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pp 1-11 | Cite as

Isolation and Culture of Adult Intestinal, Gastric, and Liver Organoids for Cre-recombinase-Mediated Gene Deletion

  • Dustin J. Flanagan
  • Renate H. M. Schwab
  • Bang M. Tran
  • Toby J. Phesse
  • Elizabeth VincanEmail author
Protocol
Part of the Methods in Molecular Biology book series

Abstract

The discovery of Lgr5 as a marker of adult stem cells meant that stem cell populations could be purified and studied in isolation. Importantly, when cultured under the appropriate conditions these stem cells form organoids in tissue culture that retain many features of the tissue of origin. The organoid cultures are accessible to genetic and biochemical manipulation, bridging the gap between in vivo mouse models and conventional tissue culture. Here we describe robust protocols to establish organoids from gastrointestinal tissues (stomach, intestine, liver) and Cre-recombinase mediated gene manipulation in vitro.

Keywords:

Intestinal organoids Gastric organoids Liver organoids Cre-recombinase 3D organoid culture Lgr5 

References

  1. 1.
    Pampaloni F, Reynaud EG, Stelzer EH (2007) The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol 8(10):839–845CrossRefPubMedGoogle Scholar
  2. 2.
    Barker N, Bartfeld S, Clevers H (2010) Tissue-resident adult stem cell populations of rapidly self-renewing organs. Cell Stem Cell 7(6):656–670CrossRefPubMedGoogle Scholar
  3. 3.
    Barker N et al (2007) Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449(7165):1003–1007ADSCrossRefPubMedGoogle Scholar
  4. 4.
    Barker N 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(1):25–36MathSciNetCrossRefPubMedGoogle Scholar
  5. 5.
    Huch M et al (2013) In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494(7436):247–250ADSCrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Sato T et al (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459(7244):262–265ADSCrossRefPubMedGoogle Scholar
  7. 7.
    Flanagan DJ et al (2015) Frizzled7 functions as a Wnt receptor in intestinal epithelial Lgr5(+) stem cells. Stem Cell Reports 4(5):759–767CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Huch M et al (2015) Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 160(1–2):299–312CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Dustin J. Flanagan
    • 1
    • 2
  • Renate H. M. Schwab
    • 1
    • 2
  • Bang M. Tran
    • 1
    • 2
  • Toby J. Phesse
    • 1
    • 2
  • Elizabeth Vincan
    • 1
    • 2
    • 3
    Email author
  1. 1.Molecular Oncology LaboratoryUniversity of MelbourneMelbourneAustralia
  2. 2.Victorian Infectious Diseases Reference LaboratoryDoherty InstituteMelbourneAustralia
  3. 3.School of Biomedical SciencesCurtin UniversityPerthAustralia

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