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.
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
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Bichat, X. General Anatomy, Applied to Physiology and Medicine. (Richardson and Lord, 1822).
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).
Sobotta, J., Huber, G. C. & De Witt, L. M. B. Atlas and Epitome of Human Histology and Microscopic Anatomy. (W. B. Saunders & company, 1903).
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).
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).
Sato, T. et al. Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).
Finkbeiner, S. R. & Spence, J. R. A gutsy task: generating intestinal tissue from human pluripotent stem cells. Dig. Dis. Sci. 58, 1176–1184 (2013).
Alberts, B. Molecular Biology of the Cell. 4th edn Ch. 19 (Garland Science, 2002).
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).
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).
Zheng, Y. et al. In vitro microvessels for the study of angiogenesis and thrombosis. Proc. Natl Acad. Sci. USA 109, 9342–9347 (2012).
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.
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).
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).
Ichinose, R. R. & Nandi, S. Lobuloalveolar differentiation in mouse mammary tissues in vitro. Science 145, 496–497 (1964).
Waymouth, C. in Biology of the Laboratory Mouse (ed. Green, Earl L.) (Dover Publications, 1966).
Guerrero, R. R., Rounds, D. E. & Booher, J. An improved organ culture method for adult mammalian lung. In Vitro 13, 517–524 (1977).
Browning, T. H. & Trier, J. S. Organ culture of mucosal biopsies of human small intestine. J. Clin. Invest. 48, 1423–1432 (1969).
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).
Autrup, H. et al. Explant culture of rat colon: a model system for studying metabolism of chemical carcinogens. In Vitro 14, 868–877 (1978).
Stoppini, L., Buchs, P. A. & Muller, D. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37, 173–182 (1991).
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).
Aplin, A. C., Fogel, E., Zorzi, P. & Nicosia, R. F. The aortic ring model of angiogenesis. Methods Enzymol. 443, 119–136 (2008).
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).
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).
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).
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.
Koo, B. K. et al. Controlled gene expression in primary Lgr5 organoid cultures. Nature Methods 9, 81–83 (2011).
Onodera, T. et al. Btbd7 regulates epithelial cell dynamics and branching morphogenesis. Science 329, 562–565 (2010).
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.
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).
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).
Steinberg, Z. et al. FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis. Development 132, 1223–1234 (2005).
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).
Liu, Y. et al. Novel role for Netrins in regulating epithelial behavior during lung branching morphogenesis. Curr. Biol. 14, 897–905 (2004).
Ootani, A. et al. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nature Med. 15, 701–706 (2009).
Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141, 1762–1772 (2011).
Huch, M. et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494, 247–250 (2013).
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).
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).
Ghosh, S. et al. PI3K/mTOR signaling regulates prostatic branching morphogenesis. Dev. Biol. 360, 329–342 (2011).
Kleinman, H. K. & Martin, G. R. Matrigel: basement membrane matrix with biological activity. Semin. Cancer Biol. 15, 378–386 (2005).
Wolf, K. et al. Collagen-based cell migration models in vitro and in vivo. Semin. Cell Dev. Biol. 20, 931–941 (2009).
Eiraku, M. et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472, 51–56 (2011).
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.
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).
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.
Suga, H. et al. Self-formation of functional adenohypophysis in three-dimensional culture. Nature 480, 57–62 (2011).
Townes, P. L. & Holtfreter, J. Directed movements and selective adhesion of embryonic amphibian cells. J. Exp. Zool. 128, 53–120 (1955).
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).
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).
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).
Fuchs, E. Epidermal differentiation: the bare essentials. J. Cell Biol. 111, 2807–2814 (1990).
Kalabis, J. et al. Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture. Nature Protoc. 7, 235–246 (2012).
Unbekandt, M. & Davies, J. A. Dissociation of embryonic kidneys followed by reaggregation allows the formation of renal tissues. Kidney Int. 77, 407–416 (2009).
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).
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).
O'Brien, L. E. et al. Rac1 orientates epithelial apical polarity through effects on basolateral laminin assembly. Nature Cell Biol. 3, 831–838 (2001).
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).
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).
Debnath, J. & Brugge, J. S. Modelling glandular epithelial cancers in three-dimensional cultures. Nature Rev. Cancer 5, 675–688 (2005).
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).
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).
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).
Wescott, M. P. et al. Pancreatic ductal morphogenesis and the Pdx1 homeodomain transcription factor. Mol. Biol. Cell 20, 4838–4844 (2009).
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.
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).
Brownfield, D. G. et al. Patterned collagen fibers orient branching mammary epithelium through distinct signaling modules. Curr. Biol. 23, 703–709 (2013).
Levental, K. R. et al. Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell 139, 891–906 (2009).
Provenzano, P. P. et al. Collagen reorganization at the tumor–stromal interface facilitates local invasion. BMC Med. 4, 38 (2006).
Provenzano, P. P. et al. Collagen density promotes mammary tumor initiation and progression. BMC Med. 6, 11 (2008).
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).
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).
Baker, B. M. & Chen, C. S. Deconstructing the third dimension: how 3D culture microenvironments alter cellular cues. J. Cell Sci. 125, 3015–3024 (2012).
Dvir, T., Timko, B. P., Kohane, D. S. & Langer, R. Nanotechnological strategies for engineering complex tissues. Nature Nanotechnol. 6, 13–22 (2010).
Singh, A. & Elisseeff, J. Biomaterials for stem cell differentiation. J. Mater. Chem. 20, 8832–8847 (2010).
Young, E. W. & Beebe, D. J. Fundamentals of microfluidic cell culture in controlled microenvironments. Chem. Soc. Rev. 39, 1036–1048 (2010).
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.
Stroock, A. D. & Fischbach, C. Microfluidic culture models of tumor angiogenesis. Tissue Engineer. Part A 16, 2143–2146 (2010).
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).
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).
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).
Larsen, M. et al. Role of PI 3-kinase and PIP3 in submandibular gland branching morphogenesis. Dev. Biol. 255, 178–191 (2003).
Puri, S. & Hebrok, M. Dynamics of embryonic pancreas development using real-time imaging. Dev. Biol. 306, 82–93 (2007).
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).
Provenzano, P. P. et al. Nonlinear optical imaging of cellular processes in breast cancer. Microsc. Microanal. 14, 532–548 (2008).
Underwood, J. M. et al. The ultrastructure of MCF-10A acini. J. Cell. Physiol. 208, 141–148 (2006).
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).
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.
Ghabrial, A. S. & Krasnow, M. A. Social interactions among epithelial cells during tracheal branching morphogenesis. Nature 441, 746–749 (2006).
Lu, P. & Werb, Z. Patterning mechanisms of branched organs. Science 322, 1506–1509 (2008).
Larsen, M., Wei, C. & Yamada, K. M. Cell and fibronectin dynamics during branching morphogenesis. J. Cell Sci. 119, 3376–3384 (2006).
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).
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.
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).
Packard, A. et al. Luminal mitosis drives epithelial cell dispersal within the branching ureteric bud. Dev. Cell 27, 319–330 (2013).
Schnatwinkel, C. & Niswander, L. Multiparametric image analysis of lung-branching morphogenesis. Dev. Dyn. 242, 622–637 (2013).
Cukierman, E., Pankov, R., Stevens, D. R. & Yamada, K. M. Taking cell-matrix adhesions to the third dimension. Science 294, 1708–1712 (2001).
Hsu, J. C. et al. Viral gene transfer to developing mouse salivary glands. J. Dent. Res. 91, 197–202 (2012).
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).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
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.
Yu, W. et al. β1-integrin orients epithelial polarity via Rac1 and laminin. Mol. Biol. Cell 16, 433–445 (2005).
Martin-Belmonte, F. et al. Cell-polarity dynamics controls the mechanism of lumen formation in epithelial morphogenesis. Curr. Biol. 18, 507–513 (2008).
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).
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).
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).
Aranda, V. et al. Par6–aPKC uncouples ErbB2 induced disruption of polarized epithelial organization from proliferation control. Nature Cell Biol. 8, 1235–1245 (2006).
Zhan, L. et al. Deregulation of scribble promotes mammary tumorigenesis and reveals a role for cell polarity in carcinoma. Cell 135, 865–878 (2008).
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).
Leung, C. T. & Brugge, J. S. Outgrowth of single oncogene-expressing cells from suppressive epithelial environments. Nature 482, 410–413 (2012).
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).
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).
Kretz, M. et al. Control of somatic tissue differentiation by the long non-coding RNA TINCR. Nature 493, 231–235 (2013).
Sakai, T., Larsen, M. & Yamada, K. M. Fibronectin requirement in branching morphogenesis. Nature 423, 876–881 (2003).
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).
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).
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).
Egeblad, M., Nakasone, E. S. & Werb, Z. Tumors as organs: complex tissues that interface with the entire organism. Dev. Cell 18, 884–901 (2010).
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).
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).
Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).
Condeelis, J. & Pollard, J. W. Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell 124, 263–266 (2006).
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).
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).
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).
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).
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).
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.
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).
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.
Knox, S. M. et al. Parasympathetic stimulation improves epithelial organ regeneration. Nature Commun. 4, 1494 (2013).
Marusyk, A., Almendro, V. & Polyak, K. Intra-tumour heterogeneity: a looking glass for cancer? Nature Rev. Cancer 12, 323–334 (2012).
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).
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).
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).
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).
Conklin, M. W. et al. Aligned collagen is a prognostic signature for survival in human breast carcinoma. Am. J. Pathol. 178, 1221–1232 (2011).
Paszek, M. J. et al. Tensional homeostasis and the malignant phenotype. Cancer Cell 8, 241–254 (2005).
Friedl, P. & Alexander, S. Cancer invasion and the microenvironment: plasticity and reciprocity. Cell 147, 992–1009 (2011).
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).
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).
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.
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.
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).
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).
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.
Antonica, F. et al. Generation of functional thyroid from embryonic stem cells. Nature 491, 66–71 (2012).
Sasai, Y. Next-generation regenerative medicine: organogenesis from stem cells in 3D culture. Cell Stem Cell 12, 520–530 (2013).
Schayowitz, A. et al. Functional profiling of live melanoma samples using a novel automated platform. PLoS ONE 7, e52760 (2013).
Miller, J. S. et al. Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues. Nature Mater. 11, 768–774 (2012).
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.
Sudo, R. Multiscale tissue engineering for liver reconstruction. Organogenesis http://dx.doi.org/10.4161/org.27968 (2014).
Schrag, D. et al. American society of clinical oncology technology assessment: chemotherapy sensitivity and resistance assays. J. Clin. Oncol. 22, 3631–3638 (2004).
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).
Vaira, V. et al. Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors. Proc. Natl Acad. Sci. USA 107, 8352–8356 (2010).
Merz, F. et al. Organotypic slice cultures of human glioblastoma reveal different susceptibilities to treatments. Neuro. Oncol. 15, 670–681 (2013).
Dekkers, J. F. et al. A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nature Med. 19, 939–945 (2013).
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.
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).
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).
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).
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).
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).
Daley, W. P. et al. ROCK1-directed basement membrane positioning coordinates epithelial tissue polarity. Development 139, 411–422 (2011).
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).
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).
O'Brien, L. E. et al. Morphological and biochemical analysis of Rac1 in three-dimensional epithelial cell cultures. Methods Enzymol. 406, 676–691 (2006).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Parrish, A. R., Gandolfi, A. J. & Brendel, K. Precision-cut tissue slices: applications in pharmacology and toxicology. Life Sci. 57, 1887–1901 (1995).
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).
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).
Mondrinos, M. J. et al. Engineering three-dimensional pulmonary tissue constructs. Tissue Eng. 12, 717–728 (2006).
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).
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).
Kovbasnjuk, O. et al. Human enteroids: preclinical models of non-inflammatory diarrhea. Stem Cell Res. Ther. 4, S3 (2014).
Spence, J. R. et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105–109 (2011).
Li, X. et al. Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture. Nature Med. 20, 769–777 (2014).
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).
Reichert, M. et al. Isolation, culture and genetic manipulation of mouse pancreatic ductal cells. Nature Protoc. 8, 1354–1365 (2013).
Okugawa, Y. A. Novel three-dimensional cell culture method to analyze epidermal cell differentiation in vitro. Methods Mol. Biol. (2013).
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).
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).
Arnaoutova, I. & Kleinman, H. K. In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. Nature Protoc. 5, 628–635 (2010).
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).
Morgan, J. P. et al. Formation of microvascular networks in vitro. Nature Protocols 8, 1820–1836 (2013).
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.
The authors declare no competing financial interests.
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.
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.
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.
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.
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.
(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.
Abnormal tissue or organ development owing to a deficient number of cells.
Abnormal tissue or organ development owing to an excess number of cells.
- Tumour xenografts
Human tumours that are implanted into immunocompromised animal hosts.
About this article
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
An ARF GTPase module promoting invasion and metastasis through regulating phosphoinositide metabolism
Nature Communications (2021)
Nature Communications (2021)
Gastric organoids—an in vitro model system for the study of gastric development and road to personalized medicine
Cell Death & Differentiation (2021)
Nature Reviews Gastroenterology & Hepatology (2021)
Nature Materials (2021)