Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Three-dimensional organotypic culture: experimental models of mammalian biology and disease

Nature Reviews Molecular Cell Biology volume 15, pages 647–664 (2014)Cite this article

Subjects

Key Points

  • Three-dimensional (3D) culture protocols have been developed for diverse tissues, organs and disease states.

  • 3D culture enables imaging of mammalian organogenesis at the cellular level.

  • Genetic manipulation within 3D cultures can resolve the cellular and molecular basis of tissue-level phenotypes.

  • 3D culture enables the independent evaluation of how distinct features of the microenvironment regulate organogenesis and disease.

  • Induced pluripotent stem (iPS) cell-derived 3D cultures enable the generation and study of tissues derived from the somatic cells of a patient.

  • 3D culture is a natural point of integration for fundamental, translational and clinical research.

Abstract

Mammalian organs are challenging to study as they are fairly inaccessible to experimental manipulation and optical observation. Recent advances in three-dimensional (3D) culture techniques, coupled with the ability to independently manipulate genetic and microenvironmental factors, have enabled the real-time study of mammalian tissues. These systems have been used to visualize the cellular basis of epithelial morphogenesis, to test the roles of specific genes in regulating cell behaviours within epithelial tissues and to elucidate the contribution of microenvironmental factors to normal and disease processes. Collectively, these novel models can be used to answer fundamental biological questions and generate replacement human tissues, and they enable testing of novel therapeutic approaches, often using patient-derived cells.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Cellular inputs to organotypic cultures.
Figure 2: The major categories of cell culture.
Figure 3: The cellular basis of epithelial tube elongation.
Figure 4: Genetic regulation of cell behaviours in mammalian tissues.
Figure 5: The role of the microenvironment in regulating epithelial function.

Similar content being viewed by others

References

  1. Bichat, X. General Anatomy, Applied to Physiology and Medicine. (Richardson and Lord, 1822).

    Google Scholar 

  2. Virchow, R. Cellular Pathology, as Based upon Physiological and Pathological Histology. Twenty Lectures Delivered in the Pathological Institute of Berlin During the Months of February, March and April, 1858. (R. M. De Witt, 1860).

    Book  Google Scholar 

  3. Sobotta, J., Huber, G. C. & De Witt, L. M. B. Atlas and Epitome of Human Histology and Microscopic Anatomy. (W. B. Saunders & company, 1903).

    Book  Google Scholar 

  4. Harrison, R. G., Greenman, M. J., Mall, F. P. & Jackson, C. M. Observations on the living developing nerve fiber. Anat. Rec. 1, 116–128 (1907).

    Article  Google Scholar 

  5. Simian, M. et al. The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells. Development 128, 3117–3131 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Sato, T. et al. Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

    Article  CAS  PubMed  Google Scholar 

  7. Finkbeiner, S. R. & Spence, J. R. A gutsy task: generating intestinal tissue from human pluripotent stem cells. Dig. Dis. Sci. 58, 1176–1184 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Alberts, B. Molecular Biology of the Cell. 4th edn Ch. 19 (Garland Science, 2002).

    Google Scholar 

  9. Nelson, C. M. & Bissell, M. J. Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. Annu. Rev. Cell Dev. Biol. 22, 287–309 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Baranski, J. D. et al. Geometric control of vascular networks to enhance engineered tissue integration and function. Proc. Natl Acad. Sci. USA 110, 7586–7591 (2013).

    Article  CAS  PubMed  Google Scholar 

  11. Zheng, Y. et al. In vitro microvessels for the study of angiogenesis and thrombosis. Proc. Natl Acad. Sci. USA 109, 9342–9347 (2012).

    Article  CAS  PubMed  Google Scholar 

  12. Nguyen, D.-H. T. et al. Biomimetic model to reconstitute angiogenic sprouting morphogenesis in vitro. Proc. Natl Acad. Sci. USA 110, 6712–6717 (2013). Introduces a 3D in vitro model of angiogenic sprouting from preformed vessels to define the morphogenetic and molecular requirements for neovascularization.

    Article  CAS  PubMed  Google Scholar 

  13. Fell, H. B. & Robison, R. The growth, development and phosphatase activity of embryonic avian femora and limb-buds cultivated in vitro. Biochem. J. 23, 767–784 (1929).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Chen, J. M. The cultivation in fluid medium of organised liver, pancreas and other tissues of foetal rats. Exp. Cell Res. 7, 518–529 (1954).

    Article  CAS  PubMed  Google Scholar 

  15. Ichinose, R. R. & Nandi, S. Lobuloalveolar differentiation in mouse mammary tissues in vitro. Science 145, 496–497 (1964).

    Article  CAS  PubMed  Google Scholar 

  16. Waymouth, C. in Biology of the Laboratory Mouse (ed. Green, Earl L.) (Dover Publications, 1966).

    Google Scholar 

  17. Guerrero, R. R., Rounds, D. E. & Booher, J. An improved organ culture method for adult mammalian lung. In Vitro 13, 517–524 (1977).

    Article  CAS  PubMed  Google Scholar 

  18. Browning, T. H. & Trier, J. S. Organ culture of mucosal biopsies of human small intestine. J. Clin. Invest. 48, 1423–1432 (1969).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Randall, K. J., Turton, J. & Foster, J. R. Explant culture of gastrointestinal tissue: a review of methods and applications. Cell Biol. Toxicol. 27, 267–284 (2011).

    Article  PubMed  Google Scholar 

  20. Autrup, H. et al. Explant culture of rat colon: a model system for studying metabolism of chemical carcinogens. In Vitro 14, 868–877 (1978).

    Article  CAS  PubMed  Google Scholar 

  21. Stoppini, L., Buchs, P. A. & Muller, D. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37, 173–182 (1991).

    Article  CAS  PubMed  Google Scholar 

  22. Gähwiler, B. H., Capogna, M., Debanne, D., McKinney, R. A. & Thompson, S. M. Organotypic slice cultures: a technique has come of age. Trends Neurosci. 20, 471–477 (1997).

    Article  PubMed  Google Scholar 

  23. Aplin, A. C., Fogel, E., Zorzi, P. & Nicosia, R. F. The aortic ring model of angiogenesis. Methods Enzymol. 443, 119–136 (2008).

    Article  CAS  PubMed  Google Scholar 

  24. Topper, R. J., Oka, T. & Vonderhaar, B. K. Techniques for studying development of normal mammary epithelial cells in organ culture. Methods Enzymol. 39, 443–454 (1975).

    Article  CAS  PubMed  Google Scholar 

  25. Hardman, P., Klement, B. J. & Spooner, B. S. Growth and morphogenesis of embryonic mouse organs on non-coated and extracellular matrix-coated Biopore membrane. Dev. Growth Differ. 35, 683–690 (1993).

    Article  CAS  PubMed  Google Scholar 

  26. Trott, J. F., Vonderhaar, B. K. & Hovey, R. C. Historical perspectives of prolactin and growth hormone as mammogens, lactogens and galactagogues — agog for the future! J. Mammary Gland Biol. Neoplasia 13, 3–11 (2008).

    Article  PubMed  Google Scholar 

  27. Shamir, E. R. et al. Twist1-induced dissemination preserves epithelial identity and requires E-cadherin. J. Cell Biol. 204, 839–856 (2014). Demonstrates, using genetic manipulation of primary normal mammary tissue, that epithelial cells can disseminate while retaining epithelial-specific proteins and gene expression. Shows that E-cadherin is required for efficient single-cell dissemination.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Koo, B. K. et al. Controlled gene expression in primary Lgr5 organoid cultures. Nature Methods 9, 81–83 (2011).

    Article  CAS  PubMed  Google Scholar 

  29. Onodera, T. et al. Btbd7 regulates epithelial cell dynamics and branching morphogenesis. Science 329, 562–565 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Cheung, K. J., Gabrielson, E., Werb, Z. & Ewald, A. J. Collective invasion in breast cancer requires a conserved basal epithelial program. Cell 155, 1639–1651 (2013). Uses organotypic culture of primary tumour organoids to identify a common subpopulation of cells that leads collective invasion across distinct breast cancer subtypes.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Daley, W. P., Gulfo, K. M., Sequeira, S. J. & Larsen, M. Identification of a mechanochemical checkpoint and negative feedback loop regulating branching morphogenesis. Dev. Biol. 336, 169–182 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Fata, J. E. et al. The MAPK(ERK-1,2) pathway integrates distinct and antagonistic signals from TGFα and FGF7 in morphogenesis of mouse mammary epithelium. Dev. Biol. 306, 193–207 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Steinberg, Z. et al. FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis. Development 132, 1223–1234 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. Zhang, X., Bush, K. T. & Nigam, S. K. In vitro culture of embryonic kidney rudiments and isolated ureteric buds. Methods Mol. Biol. 886, 13–21 (2012).

    Article  CAS  PubMed  Google Scholar 

  35. Liu, Y. et al. Novel role for Netrins in regulating epithelial behavior during lung branching morphogenesis. Curr. Biol. 14, 897–905 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Ootani, A. et al. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nature Med. 15, 701–706 (2009).

    Article  CAS  PubMed  Google Scholar 

  37. Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141, 1762–1772 (2011).

    Article  CAS  PubMed  Google Scholar 

  38. Huch, M. et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494, 247–250 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Barker, N. et al. Lgr5+ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6, 25–36 (2010).

    Article  CAS  PubMed  Google Scholar 

  40. Huch, M. et al. Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis. EMBO J. 32, 2708–2721 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Ghosh, S. et al. PI3K/mTOR signaling regulates prostatic branching morphogenesis. Dev. Biol. 360, 329–342 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kleinman, H. K. & Martin, G. R. Matrigel: basement membrane matrix with biological activity. Semin. Cancer Biol. 15, 378–386 (2005).

    Article  CAS  PubMed  Google Scholar 

  43. Wolf, K. et al. Collagen-based cell migration models in vitro and in vivo. Semin. Cell Dev. Biol. 20, 931–941 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Eiraku, M. et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472, 51–56 (2011).

    Article  CAS  PubMed  Google Scholar 

  45. Nakano, T. et al. Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell 10, 771–785 (2012). References 44 and 45 demonstrate that retinal development can be mostly recapitulated in vitro via the self-organization of ES cell-derived retinal epithelia.

    Article  CAS  PubMed  Google Scholar 

  46. Eiraku, M. et al. Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 6, 519–532 (2008).

    Article  CAS  Google Scholar 

  47. Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013). The first in vitro model of whole brain tissue, which was derived from human iPS cells, with discrete but interdependent brain domains.

    Article  CAS  PubMed  Google Scholar 

  48. Suga, H. et al. Self-formation of functional adenohypophysis in three-dimensional culture. Nature 480, 57–62 (2011).

    Article  CAS  PubMed  Google Scholar 

  49. Townes, P. L. & Holtfreter, J. Directed movements and selective adhesion of embryonic amphibian cells. J. Exp. Zool. 128, 53–120 (1955).

    Article  Google Scholar 

  50. O'Brien, L. E., Zegers, M. M. & Mostov, K. E. Building epithelial architecture: insights from three-dimensional culture models. Nature Rev. Mol. Cell Biol. 3, 531–537 (2002).

    Article  CAS  Google Scholar 

  51. Debnath, J., Muthuswamy, S. K. & Brugge, J. S. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30, 256–268 (2003).

    Article  CAS  PubMed  Google Scholar 

  52. Green, H., Kehinde, O. & Thomas, J. Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc. Natl Acad. Sci. USA 76, 5665–5668 (1979).

    Article  CAS  PubMed  Google Scholar 

  53. Fuchs, E. Epidermal differentiation: the bare essentials. J. Cell Biol. 111, 2807–2814 (1990).

    Article  CAS  PubMed  Google Scholar 

  54. Kalabis, J. et al. Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture. Nature Protoc. 7, 235–246 (2012).

    Article  CAS  Google Scholar 

  55. Unbekandt, M. & Davies, J. A. Dissociation of embryonic kidneys followed by reaggregation allows the formation of renal tissues. Kidney Int. 77, 407–416 (2009).

    Article  PubMed  Google Scholar 

  56. Ganeva, V., Unbekandt, M. & Davies, J. A. An improved kidney dissociation and reaggregation culture system results in nephrons arranged organotypically around a single collecting duct system. Organogenesis 7, 83–87 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Streuli, C. H., Bailey, N. & Bissell, M. J. Control of mammary epithelial differentiation: basement membrane induces tissue-specific gene expression in the absence of cell–cell interaction and morphological polarity. J. Cell Biol. 115, 1383–1395 (1991).

    Article  CAS  PubMed  Google Scholar 

  58. O'Brien, L. E. et al. Rac1 orientates epithelial apical polarity through effects on basolateral laminin assembly. Nature Cell Biol. 3, 831–838 (2001).

    Article  CAS  PubMed  Google Scholar 

  59. Yu, W. et al. Formation of cysts by alveolar type II cells in three-dimensional culture reveals a novel mechanism for epithelial morphogenesis. Mol. Biol. Cell 18, 1693–1700 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Greenburg, G. & Hay, E. D. Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells. J. Cell Biol. 95, 333–339 (1982).

    Article  CAS  PubMed  Google Scholar 

  61. Debnath, J. & Brugge, J. S. Modelling glandular epithelial cancers in three-dimensional cultures. Nature Rev. Cancer 5, 675–688 (2005).

    Article  CAS  Google Scholar 

  62. Ewald, A. J., Brenot, A., Duong, M., Chan, B. S. & Werb, Z. Collective epithelial migration and cell rearrangements drive mammary branching morphogenesis. Dev. Cell 14, 570–581 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Morita, K. & Nogawa, H. EGF-dependent lobule formation and FGF7-dependent stalk elongation in branching morphogenesis of mouse salivary epithelium in vitro. Dev. Dyn. 215, 148–154 (1999).

    Article  CAS  PubMed  Google Scholar 

  64. Qiao, J., Sakurai, H. & Nigam, S. K. Branching morphogenesis independent of mesenchymal–epithelial contact in the developing kidney. Proc. Natl Acad. Sci. USA 96, 7330–7335 (1999).

    Article  CAS  PubMed  Google Scholar 

  65. Wescott, M. P. et al. Pancreatic ductal morphogenesis and the Pdx1 homeodomain transcription factor. Mol. Biol. Cell 20, 4838–4844 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Nguyen-Ngoc, K.-V. et al. ECM microenvironment regulates collective migration and local dissemination in normal and malignant mammary epithelium. Proc. Natl Acad. Sci. USA 109, E2595–E2604 (2012). Demonstrates that the composition of the ECM determines the migration strategy and disseminative behaviour of both normal and tumour mammary organoids and can regulate the phenotypic consequences of molecular perturbations.

    Article  CAS  PubMed  Google Scholar 

  67. Nguyen-Ngoc, K. V. & Ewald, A. J. Mammary ductal elongation and myoepithelial migration are regulated by the composition of the extracellular matrix. J. Microsc. 251, 212–223 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Brownfield, D. G. et al. Patterned collagen fibers orient branching mammary epithelium through distinct signaling modules. Curr. Biol. 23, 703–709 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Levental, K. R. et al. Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell 139, 891–906 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Provenzano, P. P. et al. Collagen reorganization at the tumor–stromal interface facilitates local invasion. BMC Med. 4, 38 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Provenzano, P. P. et al. Collagen density promotes mammary tumor initiation and progression. BMC Med. 6, 11 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Ewald, A. J. Practical considerations for long-term time-lapse imaging of epithelial morphogenesis in three-dimensional organotypic cultures. Cold Spring Harb. Protoc. 2013, 100–117 (2013).

    PubMed  Google Scholar 

  73. Ridky, T. W., Chow, J. M., Wong, D. J. & Khavari, P. A. Invasive three-dimensional organotypic neoplasia from multiple normal human epithelia. Nature Med. 16, 1450–1455 (2010).

    Article  CAS  PubMed  Google Scholar 

  74. Baker, B. M. & Chen, C. S. Deconstructing the third dimension: how 3D culture microenvironments alter cellular cues. J. Cell Sci. 125, 3015–3024 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Dvir, T., Timko, B. P., Kohane, D. S. & Langer, R. Nanotechnological strategies for engineering complex tissues. Nature Nanotechnol. 6, 13–22 (2010).

    Article  CAS  Google Scholar 

  76. Singh, A. & Elisseeff, J. Biomaterials for stem cell differentiation. J. Mater. Chem. 20, 8832–8847 (2010).

    Article  CAS  Google Scholar 

  77. Young, E. W. & Beebe, D. J. Fundamentals of microfluidic cell culture in controlled microenvironments. Chem. Soc. Rev. 39, 1036–1048 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Huh, D. et al. Reconstituting organ-level lung functions on a chip. Science 328, 1662–1668 (2010). Introduces the concept of using microfabrication and microfluidics to construct biomimetic microsystems with tissue–tissue interfaces, efficient nutrient delivery, mechanical integrity and organ functionality.

    Article  CAS  PubMed  Google Scholar 

  79. Stroock, A. D. & Fischbach, C. Microfluidic culture models of tumor angiogenesis. Tissue Engineer. Part A 16, 2143–2146 (2010).

    Article  Google Scholar 

  80. Gartner, Z. J. & Bertozzi, C. R. Programmed assembly of 3-dimensional microtissues with defined cellular connectivity. Proc. Natl Acad. Sci. USA 106, 4606–4610 (2009).

    Article  CAS  PubMed  Google Scholar 

  81. Varner, V. D. & Nelson, C. M. Let's push things forward: disruptive technologies and the mechanics of tissue assembly. Integr. Biol. 5, 1162–1173 (2013).

    Article  CAS  Google Scholar 

  82. Huebner, R. J., Lechler, T. & Ewald, A. J. Developmental stratification of the mammary epithelium occurs through symmetry-breaking vertical divisions of apically positioned luminal cells. Development 141, 1085–1094 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Larsen, M. et al. Role of PI 3-kinase and PIP3 in submandibular gland branching morphogenesis. Dev. Biol. 255, 178–191 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Puri, S. & Hebrok, M. Dynamics of embryonic pancreas development using real-time imaging. Dev. Biol. 306, 82–93 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Kim, H. Y., Varner, V. D. & Nelson, C. M. Apical constriction initiates new bud formation during monopodial branching of the embryonic chicken lung. Development 140, 3146–3155 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Provenzano, P. P. et al. Nonlinear optical imaging of cellular processes in breast cancer. Microsc. Microanal. 14, 532–548 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Underwood, J. M. et al. The ultrastructure of MCF-10A acini. J. Cell. Physiol. 208, 141–148 (2006).

    Article  CAS  PubMed  Google Scholar 

  88. Ewald, A. J. et al. Mammary collective cell migration involves transient loss of epithelial features and individual cell migration within the epithelium. J. Cell Sci. 125, 2638–2654 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Grugan, K. D. et al. Fibroblast-secreted hepatocyte growth factor plays a functional role in esophageal squamous cell carcinoma invasion. Proc. Natl Acad. Sci. USA 107, 11026–11031 (2010). Uses organotypic culture and independent genetic manipulation of epithelial and stromal compartments to implicate fibroblast-secreted HGF and its epithelial receptor MET in the invasion of transformed oesophageal epithelial cells.

    Article  CAS  PubMed  Google Scholar 

  90. Ghabrial, A. S. & Krasnow, M. A. Social interactions among epithelial cells during tracheal branching morphogenesis. Nature 441, 746–749 (2006).

    Article  CAS  PubMed  Google Scholar 

  91. Lu, P. & Werb, Z. Patterning mechanisms of branched organs. Science 322, 1506–1509 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Larsen, M., Wei, C. & Yamada, K. M. Cell and fibronectin dynamics during branching morphogenesis. J. Cell Sci. 119, 3376–3384 (2006).

    Article  CAS  PubMed  Google Scholar 

  93. Shakya, R., Watanabe, T. & Costantini, F. The role of GDNF/Ret signaling in ureteric bud cell fate and branching morphogenesis. Dev. Cell 8, 65–74 (2005).

    Article  CAS  PubMed  Google Scholar 

  94. Chi, X. et al. Ret-dependent cell rearrangements in the Wolffian duct epithelium initiate ureteric bud morphogenesis. Dev. Cell 17, 199–209 (2009). Demonstrates, using an elegant series of chimeric embryonic kidneys, that levels of RET signalling dictate cellular contribution to the ureteric bud tip domain.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Patel, V. N. et al. Specific heparan sulfate structures modulate FGF10-mediated submandibular gland epithelial morphogenesis and differentiation. J. Biol. Chem. 283, 9308–9317 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Packard, A. et al. Luminal mitosis drives epithelial cell dispersal within the branching ureteric bud. Dev. Cell 27, 319–330 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Schnatwinkel, C. & Niswander, L. Multiparametric image analysis of lung-branching morphogenesis. Dev. Dyn. 242, 622–637 (2013).

    Article  CAS  PubMed  Google Scholar 

  98. Cukierman, E., Pankov, R., Stevens, D. R. & Yamada, K. M. Taking cell-matrix adhesions to the third dimension. Science 294, 1708–1712 (2001).

    Article  CAS  PubMed  Google Scholar 

  99. Hsu, J. C. et al. Viral gene transfer to developing mouse salivary glands. J. Dent. Res. 91, 197–202 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Sequeira, S. J., Gervais, E. M., Ray, S. & Larsen, M. Genetic modification and recombination of salivary gland organ cultures. J. Vis. Exp. 28, e50060 (2013).

    Google Scholar 

  101. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Schwank, G. et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13, 653–658 (2013). Demonstrates that CRISPR–Cas9 and organoid culture can be coupled to correct disease mutations in patient-derived cells and assay for restored tissue function.

    Article  CAS  PubMed  Google Scholar 

  103. Yu, W. et al. β1-integrin orients epithelial polarity via Rac1 and laminin. Mol. Biol. Cell 16, 433–445 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Martin-Belmonte, F. et al. Cell-polarity dynamics controls the mechanism of lumen formation in epithelial morphogenesis. Curr. Biol. 18, 507–513 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Bryant, D. M. et al. A molecular network for de novo generation of the apical surface and lumen. Nature Cell Biol. 12, 1035–1045 (2010).

    Article  CAS  PubMed  Google Scholar 

  106. Gálvez-Santisteban, M. et al. Synaptotagmin-like proteins control the formation of a single apical membrane domain in epithelial cells. Nature Cell Biol. 14, 838–849 (2012).

    Article  CAS  PubMed  Google Scholar 

  107. Muthuswamy, S. K., Li, D., Lelievre, S., Bissell, M. J. & Brugge, J. S. ErbB2, but not ErbB1, reinitiates proliferation and induces luminal repopulation in epithelial acini. Nature Cell Biol. 3, 785–792 (2001).

    Article  CAS  PubMed  Google Scholar 

  108. Aranda, V. et al. Par6–aPKC uncouples ErbB2 induced disruption of polarized epithelial organization from proliferation control. Nature Cell Biol. 8, 1235–1245 (2006).

    Article  CAS  PubMed  Google Scholar 

  109. Zhan, L. et al. Deregulation of scribble promotes mammary tumorigenesis and reveals a role for cell polarity in carcinoma. Cell 135, 865–878 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. Xue, B., Krishnamurthy, K., Allred, D. C. & Muthuswamy, S. K. Loss of Par3 promotes breast cancer metastasis by compromising cell–cell cohesion. Nature Cell Biol. 15, 1–14 (2013).

    Article  CAS  Google Scholar 

  111. Leung, C. T. & Brugge, J. S. Outgrowth of single oncogene-expressing cells from suppressive epithelial environments. Nature 482, 410–413 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Sakurai, A., Matsuda, M. & Kiyokawa, E. Activated Ras protein accelerates cell cycle progression to perturb Madin–Darby canine kidney cystogenesis. J. Biol. Chem. 287, 31703–31711 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Truong, A. B., Kretz, M., Ridky, T. W., Kimmel, R. & Khavari, P. A. p63 regulates proliferation and differentiation of developmentally mature keratinocytes. Genes Dev. 20, 3185–3197 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. Kretz, M. et al. Control of somatic tissue differentiation by the long non-coding RNA TINCR. Nature 493, 231–235 (2013).

    Article  CAS  Google Scholar 

  115. Sakai, T., Larsen, M. & Yamada, K. M. Fibronectin requirement in branching morphogenesis. Nature 423, 876–881 (2003).

    Article  CAS  PubMed  Google Scholar 

  116. Yates, L. L. et al. Scribble is required for normal epithelial cell–cell contacts and lumen morphogenesis in the mammalian lung. Dev. Biol. 373, 267–280 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. Liu, J. S., Farlow, J. T., Paulson, A. K., Labarge, M. A. & Gartner, Z. J. Programmed cell-to-cell variability in Ras activity triggers emergent behaviors during mammary epithelial morphogenesis. Cell Rep. 2, 1461–1470 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Plichta, K. A., Mathers, J. L., Gestl, S. A., Glick, A. B. & Gunther, E. J. Basal but not luminal mammary epithelial cells require PI3K/mTOR signaling for Ras-driven overgrowth. Cancer Res. 72, 5856–5866 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  119. Egeblad, M., Nakasone, E. S. & Werb, Z. Tumors as organs: complex tissues that interface with the entire organism. Dev. Cell 18, 884–901 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  120. Egeblad, M., Rasch, M. G. & Weaver, V. M. Dynamic interplay between the collagen scaffold and tumor evolution. Curr. Opin. Cell Biol. 22, 697–706 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. Beck, J. N., Singh, A., Rothenberg, A. R., Elisseeff, J. H. & Ewald, A. J. The independent roles of mechanical, structural and adhesion characteristics of 3D hydrogels on the regulation of cancer invasion and dissemination. Biomaterials 34, 9486–9495 (2013).

    Article  CAS  PubMed  Google Scholar 

  122. Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).

    Article  CAS  PubMed  Google Scholar 

  123. Condeelis, J. & Pollard, J. W. Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell 124, 263–266 (2006).

    Article  CAS  PubMed  Google Scholar 

  124. DeNardo, D. G. et al. CD4+ T cells regulate pulmonary metastasis of mammary carcinomas by enhancing protumor properties of macrophages. Cancer Cell 16, 91–102 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  125. Gaggioli, C. et al. Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nature Cell Biol. 9, 1392–1400 (2007).

    Article  CAS  PubMed  Google Scholar 

  126. Calvo, F. et al. Mechanotransduction and YAP-dependent matrix remodelling is required for the generation and maintenance of cancer-associated fibroblasts. Nature Cell Biol. 15, 637–646 (2013).

    Article  CAS  PubMed  Google Scholar 

  127. Okawa, T. et al. The functional interplay between EGFR overexpression, hTERT activation, and p53 mutation in esophageal epithelial cells with activation of stromal fibroblasts induces tumor development, invasion, and differentiation. Genes Dev. 21, 2788–2803 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  128. Lee, J.-H. et al. Lung stem cell differentiation in mice directed by endothelial cells via a BMP4–NFATc1–thrombospondin-1 axis. Cell 156, 440–455 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  129. Ghajar, C. M. et al. The perivascular niche regulates breast tumour dormancy. Nature Cell Biol. 15, 807–817 (2013). References 128 and 129 define a role for endothelial-derived TSP1 in regulating epithelial differentiation and tumour growth.

    Article  CAS  PubMed  Google Scholar 

  130. Infanger, D. W. et al. Glioblastoma stem cells are regulated by interleukin-8 signaling in a tumoral perivascular niche. Cancer Res. 73, 7079–7089 (2013).

    Article  CAS  PubMed  Google Scholar 

  131. Knox, S. M. et al. Parasympathetic innervation maintains epithelial progenitor cells during salivary organogenesis. Science 329, 1645–1647 (2010). Demonstrates, using a combination of 3D-embedded culture and whole-organ culture, that parasympathetic innervation maintains salivary epithelial progenitors and offers a therapeutic strategy for organ repair.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  132. Knox, S. M. et al. Parasympathetic stimulation improves epithelial organ regeneration. Nature Commun. 4, 1494 (2013).

    Article  CAS  Google Scholar 

  133. Marusyk, A., Almendro, V. & Polyak, K. Intra-tumour heterogeneity: a looking glass for cancer? Nature Rev. Cancer 12, 323–334 (2012).

    Article  CAS  Google Scholar 

  134. Carey, S. P., Starchenko, A., McGregor, A. L. & Reinhart-King, C. A. Leading malignant cells initiate collective epithelial cell invasion in a three-dimensional heterotypic tumor spheroid model. Clin. Exp. Metastasis 30, 615–630 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  135. Dang, T. T., Prechtl, A. M. & Pearson, G. W. Breast cancer subtype-specific interactions with the microenvironment dictate mechanisms of invasion. Cancer Res. 71, 6857–6866 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. Gudjonsson, T. et al. Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition. J. Cell Sci. 115, 39–50 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  137. Chanson, L. et al. Self-organization is a dynamic and lineage-intrinsic property of mammary epithelial cells. Proc. Natl Acad. Sci. USA 108, 3264–3269 (2011).

    Article  CAS  PubMed  Google Scholar 

  138. Conklin, M. W. et al. Aligned collagen is a prognostic signature for survival in human breast carcinoma. Am. J. Pathol. 178, 1221–1232 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  139. Paszek, M. J. et al. Tensional homeostasis and the malignant phenotype. Cancer Cell 8, 241–254 (2005).

    Article  CAS  PubMed  Google Scholar 

  140. Friedl, P. & Alexander, S. Cancer invasion and the microenvironment: plasticity and reciprocity. Cell 147, 992–1009 (2011).

    Article  CAS  PubMed  Google Scholar 

  141. Sia, S. K., Gillette, B. M. & Yang, G. J. Synthetic tissue biology: tissue engineering meets synthetic biology. Birth Defects Res. C Embryo. Today 81, 354–361 (2007).

    Article  CAS  PubMed  Google Scholar 

  142. Elliott, M. J. et al. Stem-cell-based, tissue engineered tracheal replacement in a child: a 2-year follow-up study. Lancet 380, 994–1000 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  143. Sato, T. et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469, 415–418 (2011). Demonstrates that Paneth cells, which are a differentiated stem cell progeny, function as an essential part of the stem cell niche in intestinal crypts and significantly increase the ability of LGR5+ stem cells to form long-lived organoids in vitro.

    Article  CAS  PubMed  Google Scholar 

  144. Yui, S. et al. Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell. Nature Med. 18, 618–623 (2012). Demonstrates the potential of stem cell organoids to repair experimental injuries to the colon in small-animal models and suggests that organoids could be used therapeutically in human patients.

    Article  CAS  PubMed  Google Scholar 

  145. Assawachananont, J. et al. Transplantation of embryonic and induced pluripotent stem cell-derived 3D retinal sheets into retinal degenerative mice. Stem Cell Rep. 2, 662–674 (2014).

    Article  Google Scholar 

  146. Saito, H., Takeuchi, M., Chida, K. & Miyajima, A. Generation of glucose-responsive functional islets with a three-dimensional structure from mouse fetal pancreatic cells and iPS cells in vitro. PLoS ONE 6, e28209 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  147. Sneddon, J. B., Borowiak, M. & Melton, D. A. Self-renewal of embryonic-stem-cell-derived progenitors by organ-matched mesenchyme. Nature 491, 765–768 (2012). Develops techniques to efficiently differentiate ES cells into endodermal progenitors and then, using co-culture with mesenchyme and transplantation, differentiate these progenitors into glucose-sensitive, insulin-secreting cells in vivo.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  148. Antonica, F. et al. Generation of functional thyroid from embryonic stem cells. Nature 491, 66–71 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  149. Sasai, Y. Next-generation regenerative medicine: organogenesis from stem cells in 3D culture. Cell Stem Cell 12, 520–530 (2013).

    Article  CAS  PubMed  Google Scholar 

  150. Schayowitz, A. et al. Functional profiling of live melanoma samples using a novel automated platform. PLoS ONE 7, e52760 (2013).

    Article  CAS  Google Scholar 

  151. Miller, J. S. et al. Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues. Nature Mater. 11, 768–774 (2012).

    Article  CAS  Google Scholar 

  152. Takebe, T. et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 499, 481–484 (2013). Develops a novel model for vascularized human liver from iPS cells and demonstrates the functional engraftment of these liver buds into mice. Notably, the transplanted tissue had characteristics of human liver at the level of protein production and drug metabolism.

    Article  CAS  PubMed  Google Scholar 

  153. Sudo, R. Multiscale tissue engineering for liver reconstruction. Organogenesis http://dx.doi.org/10.4161/org.27968 (2014).

  154. Schrag, D. et al. American society of clinical oncology technology assessment: chemotherapy sensitivity and resistance assays. J. Clin. Oncol. 22, 3631–3638 (2004).

    Article  CAS  PubMed  Google Scholar 

  155. Burstein, H. J. et al. American Society of Clinical Oncology clinical practice guideline update on the use of chemotherapy sensitivity and resistance assays. J. Clin. Oncol. 29, 3328–3330 (2011).

    Article  PubMed  Google Scholar 

  156. Vaira, V. et al. Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors. Proc. Natl Acad. Sci. USA 107, 8352–8356 (2010).

    Article  CAS  PubMed  Google Scholar 

  157. Merz, F. et al. Organotypic slice cultures of human glioblastoma reveal different susceptibilities to treatments. Neuro. Oncol. 15, 670–681 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  158. Dekkers, J. F. et al. A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nature Med. 19, 939–945 (2013).

    Article  CAS  PubMed  Google Scholar 

  159. Muranen, T. et al. Inhibition of PI3K/mTOR leads to adaptive resistance in matrix-attached cancer cells. Cancer Cell 21, 227–239 (2012). Shows that the dimensionality of cancer spheroids is relevant for the rational design of drug combinations owing to distinct responses in matrix-attached and matrix-deprived cells.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  160. Walker, J. L. et al. Diverse roles of E-cadherin in the morphogenesis of the submandibular gland: insights into the formation of acinar and ductal structures. Dev. Dyn. 237, 3128–3141 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  161. Weaver, V. M. et al. Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies. J. Cell Biol. 137, 231–245 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  162. Ginsburg, E. & Vonderhaar, B. K. in Methods in Mammary Gland Biology and Breast Cancer Research (eds. Ip, M.M. & Asch, B.B.) 147–154 (Springer US, 2000).

    Book  Google Scholar 

  163. Nguyen-Ngoc, K. V. et al. in Tissue Morphogenesis: Methods and Protocols Vol. 1189 Methods in Molecular Biology (ed. Nelson, C. M.) (Springer Science and Business Media, 2014).

    Google Scholar 

  164. Akhtar, N. & Streuli, C. H. An integrin–ILK–microtubule network orients cell polarity and lumen formation in glandular epithelium. Nature Cell Biol. 15, 17–27 (2012).

    Article  CAS  Google Scholar 

  165. Daley, W. P. et al. ROCK1-directed basement membrane positioning coordinates epithelial tissue polarity. Development 139, 411–422 (2011).

    Article  CAS  Google Scholar 

  166. Pradhan-Bhatt, S. et al. Implantable three-dimensional salivary spheroid assemblies demonstrate fluid and protein secretory responses to neurotransmitters. Tissue Eng. Part A 19, 1610–1620 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  167. Wei, C., Larsen, M., Hoffman, M. P. & Yamada, K. M. Self-organization and branching morphogenesis of primary salivary epithelial cells. Tissue Eng. 13, 721–735 (2007).

    Article  CAS  PubMed  Google Scholar 

  168. O'Brien, L. E. et al. Morphological and biochemical analysis of Rac1 in three-dimensional epithelial cell cultures. Methods Enzymol. 406, 676–691 (2006).

    Article  CAS  PubMed  Google Scholar 

  169. Yagi, S., Matsuda, M. & Kiyokawa, E. Suppression of Rac1 activity at the apical membrane of MDCK cells is essential for cyst structure maintenance. EMBO Rep. 13, 237–243 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  170. Srinivas, S. et al. Expression of green fluorescent protein in the ureteric bud of transgenic mice: a new tool for the analysis of ureteric bud morphogenesis. Dev. Genet. 24, 241–251 (1999).

    Article  CAS  PubMed  Google Scholar 

  171. Costantini, F., Watanabe, T., Lu, B., Chi, X. & Srinivas, S. Dissection of embryonic mouse kidney, culture in vitro, and imaging of the developing organ. Cold Spring Harb. Protoc. 2011, http://dx.doi.org/10.1101/pdb.prot5613 (2011).

  172. Rosines, E. et al. Constructing kidney-like tissues from cells based on programs for organ development: toward a method of in vitro tissue engineering of the kidney. Tissue Eng. Part A 16, 2441–2455 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  173. Steer, D. L., Bush, K. T., Meyer, T. N., Schwesinger, C. & Nigam, S. K. A strategy for in vitro propagation of rat nephrons. Kidney Int. 62, 1958–1965 (2002).

    Article  PubMed  Google Scholar 

  174. Taub, M., Wang, Y., Szczesny, T. M. & Kleinman, H. K. Epidermal growth factor or transforming growth factor α is required for kidney tubulogenesis in matrigel cultures in serum-free medium. Proc. Natl Acad. Sci. USA 87, 4002–4006 (1990).

    Article  CAS  PubMed  Google Scholar 

  175. Morizane, R. et al. Kidney specific protein-positive cells derived from embryonic stem cells reproduce tubular structures in vitro and differentiate into renal tubular cells. PLoS ONE 8, e64843 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  176. Taguchi, A. et al. Redefining the in vivo origin of metanephric nephron progenitors enables generation of complex kidney structures from pluripotent stem cells. Cell Stem Cell 14, 53–67 (2014).

    Article  CAS  PubMed  Google Scholar 

  177. Takasato, M., Little, M. H. & Elefanty, A. G. Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney. Nature Cell Biol. 16, 118–126 (2013).

    Article  CAS  PubMed  Google Scholar 

  178. Parrish, A. R., Gandolfi, A. J. & Brendel, K. Precision-cut tissue slices: applications in pharmacology and toxicology. Life Sci. 57, 1887–1901 (1995).

    Article  CAS  PubMed  Google Scholar 

  179. del Moral, P.-M. & Warburton, D. Explant culture of mouse embryonic whole lung, isolated epithelium, or mesenchyme under chemically defined conditions as a system to evaluate the molecular mechanism of branching morphogenesis and cellular differentiation. Methods Mol. Biol. 633, 71–79 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  180. Rock, J. R. et al. Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc. Natl Acad. Sci. 106, 12771–12775 (2009).

    Article  CAS  PubMed  Google Scholar 

  181. Mondrinos, M. J. et al. Engineering three-dimensional pulmonary tissue constructs. Tissue Eng. 12, 717–728 (2006).

    Article  CAS  PubMed  Google Scholar 

  182. Jaffe, A. B., Kaji, N., Durgan, J. & Hall, A. Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis. J. Cell Biol. 183, 625–633 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  183. Magudia, K., Lahoz, A. & Hall, A. K-Ras and B-Raf oncogenes inhibit colon epithelial polarity establishment through up-regulation of c-myc. J. Cell Biol. 198, 185–194 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  184. Kovbasnjuk, O. et al. Human enteroids: preclinical models of non-inflammatory diarrhea. Stem Cell Res. Ther. 4, S3 (2014).

    Article  Google Scholar 

  185. Spence, J. R. et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105–109 (2011).

    Article  CAS  PubMed  Google Scholar 

  186. Li, X. et al. Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture. Nature Med. 20, 769–777 (2014).

    Article  CAS  PubMed  Google Scholar 

  187. Stange, D. E. et al. Differentiated Troy+ chief cells act as reserve stem cells to generate all lineages of the stomach epithelium. Cell 155, 357–368 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  188. Reichert, M. et al. Isolation, culture and genetic manipulation of mouse pancreatic ductal cells. Nature Protoc. 8, 1354–1365 (2013).

    Article  CAS  Google Scholar 

  189. Okugawa, Y. A. Novel three-dimensional cell culture method to analyze epidermal cell differentiation in vitro. Methods Mol. Biol. (2013).

  190. Lang, S. H. et al. Experimental prostate epithelial morphogenesis in response to stroma and three-dimensional matrigel culture. Cell Growth Differ. 12, 631–640 (2001).

    CAS  PubMed  Google Scholar 

  191. Kubota, Y., Kleinman, H. K., Martin, G. R. & Lawley, T. J. Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J. Cell Biol. 107, 1589–1598 (1988).

    Article  CAS  PubMed  Google Scholar 

  192. Arnaoutova, I. & Kleinman, H. K. In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. Nature Protoc. 5, 628–635 (2010).

    Article  CAS  Google Scholar 

  193. Davis, G. E. et al. Control of vascular tube morphogenesis and maturation in 3D extracellular matrices by endothelial cells and pericytes. Methods Mol. Biol. 1066, 17–28 (2013).

    Article  CAS  PubMed  Google Scholar 

  194. Morgan, J. P. et al. Formation of microvascular networks in vitro. Nature Protocols 8, 1820–1836 (2013).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors apologize to the many scientists whose outstanding work could not be cited owing to space limitations. A.J.E. and E.R.S. were supported by a Research Scholar Grant (RSG-12-141-01-CSM) from the American Cancer Society. A.J.E. was also supported in part by funds from the National Institutes of Health National Cancer Institute (NIH–NCI) (U01 CA155758), by a Jerome L. Greene Foundation Discovery Project, by a grant from the Mary Kay Ash Foundation (036-13), by funds from the Cindy Rosencrans Fund for Triple Negative Breast Cancer Research and by a grant from the Breast Cancer Research Foundation.

Author information

Authors and Affiliations

  1. Departments of Cell Biology and Oncology, Center for Cell Dynamics, School of Medicine, Johns Hopkins University, 855 North Wolfe Street, Baltimore, 21205, Maryland, USA

    Eliah R. Shamir & Andrew J. Ewald

Authors
  1. Eliah R. Shamir
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrew J. Ewald
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Andrew J. Ewald.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

PowerPoint slides

Glossary

Epithelium

A type of animal tissue derived from ectoderm or endoderm that lines all cavities and body surfaces and consists of one or more layers of polarized, tightly connected cells.

Connective tissue

A type of animal tissue derived from mesenchyme that provides structural and nutritional support and connectivity among other tissues; it consists of individual cells, ground substance and fibres.

Extracellular matrix

(ECM). The non-cellular component of tissues that provides both structural support and signalling cues to cells; it is composed of a network of proteins such as collagen, fibronectin and laminin.

Mesenchyme

Loosely organized, undifferentiated cells derived from embryonic mesoderm that give rise to the connective tissues of the body and the lymphatic and circulatory systems.

Induced pluripotent stem cells

(iPS cells). Adult somatic cells that are genetically reprogrammed to generate embryonic-like pluripotent stem cells.

Basement membrane

An organized thin layer of extracellular matrix proteins that separates the epithelium from the surrounding connective tissue.

Stromal cells

The connective tissue cells of an organ (for example, fibroblasts), which support the function of the parenchymal cells of the organ.

Tissue stem cells

Adult stem cells that can give rise to some or all of the specialized cells of the tissue or organ from which they originate.

Matrigel

A gelatinous basement membrane matrix derived from Engelbreth–Holm–Swarm mouse sarcoma cells; it promotes cell differentiation and models the in vivo microenvironment of many tissues.

Self-organization

In tissues, the spontaneous formation of a highly ordered structure from a population of cells in the absence of pre-patterns.

Stratified epithelium

An epithelium that is composed of two or more layers of cells; it is often found in locations that require increased protection, such as exterior body surfaces.

Microfluidic systems

Devices that comprise submillimetre channels, pumps and valves that enable controlled, reproducible analysis of small samples of cells in nanolitre or picolitre volumes.

Asymmetric divisions

Cell divisions that result in two daughter cells with different fates, such as localization into distinct epithelial cell layers with unequal inheritance of polarity proteins.

Cre–lox

A site-specific recombination tool that uses the enzyme Cre recombinase to induce deletions, translocations or inversions in segments of genomic DNA that are flanked by loxP sites.

CRISPR–Cas9

(Clustered, regularly interspaced short palindromic repeats–CRISPR-associated protein 9). A genome-editing tool that uses the microbial RNA-guided Cas9 nuclease to make targeted changes in the DNA of eukaryotic cells.

Hypoplasia

Abnormal tissue or organ development owing to a deficient number of cells.

Hyperplasia

Abnormal tissue or organ development owing to an excess number of cells.

Tumour xenografts

Human tumours that are implanted into immunocompromised animal hosts.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shamir, E., Ewald, A. Three-dimensional organotypic culture: experimental models of mammalian biology and disease. Nat Rev Mol Cell Biol 15, 647–664 (2014). https://doi.org/10.1038/nrm3873

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrm3873

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing