 3097358‐1004)
  • Dissecting microscope
  • Micro‐dissecting curved scissors
  • Two pairs of #7 fine point curved forceps

Alternate Protocol 2: Culture of Colonoids Derived from Colonic Crypts

For materials, see protocol 1.

Basic Protocol 2: Lgr5‐GFP+ve Gastrointestinal Stem Cell Sorting and Culture

  Materials
  • Lgr5‐GFP+ve‐ires‐CreER C57BL6/J mouse (The Jackson laboratory) aged 6 to 8 weeks
  • TryPLE Express (Invitrogen).
  • Y‐27632 (Sigma‐Aldrich, 10 mM in ultrapure H 2O, filter sterilized with 0.22‐µm filter)
  • Basal minigut medium (see recipe)
  • N‐acetylcysteine (Sigma‐Aldrich, 500× stock; 500 mM in ultrapure water, filter sterilized with 0.22‐µm filter)
  • Bovine serum albumin (BSA)
  • 7‐Aminoactinomycin D (100× stock; 500 µg/ml in sterile DPBS, Invitrogen, cat. no. A1310; Ex/Em (nm), 548/649)
  • APC‐Annexin V (Invitrogen, cat. no. A35110; Ex/Em (nm), 650/660).
  • Matrigel, growth factor reduced (GFR), phenol red free (R&D Systems)
  • Jagged‐1 Fc chimera peptide (R&D Systems, 1000× stock; 500 µg/ml in sterile DPBS)
  • Murine recombinant Wnt3a (R&D Systems, 1000× stock; 100 µg/ml in sterile DPBS/0.1%BSA)
  • Murine recombinant R‐spondin 1 (R&D Systems, 1000× stock; 1 mg/ml in sterile DPBS/0.1% BSA)
  • Murine recombinant Noggin (R&D Systems, 1000× stock; 100 µg/ml in sterile DPBS/0.1% BSA)
  • Human recombinant EGF (R&D Systems, 10,000× stock ; 500 µg/ml in sterile DPBS/0.1% BSA)
  • CHIR99021 (4000× stock, 10 mM in DMSO; Stemgent, https://www.stemgent.com)
  • Thiazovivin (4000× stock, 10 mM in DMSO; Stemgent, https://www.stemgent.com)
  • MACS C‐Tubes (Miltenyi Biotec)
  • GentleMACS Dissociator (Miltenyi Biotec)
  • 50‐ml conical polypropylene tubes (e.g., BD Falcon)
  • 40‐µm cell strainer
  • Refrigerated centrifuge
  • Hemacytometer
  • FACS tube with 35‐µm mesh cap
  • Cell sorter (BD FACSAria II; Beckman‐Coulter MoFlo XDP)
  • 96‐well plate
  • Additional reagents and equipment for isolation of crypts from mouse ( protocol 1, steps 1 to 10), and counting cells using a hemacytometer and trypan blue exclusion test for cell viability (Sandell and Sakai, )

Basic Protocol 3: Imaging of the Gastrointestingal Epithelial Organoids

  Materials
  • Epithelial organoids ( protocol 1)
  • Complete minigut medium (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 4% paraformaldehyde (PFA)
  • 50 mM NH 4Cl in PBS
  • 0.1% (v/v) Triton X‐100 in PBS
  • 5% (w/v) bovine serum albumin (BSA) or fetal bovine serum (FBS)
  • Primary antibody (E‐cadherin for epithelial cells; see Table 13.1.7900)
  • Secondary antibody (see Table 13.1.7900)
  • 10 µg/ml Hoechst 33342 (Invitrogen)
  • Dulbecco's Phosphate‐Buffered Saline (DPBS) without Ca2+ and Mg2+ (DPBS: Thermo Fisher Scientific, cat. no. SH3002802)
  • 2% methylene blue in PBS
  • 30% (w/v) sucrose
  • OCT compound (Tissue‐Tek)
  • 70% ethanol
  • 8‐well Lab‐Tek chamber with #1.0 borosilicate coverglass (Thermo Scientific)
  • CO 2 module S/temperature module S/humidifier S unit (PeCon incubation system, http://www.pecon.biz/)
  • Heating insert P‐Labtek S1 (PeCon incubation chamber, http://www.pecon.biz/)
  • Inverted confocal microscope (Zeiss LSM710)
  • EC Plan‐Neofluar 10 × 0.3 (dry) or Plan‐Apochromat 20×/0.8 (dry) objective lens
  • C‐Achroplan NIR 40×/0.8 (water) objective lens
  • 1 × 1–cm cryomold
  • Additional reagents and equipment for passaging epithelial organoids ( protocol 1)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Literature Cited

  Akcora, D., Huynh, D., Lightowler, S., Germann, M., Robine, S., de May, J.R., Pollard, J.W., Stanley, E.R., Malaterre, J., and Ramsay, R.G. 2013. The CSF‐1 receptor fashions the intestinal stem cell niche. Stem Cell Res. 10:203‐212.
  Barker, N., Huch, M., Kujala, P., van de Wetering, M., Snippert, H.J., van Es, J.H., Sato, T., Stange, D.E., Begthel, H., van den Born, M., Danenberg, E., van den Brink, S., Korving, J., Abo, A., Peters, P.J., Wright, N., Poulsom, R., and Clevers, H. 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.
  Donovan, J. and Brown, P. 2006. Euthenasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
  Durand, A., Donahue, B., Peignon, G., Letourneur, F., Cagnard, N., Slomianny, C., Perret, C., Shroyer, N.F., and Romagnolo, B. 2012. Functional intestinal stem cells after Paneth cell ablation induced by the loss of transcription factor Math1 (Atoh1). Proc. Natl. Acad. Sci. U.S.A. 109:8965‐8970.
  Farin, H.F., Van Es, J.H., and Clevers, H. 2012. Redundant sources of Wnt regulate intestinal stem cells and promote formation of Paneth cells. Gastroenterology 143:1518‐1529.
  Fuller, M.K., Faulk, D.M., Sundaram, N., Mahe, M.M., Stout, K.M., von Furstenberg, R.J., Smith, B.J., McNaughton, K.K., Shroyer, N.F., Helmrath, M.A., and Henning, S.J. 2013. Intestinal stem cells remain viable after prolonged tissue storage. Cell Tissue Res. 354:441‐450.
  Guan, Y., Watson, A.J., Marchiando, A.M., Bradford, E., Shen, L., Turner, J.R., and Montrose, M.H. 2011. Redistribution of the tight junction protein ZO‐1 during physiological shedding of mouse intestinal epithelial cells. Am. J. Physiol. Cell. Physiol 300:C1404‐C1414.
  Jung, P., Sato, T., Merlos‐Suarez, A., Barriga, F.M., Iglesias, M., Rossell, D., Auer, H., Gallardo, M., Blasco, M.A., Sancho, E., Clevers, H., and Batlle, E. 2011. Isolation and in vitro expansion of human colonic stem cells. Nature Med. 17:1225‐1227.
  Levin, D.E., Sala, F.G., Barthel, E.R., Speer, A.L., Hou, X., Torashima, Y., and Grikscheit, T.C. 2013. A “living bioreactor” for the production of tissue‐engineered small intestine. Methods Mol Biol. 1001:299‐309.
  Liu, J., Walker, N.M., Cook, M.T., Ootani, A., and Clarke, L.L. 2012. Functional Cftr in crypt epithelium of organotypic enteroid cultures from murine small intestine. Am. J. Physiol. Cell Physiol. 302:C1492‐C1503.
  Miyoshi, H., Ajima, R., Luo, C.T., Yamaguchi, T.P., and Stappenbeck, T.S. 2012. Wnt5a potentiates TGF‐beta signaling to promote colonic crypt regeneration after tissue injury. Science 338:108‐113.
  Mizutani, T., Nakamura, T., Morikawa, R., Fukuda, M., Mochizuki, W., Yamauchi, Y., Nozaki, K., Yui, S., Nemoto, Y., Nagaishi, T., Okamoto, R., Tsuchiya, K., and Watanabe, M. 2012. Real‐time analysis of P‐glycoprotein‐mediated drug transport across primary intestinal epithelium three‐dimensionally cultured in vitro. Biochem. Biophys. Res. Commun. 419:238‐243.
  Noah, T.K., Donahue, B., and Shroyer, N.F. 2011. Intestinal development and differentiation. Exp. Cell Res. 317:2702‐2710.
  Ootani, A., Li, X., Sangiorgi, E., Ho, Q.T., Ueno, H., Toda, S., Sugihara, H., Fujimoto, K., Weissman, I.L., Capecchi, M.R., and Kuo, C.J. 2009. Sustained in vitro intestinal epithelial culture within a Wnt‐dependent stem cell niche. Nat. Med. 15:701‐706.
  Ramalingam, S., Daughtridge, G.W., Johnston, M.J., Gracz, A.D., and Magness, S.T. 2012. Distinct levels of Sox9 expression mark colon epithelial stem cells that form colonoids in culture. Am. J. Physiol. Gastrointest. Liver Physiol. 302:G10‐G20.
  Rothenberg, M.E., Nusse, Y., Kalisky, T., Lee, J.J., Dalerba, P., Scheeren, F., Lobo, N., Kulkarni, S., Sim, S., Qian, D., Beachy, P.A., Pasricha, P.J., Quake, S.R., and Clarke, M.F. 2012. Identification of a cKit(+) colonic crypt base secretory cell that supports Lgr5(+) stem cells in mice. Gastroenterology 142:1195‐1205.
  Sandell, A. and Sakai, D. 2011. Mammalian cell culture. Curr. Protoc. Essen. Lab. Tech. 5:4.3.1‐4.3.32.
  Sato, T., Vries, R.G., Snippert, H.J., van de Wetering, M., Barker, N., Stange, D.E., van Es, J.H., Abo, A., Kujala, P., Peters, P.J., and Clevers, H. 2009. Single Lgr5 stem cells build crypt‐villus structures in vitro without a mesenchymal niche. Nature 459:262‐265.
  Sato, T., Stange, D.E., Ferrante, M., Vries, R.G., Van Es, J.H., Van den Brink, S., Van Houdt, W.J., Pronk, A., Van Gorp, J., Siersema, P.D., and Clevers, H. 2011. Long‐term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141:1762‐1772.
  Simon‐Assmann, P., Turck, N., Sidhoum‐Jenny, M., Gradwohl, G., and Kedinger, M. 2007. In vitro models of intestinal epithelial cell differentiation. Cell Biol. Toxicol. 23:241‐256.
  Spence, J.R., Mayhew, C.N., Rankin, S.A., Kuhar, M.F., Vallance, J.E., Tolle, K., Hoskins, E.E., Kalinichenko, V.V., Wells, S.I., Zorn, A.M., Shroyer, N.F., and Wells, J.M. 2011. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470:105‐109.
  Stelzner, M., Helmrath, M., Dunn, J.C., Henning, S.J., Houchen, C.W., Kuo, C., Lynch, J., Li, L., Magness, S.T., Martin, M.G., Wong, M.H., and Yu, J. 2012. A nomenclature for intestinal in vitro cultures. Am. J. Physiol. Gastrointest. Liver Physiol. 302:G1359‐G1363.
  Tait, I.S., Flint, N., Campbell, F.C., and Evans, G.S. 1994. Generation of neomucosa in vivo by transplantation of dissociated rat postnatal small intestinal epithelium. Differentiation 56:91‐100.
  van Es, J.H., Sato, T., van de Wetering, M., Lyubimova, A., Nee, A.N., Gregorieff, A., Sasaki, N., Zeinstra, L., van den Born, M., Korving, J., Martens, A.C., Barker, N., van Oudenaarden, A., and Clevers, H. 2012. Dll1+ secretory progenitor cells revert to stem cells upon crypt damage. Nat. Cell Biol. 14:1099‐1104.
  von Furstenberg, R.J., Gulati, A.S., Baxi, A., Doherty, J.M., Stappenbeck, T.S., Gracz, A.D., Magness, S.T., and Henning, S.J. 2011. Sorting mouse jejunal epithelial cells with CD24 yields a population with characteristics of intestinal stem cells. Am. J. Physiol. Gastrointest. Liver Physiol. 300:G409‐G417.
  Wang, F., Scoville, D., He, X.C., Mahe, M., Box, A., Perry, J., Smith, N.R., Lei Nanye, N., Davies, P.S., Fuller, M.K., Haug, J.S., McClain, M., Gracz, A.D., Ding, S., Stelzner, M., Dunn, J.C., Magness, S.T., Wong, M.H., Martin, M., Helmrath, M., and Li, L. 2013. Isolation and characterization of intestinal stem cells based on surface marker combinations and colony‐formation assay. Gastroenterology. 145:383‐395.
  Yan, K.S., Chia, L.A., Li, X., Ootani, A., Su, J., Lee, J.Y., Su, N., Luo, Y., Heilshorn, S.C., Amieva, M.R., Sangiorgi, E., Capecchi, M.R., and Kuo, C.J. 2012. The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations. Proc. Natl. Acad. Sci. U.S.A. 109:466‐471.
  Yui, S., Nakamura, T., Sato, T., Nemoto, Y., Mizutani, T., Zheng, X., Ichinose, S., Nagaishi, T., Okamoto, R., Tsuchiya, K., Clevers, H., and Watanabe, M. 2012. Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5(+) stem cell. Nat. Med. 18:618‐623.
Key References
  Barker et al., 2010. See above.
  This paper describes, for the first time, the establishment of gastric epithelial organoids (gastroids).
  Sato et al., 2009. See above.
  The authors developed the conditions for a long‐term culture of intestinal crypt‐derived enteroids as well as the establishment of single Lgr5+ve+ve cell‐derived enteroids. Methods described in this article are based on this paper.
  Sato et al., 2011. See above.
  In this study, colonic crypt‐derived colonoids are generated based on the method developed by Sato et al. in 2009.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library