Analysis of Cell Death Induction in Intestinal Organoids In Vitro

  • Thomas Grabinger
  • Eugenia Delgado
  • Thomas Brunner
Part of the Methods in Molecular Biology book series (MIMB, volume 1419)


The intestinal epithelium has an important function in the absorption of nutrients contained in the food. Furthermore, it also has an important barrier function, preventing luminal pathogens from entering the bloodstream. This single cell layer epithelium is quite sensitive to various cell death-promoting triggers, including drugs, irradiation, and TNF family members, leading to loss of barrier integrity, epithelial erosion, inflammation, malabsorption, and diarrhea. In order to assess the intestinal epithelium-damaging potential of treatments and substances specific test systems are required. As intestinal tumor cell lines are a poor substitute for primary intestinal epithelial cells, and in vivo experiments in mice are costly and often unethical, the use of intestinal organoids cultured from intestinal crypts provide an ideal tool to study cell death induction and mechanisms in primary intestinal epithelial cells. This protocol describes the isolation and culture of intestinal organoids from murine small intestinal crypts, and the quantitative assessment of cell death induction in these organoids.

Key words

Apoptosis Intestinal epithelial cells Crypts TNFα Chemotherapy MTT Irradiation Organoids Enteroids 



This work was supported by Research Grants from the German Science Foundation to TB. Thomas Grabinger received a fellowship from the RTG 1331 graduate school (supported by the German Science Foundation).


  1. 1.
    Kato T, Owen RL (2004) Structure and function of the intestinal mucosal epithelium. In: Mestecky J, Lamm ME, Strober W, Bienenstock J, McGhee JR, Mayer L (eds) Mucosal immunology, vol 1. Elsevier, San Diego, pp 131–151Google Scholar
  2. 2.
    Cheroutre H, Lambolez F, Mucida D (2011) The light and dark sides of intestinal intraepithelial lymphocytes. Nat Rev Immunol 11(7):445–456CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Stappenbeck TS, Wong MH, Saam JR, Mysorekar IU, Gordon JI (1998) Notes from some crypt watchers: regulation of renewal in the mouse intestinal epithelium. Curr Opin Cell Biol 10(6):702–709CrossRefPubMedGoogle Scholar
  4. 4.
    Clevers H (2013) The intestinal crypt, a prototype stem cell compartment. Cell 154(2):274–284CrossRefPubMedGoogle Scholar
  5. 5.
    Bowen JM, Gibson RJ, Cummins AG, Keefe DM (2006) Intestinal mucositis: the role of the Bcl-2 family, p53 and caspases in chemotherapy-induced damage. Support Care Cancer 14(7):713–731CrossRefPubMedGoogle Scholar
  6. 6.
    Piguet PF, Vesin C, Guo J, Donati Y, Barazzone C (1998) TNF-induced enterocyte apoptosis in mice is mediated by the TNF receptor 1 and does not require p53. Eur J Immunol 28(11):3499–3505CrossRefPubMedGoogle Scholar
  7. 7.
    Piguet PF, Vesin C, Donati Y, Barazzone C (1999) TNF-induced enterocyte apoptosis and detachment in mice: induction of caspases and prevention by a caspase inhibitor, ZVAD-fmk. Lab Invest 79(4):495–500PubMedGoogle Scholar
  8. 8.
    Grabinger T, Luks L, Kostadinova F, Zimberlin C, Medema JP, Leist M, Brunner T (2014) Ex vivo culture of intestinal crypt organoids as a model system for assessing cell death induction in intestinal epithelial cells and enteropathy. Cell Death Dis 5, e1228CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Lin T, Brunner T, Tietz B, Madsen J, Bonfoco E, Reaves M, Huflejt M, Green DR (1998) Fas ligand- mediated killing by intestinal intraepithelial lymphocytes. Participation in intestinal graft-versus-host disease. J Clin Invest 101(3):570–577CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Dagenais M, Douglas T, Saleh M (2014) Role of programmed necrosis and cell death in intestinal inflammation. Curr Opin Gastroenterol 30(6):566–575CrossRefPubMedGoogle Scholar
  11. 11.
    Brunner T, Mueller C (2003) Apoptosis in disease: about shortage and excess. Essays Biochem 39:119–130CrossRefPubMedGoogle Scholar
  12. 12.
    Miyoshi H, Stappenbeck TS (2013) In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nat Protoc 8(12):2471–2482CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Sato T, Clevers H (2013) Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340(6137):1190–1194CrossRefPubMedGoogle Scholar
  14. 14.
    Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, van Es JH, Abo A, Kujala P, Peters PJ, Clevers H (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459(7244):262–265CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Thomas Grabinger
    • 1
  • Eugenia Delgado
    • 1
  • Thomas Brunner
    • 1
  1. 1.Biochemical PharmacologyUniversity of KonstanzKonstanzGermany

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