3D Cell Culture Models of Epithelial Tissues

  • Kai ZhangEmail author
  • Aki ManninenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1926)


Cells in tissues in vivo face a very different microenvironment than typical cultured cells plated on a plastic dish. Already several decades ago, cell biologists observed that cell lines show dramatically different morphology and growth characteristics when embedded into three-dimensional (3D) substrates or standard tissue culture plates (Montesano R, Schaller G, Orci L, Cell. 66:697–711, 1991; Barcellos-Hoff MH, Aggeler J, Ram TG, Bissell MJ, Development. 105:223–235, 1989; Simian M, Bissell MJ, J Cell Biol. 216:31–40, 2017). Despite its imminent benefit for cell biological studies, suspicion and prejudice toward more complicated sample preparation requirements limited the popularity of 3D culture techniques until recently, when it was shown that soft 3D gels made of basement membrane extracts (BME) allow prolonged culture of many types of primary epithelial cells (Clevers H, Cell. 165:1586–1597, 2016; Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, et al., Nature. 459:262–265, 2009). These observations have brought 3D organoid culture systems into the mainstream. Here we describe two protocols for culturing epithelial cells in 3D substrates, the “blob culture” setup where cells are fully embedded into BME gel and the “overlay setup” where cells are seeded on top of BME gel and then overlaid with a thin layer of BME (Debnath J, Brugge JS, Nat Rev Cancer. 5:675–688, 2005; Bryant DM, Datta A, Rodriguez-Fraticelli AE, Peranen J, Martin-Belmonte F, Mostov KE, Nat Cell Biol. 12:1035–1045, 2010).

Key words

MDCK 3D culture Organoid 



Riitta Jokela is acknowledged for overall expert technical assistance, Jaana Träskelin for expert technical assistance at Biocenter Oulu Virus Core Laboratory, and Veli-Pekka Ronkainen for expert assistance in microscopy at Biocenter Oulu Tissue Imaging Center. This work was funded by the Academy of Finland (251314, 135560, 263770, and 140974/AM).


  1. 1.
    Montesano R, Schaller G, Orci L (1991) Induction of epithelial tubular morphogenesis in vitro by fibroblast-derived soluble factors. Cell 66:697–711CrossRefGoogle Scholar
  2. 2.
    Barcellos-Hoff MH, Aggeler J, Ram TG, Bissell MJ (1989) Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane. Development 105:223–235PubMedPubMedCentralGoogle Scholar
  3. 3.
    Debnath J, Brugge JS (2005) Modelling glandular epithelial cancers in three-dimensional cultures. Nat Rev Cancer 5:675–688CrossRefGoogle Scholar
  4. 4.
    Bryant DM, Datta A, Rodriguez-Fraticelli AE, Peranen J, Martin-Belmonte F, Mostov KE (2010) A molecular network for de novo generation of the apical surface and lumen. Nat Cell Biol 12:1035–1045CrossRefGoogle Scholar
  5. 5.
    Simian M, Bissell MJ (2017) Organoids: a historical perspective of thinking in three dimensions. J Cell Biol 216:31–40CrossRefGoogle Scholar
  6. 6.
    Manninen A (2015) Epithelial polarity—generating and integrating signals from the ECM with integrins. Exp Cell Res 334:337–349CrossRefGoogle Scholar
  7. 7.
    Clevers H (2016) Modeling development and disease with organoids. Cell 165:1586–1597CrossRefGoogle Scholar
  8. 8.
    Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE et al (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459:262–265CrossRefGoogle Scholar
  9. 9.
    Myllymäki SM, Teräväinen TP, Manninen A (2011) Two distinct integrin-mediated mechanisms contribute to apical lumen formation in epithelial cells. PLoS One 6:e19453CrossRefGoogle Scholar
  10. 10.
    Zhang K, Myllymaki SM, Gao P, Devarajan R, Kytola V, Nykter M et al (2017) Oncogenic K-Ras upregulates ITGA6 expression via FOSL1 to induce anoikis resistance and synergizes with αV-class integrins to promote EMT. Oncogene 36:5681–5694CrossRefGoogle Scholar
  11. 11.
    Welm BE, Dijkgraaf GJ, Bledau AS, Welm AL, Werb Z (2008) Lentiviral transduction of mammary stem cells for analysis of gene function during development and cancer. Cell Stem Cell 2:90–10CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Center for Cellular Immunotherapies, Department of Pathology and Laboratory Medicine, Perelman School of MedicineUniversity of PennsylvaniaPhiladelphiaUSA
  2. 2.Faculty of Biochemistry and Molecular Medicine, Biocenter Oulu, Oulu Center for Cell-Matrix ResearchUniversity of OuluOuluFinland

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