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

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.

Figure 1. Cellular inputs to organotypic cultures

a Whole-organ and organ-slice cultures. Tissues that develop during embryogenesis, such as the salivary gland, kidney and lung, are commonly explanted as whole organs; for example, explants of metanephric kidney that have been isolated from the embryonic urogenital ridge will undergo vigorous branching morphogenesis in three-dimensional (3D) culture. Tissues that develop postnatally, such as the mammary gland, intestine, brain and aorta, can be sectioned into tissue ‘slices’ owing to their large size. b Tissue organoid cultures. Primary organs can be harvested and processed by mechanical disruption and enzymatic digestion into tissue fragments (known as tissue organoids). The native stromal cells and extracellular matrix are typically removed, which enables isolated culture of the epithelial tissues. The resulting organoids contain diverse epithelial cell types organized in their normal spatial configuration and are typically explanted into commercial extracellular matrices, such as Matrigel or collagen I; for example, mammary epithelial organoids will undergo branching morphogenesis in 3D Matrigel. c Stem cell organoid cultures. Stem cells can be used to generate organoids that model the architecture and cellular composition of a larger organ. Both embryonic and adult induced pluripotent stem (iPS) cells have been used to generate organoids of the kidney, lung, intestine, liver, optic cup and brain; for example, embryonic stem (ES) cells that are cultured in the presence of Matrigel and differentiation factors will aggregate and self-organize into optic cup-like structures. Alternatively, single tissue stem cells that have been isolated from an adult organ can be used; for example, Leu-rich repeat-containing G protein-coupled receptor (LGR5) - expressing (LGR5⁺) tissue stem cells that are embedded within Matrigel will generate many tissues of the digestive tract. d Primary cell cultures. Primary keratinocytes from the skin and oesophagus have been cultured on cell culture inserts to organize into stratified epithelium. In addition, primary epithelial cells from the salivary gland, lung, kidney and pancreas, as well as endothelial cells, have been used in two and one-half-dimensional (2.5D) or 3D culture.

Figure 2. The major categories of cell culture

a Two-dimensional (2D) culture. Cells are typically cultured directly on a highly rigid substrate such as glass or plastic. The medium can be supplemented with extracellular matrix (ECM) proteins to induce a more differentiated cell state; for example, addition of laminin I will induce the differentiation of mammary epithelial cells. b Two and one-half-dimensional (2.5D) culture (also known as drip culture). Cells are cultured on top of a thin, organized layer of ECM, and diluted ECM proteins (such as laminins) are present in the medium. This format is ideal for imaging and antibody staining and is sufficient for epithelial acinar formation (for example, in MCF-10A and MDCK (Madin–Darby canine kidney) cell lines). 2.5D cultures have also been used to generate endothelial networks. The mechanical or structural properties of the ECM layer can be varied, and microfluidics can be used to generate flow-over gradients. c Three-dimensional (3D)-embedded culture. Cells are cultured within a gel of ECM proteins. Cells are uniformly exposed to an organized ECM and can further remodel and restructure the ECM over time; for example, mammary tissue organoids will undergo branching morphogenesis in 3D Matrigel. This format requires that the ECM solution is cell-compatible both in liquid and in gel form, and it enables the incorporation of different ECM components, multicellular tissues and stromal cells. If constructed within microfluidic devices, these cultures can be subjected to in- or through-gel gradients. ECMs can also be precisely patterned in 3D. d Mechanically supported culture. Cells, organ slices or whole organs are cultured on a tissue culture insert that is either submerged in medium or maintained at an air–liquid interface. Histologically realistic epithelial tissues can be constructed in stages, with initial assembly of keratinocytes into an epithelial cell layer on a submerged culture insert, followed by exposure of these cells to an air–liquid interface to induce the formation of stratified epidermis. Stromal cells can be co-cultured with the epithelial cells or added to a separate compartment within the culture dish to study paracrine effects without direct physical contact between cell types. Slices of large organs, such as the brain, can be cultured on these inserts.

Figure 3. The cellular basis of epithelial tube elongation

a Schematic diagram showing epithelial bud initiation and tube elongation. Although the process is conceptually similar across the various organs, it was unclear whether tube elongation was accomplished by conserved cellular mechanisms. b Mammary epithelium elongates from a mammary placode into a surrounding fat pad starting at 3 weeks after birth. Branching morphogenesis involves transitions from simple to stratified to simple epithelium. The terminal end bud initiates and elongates as a multilayered structure at the growing front and eventually repolarizes into a simple bilayer (inset). c Salivary gland epithelium develops embryonically from a single stratified bud that undergoes successive clefting and extracellular matrix remodelling to form a branched network with simple architecture. At the single bud stage (at embryonic day 13.5), the epithelium already contains a morphologically distinct outer layer of columnar cells, which form the acinar epithelium of the gland, and many inner rounded cells, which form the ductal epithelium (inset). d Kidney branching morphogenesis initiates embryonically when the Wolffian duct evaginates into the surrounding metanephric mesenchyme as the ureteric bud. The epithelial bud transitions from simple to pseudostratified to simple architecture (inset). e Lung development occurs embryonically. Avian lung maintains simple organization throughout branching morphogenesis and initiates new buds via apical constriction (inset). f Salivary gland epithelium requires fibroblast growth factor 10 (FGF10) for branching morphogenesis, and heparan sulphate increases the affinity of FGF10 for its receptor. Specific sulphation patterns of heparan sulphate regulate FGF10-mediated morphogenetic events, such as proliferation, end-bud expansion and duct elongation. g In branching ureteric bud tips, pre-mitotic epithelial cells delaminate into the lumen to undergo cell division while maintaining a thin basal process at the site of origin. One daughter cell (blue) reinserts at the original site and the second daughter cell (green) inserts at a position one to three cell diameters away. h In the chick lung, treatment with the proliferation inhibitor aphidicolin does not block bud formation, which shows that cell proliferation is dispensable for bud initiation. i During mouse lung development, domain branching is characterized by a localized increase in cell division within the incipient bud relative to adjacent trunk cells. Within the bifurcating bud at the end of the tube, there is both an enrichment of proliferation relative to the trunk and a polarization of the plane of cell division within the future cleft region. Arrows indicate the orientation of cell division.

Figure 4. Genetic regulation of cell behaviours in mammalian tissues

a siRNA-mediated knockdown of p63 showed that this transcription factor is required for both proliferation and differentiation in regenerating organotypic postnatal epidermis. Depletion of p63 in all cells leads to tissue hypoplasia, defects in epidermal stratification and differentiation, and loss of simple epithelial markers. Mosaic mixtures of control cells and p63 siRNA-treated cells leads to a cell-autonomous failure of differentiation in the p63 knockdown cells. b Fibronectin was known to accumulate within the forming clefts in the salivary gland. Gene expression analysis showed that fibronectin binding induces expression of Btbd7 in epithelial cells within the clefts. BTBD7 regulates cleft progression by reducing cell–cell adhesion and promoting the formation of transient intercellular gaps. c Labelled cells within chimeric embryonic kidneys compete for contribution to the ureteric bud, depending on their individual level of RET signalling. In a chimeric Wolffian duct, labelled epithelial cells that lack the receptor tyrosine kinase (RTK) RET (Ret^−/− cells) are excluded from the tips of elongating ureteric buds in favour of wild-type cells. By contrast, cells that are depleted of Sprouty1 (Spry1^−/−), which is a repressor of RTK signalling, have increased levels of RET and accumulate at the ureteric bud tip domain instead of wild-type cells. d Tet-inducible Twist1 expression leads to loss of tissue polarity and the rapid dissemination of otherwise normal mammary epithelial cells. Disseminated cells retain epithelial gene expression (for example, cytokeratin 8), localize epithelial cadherin (E-cadherin) and β-catenin to the membrane and require E-cadherin protein to disseminate as single cells. Part b of the figure from Onodera, T. et al. Btbd7 regulates epithelial cell dynamics and branching morphogenesis. Science 329, 562–565 (2010). Adapted with permission from AAAS. Part c of the figure adapted from Dev. Cell, 17, Chi, X. et al., Ret-dependent cell rearrangements in the Wolffian duct epithelium initiate ureteric bud morphogenesis, 199–209, Copyright (2009), with permission from Elsevier.

Figure 5. The role of the microenvironment in regulating epithelial function

a Schematic overview of different components of the tissue microenvironment, including immune cells (for example, macrophages), blood vessels, fibroblasts and extracellular matrix (for example, collagen I). The components and properties of the microenvironment can be readily modified in three-dimensional (3D) culture. b Co-cultures of lung or bone marrow stroma mixed with endothelial cells were used to generate a 3D organotypic microvascular niche. The angiogenesis inhibitor thrombospondin 1 (TSP1) induces breast tumour cell dormancy in mature endothelium, whereas transforming growth factor-β1 (TGFβ1) and periostin promote tumour cell growth in neovascular tips, which lack TSP1 (REF. 129). c Direct comparisons of the same tissue in different microenvironments (that is, Matrigel or collagen I) shows that the composition of the ECM regulates invasive and disseminative behaviours of both normal and malignant mammary epithelium. d The mechanical properties of the microenvironment can affect cell and tissue function. High rigidity (which is achieved by crosslinking poly(ethylene) glycol (PEG) networks within Matrigel scaffolds) suppresses the growth of both normal and neoplastic tissue but does not induce invasion or dissemination. The addition of adhesive peptides promotes dissemination of both normal and tumour cells. Part b of the figure adapted from REF. , Nature Publishing Group. Part c of the figure adapted from 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).