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Capturing complex 3D tissue physiology in vitro

Key Points

  • Over the past two decades, the field of tissue engineering has focused primarily on the creation of tissues for patients. The emphasis of the field is now shifting to include the creation of complex in vitro tissue models that help to explain disease processes (for example, breast cancer) and serve as tools to assess the safety and efficacy of therapies (for example, screens of liver toxicity).

  • The 3D extracellular-matrix (ECM) environment in vivo provides both chemical and physical cues to regulate cell behaviour, serving not only as a structural support, but as a depot of many effector molecules. New synthetic matrices that include well-defined adhesion, growth factor and degradation moieties are being developed to mimic these cues, thereby allowing the quantitative analysis of cell migration, differentiation, survival and growth.

  • Gradients of nutrients and effector molecules are present in 3D cultures. The magnitude of gradients for vital molecules such as oxygen can be predicted for a given experimental arrangement, but data are just emerging for the rates of production and consumption of growth factors, cytokines and other effector molecules.

  • All tissues are subjected to mechanical forces that arise from interstitial flow and tissue movement. These mechanical forces can redistribute effector molecules that are secreted by cells, resulting in the coupling of chemical and mechanical signalling.

  • Microfabrication methods that have been adapted from the microelectronics industry and applied to miniaturize biochemical analyses are now being applied to create complex 3D tissue structures for in vitro studies, and are being combined with microfluidic pumps that can provide microscale fluid flows through tissues for long-term culture.

  • Experimental systems must be developed hand-in-hand with mathematical models that take into account the integration of numerous cues that influence downstream signals and, ultimately, cell responses.

Abstract

The emergence of tissue engineering raises new possibilities for the study of complex physiological and pathophysiological processes in vitro. Many tools are now available to create 3D tissue models in vitro, but the blueprints for what to make have been slower to arrive. We discuss here some of the 'design principles' for recreating the interwoven set of biochemical and mechanical cues in the cellular microenvironment, and the methods for implementing them. We emphasize applications that involve epithelial tissues for which 3D models could explain mechanisms of disease or aid in drug development.

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Figure 1: The importance of the 3D environment for engineering cell function.
Figure 2: Coupling between biophysical and biochemical cues.
Figure 3: Biophysical influences on interactions between tumour cells.
Figure 4: Microscale 3D model of liver.

References

  1. 1

    Lysaght, M. J. & Hazlehurst, A. L. Tissue engineering: the end of the beginning. Tissue Eng. 10, 309?320 (2004).

    Article  Google Scholar 

  2. 2

    Griffith, L. G. & Naughton, G. Tissue engineering ?current challenges and expanding opportunities. Science 295, 1009?1014 (2002).

    Article  CAS  Google Scholar 

  3. 3

    Suuronen, E. J., Sheardown, H., Newman, K. D., McLaughlin, C. R. & Griffith, M. Building in vitro models of organs. Int. Rev. Cytol. 244, 137?173 (2005).

    Article  CAS  Google Scholar 

  4. 4

    Sivaraman, A. et al. A microscale in vitro physiological model of the liver: predictive screens for drug metabolism and enzyme induction. Curr. Drug Metab. 6, 569?592 (2005).

    Article  CAS  Google Scholar 

  5. 5

    Kuperwasser, C. et al. Reconstruction of functionally normal and malignant human breast tissues in mice. Proc. Natl Acad. Sci. USA 101, 4966?4971 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Katoh, M. et al. Expression of human phase II enzymes in chimeric mice with humanized liver. Drug Metab. Dispos. 33, 1333?1340 (2005).

    Article  CAS  Google Scholar 

  7. 7

    Rangarajan, A., Hong, S. J., Gifford, A. & Weinberg, R. A. Species- and cell type-specific requirements for cellular transformation. Cancer Cell 6, 171?183 (2004).

    Article  CAS  Google Scholar 

  8. 8

    Watt, F. M. Selective migration of terminally differentiating cells from the basal layer of cultured human epidermis. J. Cell Biol. 98, 16?21 (1984).

    Article  CAS  Google Scholar 

  9. 9

    Louekari, K. Status and prospects of in vitro tests in risk assessment. Altern. Lab. Anim. 32, 431?435 (2004).

    CAS  PubMed  Google Scholar 

  10. 10

    Knight, B. et al. Visualizing muscle cell migration in situ. Curr. Biol. 10, 576?585 (2000).

    Article  CAS  Google Scholar 

  11. 11

    Roskelley, C. D., Desprez, P. Y. & Bissell, M. J. Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction. Proc. Natl Acad. Sci. USA 91, 12378?12382 (1994). Seminal work that links mammary phenotype to 3D-culture conditions.

    Article  CAS  Google Scholar 

  12. 12

    Bissell, M. J., Rizki, A. & Mian, I. S. Tissue architecture: the ultimate regulator of breast epithelial function. Curr. Opin. Cell Biol. 15, 753?762 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

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

    Article  CAS  Google Scholar 

  14. 14

    Paszek, M. J. & Weaver, V. M. The tension mounts: mechanics meets morphogenesis and malignancy. J. Mammary Gland Biol. Neoplasia 9, 325?342 (2004).

    Article  Google Scholar 

  15. 15

    Wozniak, M. A., Desai, R., Solski, P. A., Der, C. J. & Keely, P. J. ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix. J. Cell Biol. 163, 583?595 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Zegers, M. M., O'Brien, L. E., Yu, W., Datta, A. & Mostov, K. E. Epithelial polarity and tubulogenesis in vitro. Trends Cell Biol. 13, 169?176 (2003).

    Article  CAS  Google Scholar 

  17. 17

    Grinnell, F., Ho, C. H., Lin, Y.-C. & Skuta, G. Differences in the regulation of fibroblast contraction of floating versus stressed collagen matrices. J. Biol. Chem. 274, 918?923 (1999).

    Article  CAS  Google Scholar 

  18. 18

    Zaman, M. H., Kamm, R. D., Matsudaira, P. & Lauffenburger, D. A. Computational model for cell migration in three-dimensional matrices. Biophys. J. 89, 1389?1397 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Lutolf, M. P. et al. Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: engineering cell-invasion characteristics. Proc. Natl Acad. Sci. USA 100, 5413?5418 (2003). Pioneering demonstration of how synthetic extracellular matrices can be tuned to control many facets of cell behaviour in tissue remodelling, using approaches that are accessible to the general cell-biology laboratory.

    Article  CAS  Google Scholar 

  20. 20

    Lo, C. M., Wang, H. B., Dembo, M. & Wang, Y. L. Cell movement is guided by the rigidity of the substrate. Biophys. J. 79, 144?152 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Peyton, S. R. & Putnam, A. J. Extracellular matrix rigidity governs smooth muscle cell motility in a biphasic fashion. J. Cell Physiol. 204, 198?209 (2005).

    Article  CAS  Google Scholar 

  22. 22

    Sieminski, A. L., Hebbel, R. P. & Gooch, K. J. The relative magnitudes of endothelial force generation and matrix stiffness modulate capillary morphogenesis in vitro. Exp. Cell Res. 297, 574?584 (2004).

    Article  CAS  Google Scholar 

  23. 23

    Muschler, J. et al. A role for dystroglycan in epithelial polarization: loss of function in breast tumor cells. Cancer Res. 62, 7102?7109 (2002).

    CAS  PubMed  Google Scholar 

  24. 24

    Paszek, M. J. et al. Tensional homeostasis and the malignant phenotype. Cancer Cell 8, 241?254 (2005). Elegant demonstration of a link between matrix mechanics and phenotype in normal and malignant mammary tissue, using a comprehensive range of innovative methods to systematically vary matrix properties in vitro to match measured in vivo properties, as well as to quantify cell responses.

    Article  CAS  Google Scholar 

  25. 25

    Wozniak, M. A. & Keely, P. J. Use of three-dimensional collagen gels to study mechanotransduction in T47D breast epithelial cells. Biol. Proced. Online 7, 144?161 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Tomasek, J. J., Gabbiani, G., Hinz, B., Chaponnier, C. & Brown, R. A. Myofibroblasts and mechanoregulation of connective tissue remodelling. Nature Rev. Mol. Cell Biol. 3, 349?363 (2002).

    Article  CAS  Google Scholar 

  27. 27

    Shreiber, D. I., Barocas, V. H. & Tranquillo, R. T. Temporal variations in cell migration and traction during fibroblast-mediated gel compaction. Biophys. J. 84, 4102?4114 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Tsai, K. K., Chuang, E. Y., Little, J. B. & Yuan, Z. M. Cellular mechanisms for low-dose ionizing radiation-induced perturbation of the breast tissue microenvironment. Cancer Res. 65, 6734?6744 (2005).

    Article  CAS  Google Scholar 

  29. 29

    Paralkar, V. M., Vukicevic, S. & Reddi, A. H. Transforming growth factor β type 1 binds to collagen IV of basement membrane matrix: implications for development. Dev. Biol. 143, 303?308 (1991).

    Article  CAS  Google Scholar 

  30. 30

    Ruhrberg, C. et al. Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis. Genes Dev. 16, 2684?2698 (2002). This paper showed that heparin-binding VEGF isoforms were necessary for endothelial branching, using transgenic mice that expressed only either matrix-interacting or non-interacting VEGF isoforms.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Tschumperlin, D. et al. Mechanotransduction through growth factor shedding into the extracellular space. Nature 429, 83?86 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Swartz, M. A. Signaling in morphogenesis: transport cues in morphogenesis. Curr. Opin. Biotech. 14, 547?550 (2003).

    Article  CAS  Google Scholar 

  33. 33

    Lutolf, M. P. & Hubbell, J. A. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nature Biotechnol. 23, 47?55 (2005).

    Article  CAS  Google Scholar 

  34. 34

    Wang, Y. L. & Pelham, R. J. Jr . Preparation of a flexible, porous polyacrylamide substrate for mechanical studies of cultured cells. Methods Enzymol. 298, 489?496 (1998).

    Article  CAS  Google Scholar 

  35. 35

    Reinhart-King, C. A., Dembo, M. & Hammer, D. A. The dynamics and mechanics of endothelial cell spreading. Biophys. J. 89, 676?689 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Semler, E. J., Lancin, P. A., Dasgupta, A. & Moghe, P. V. Engineering hepatocellular morphogenesis and function via ligand-presenting hydrogels with graded mechanical compliance. Biotechnol. Bioeng. 89, 296?307 (2005).

    Article  CAS  Google Scholar 

  37. 37

    Kong, H. J., Polte, T. R., Alsberg, E. & Mooney, D. J. FRET measurements of cell-traction forces and nano-scale clustering of adhesion ligands varied by substrate stiffness. Proc. Natl Acad. Sci. USA 102, 4300?4305 (2005).

    Article  CAS  Google Scholar 

  38. 38

    Semino, C. E., Merok, J. R., Crane, G. G., Panagiotakos, G. & Zhang, S. Functional differentiation of hepatocyte-like spheroid structures from putative liver progenitor cells in three-dimensional peptide scaffolds. Differentiation 71, 262?270 (2003).

    Article  CAS  Google Scholar 

  39. 39

    Kisiday, J. et al. Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: implications for cartilage tissue repair. Proc. Natl Acad. Sci. USA 99, 9996?10001 (2002).

    Article  CAS  Google Scholar 

  40. 40

    Raeber, G. P., Lutolf, M. P. & Hubbell, J. A. Molecularly engineered PEG hydrogels: a novel model system for proteolytically mediated cell migration. Biophys. J. 89, 1374?1388 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Maheshwari, G., Brown, G., Lauffenburger, D. A., Wells, A. & Griffith, L. G. Cell adhesion and motility depend on nanoscale RGD clustering. J. Cell Sci. 113, 1677?1686 (2000).

    CAS  PubMed  Google Scholar 

  42. 42

    Coussen, F., Choquet, D., Sheetz, M. P. & Erickson, H. P. Trimers of the fibronectin cell adhesion domain localize to actin filament bundles and undergo rearward translocation. J. Cell Sci. 115, 2581?2590 (2002).

    CAS  PubMed  Google Scholar 

  43. 43

    Orend, G. Potential oncogenic action of tenascin-C in tumorigenesis. Int. J. Biochem. Cell Biol. 37, 1066?1083 (2005).

    Article  CAS  Google Scholar 

  44. 44

    Ehrbar, M. et al. Cell-demanded liberation of VEGF121 from fibrin implants induces local and controlled blood vessel growth. Circ. Res. 94, 1124?1132 (2004). Using protein engineering, this study showed that matrix-binding forms of growth factors led to different signalling patterns, compared with free or unbound growth factors.

    Article  CAS  Google Scholar 

  45. 45

    Helm, C. E., Fleury, M. E., Zisch, A. H., Boschetti, F. & Swartz, M. A. Synergy between interstitial flow and VEGF directs capillary morphogenesis in vitro through a gradient amplification mechanism. Proc. Natl Acad. Sci. USA 44, 15779?15784 (2005).

    Article  CAS  Google Scholar 

  46. 46

    Gobin, A. S. & West, J. L. Effects of epidermal growth factor on fibroblast migration through biomimetic hydrogels. Biotechnol. Prog. 19, 1781?1785 (2003).

    Article  CAS  Google Scholar 

  47. 47

    Rosner, B. I., Hang, T. & Tranquillo, R. T. Schwann cell behavior in three-dimensional collagen gels: evidence for differential mechano-transduction and the influence of TGF-β1 in morphological polarization and differentiation. Exp. Neurol. 195, 81?91 (2005).

    Article  CAS  Google Scholar 

  48. 48

    Kellner, K. et al. Determination of oxygen gradients in engineered tissue using a fluorescent sensor. Biotechnol. Bioeng. 80, 73?83 (2002).

    Article  CAS  Google Scholar 

  49. 49

    Glicklis, R., Merchuk, J. C. & Cohen, S. Modeling mass transfer in hepatocyte spheroids via cell viability, spheroid size, and hepatocellular functions. Biotechnol. Bioeng. 86, 672?680 (2004).

    Article  CAS  Google Scholar 

  50. 50

    Gebhardt, R. et al. New hepatocyte in vitro systems for drug metabolism: metabolic capacity and recommendations for application in basic research and drug development, standard operation procedures. Drug Metab. Rev. 35, 145?213 (2003).

    Article  CAS  Google Scholar 

  51. 51

    Martin, Y. & Vermette, P. Bioreactors for tissue mass culture: design, characterization, and recent advances. Biomaterials 26, 7481?7503 (2005).

    Article  CAS  Google Scholar 

  52. 52

    Guppy, M., Leedman, P., Zu, X. & Russell, V. Contribution by different fuels and metabolic pathways to the total ATP turnover of proliferating MCF-7 breast cancer cells. Biochem. J. 364, 309?315 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. 53

    Hermitte, F., Brunet de la Grange, P., Belloc, F., Praloran, V. & Ivanovic, Z. Very low O2 concentration (0.1%) favors G0 return of dividing CD34+ cells. Stem Cells 24, 65?73 (2006).

    Article  Google Scholar 

  54. 54

    Ezashi, T., Das, P. & Roberts, R. M. Low O2 tensions and the prevention of differentiation of hES cells. Proc. Natl Acad. Sci. USA 102, 4783?4788 (2005).

    Article  CAS  Google Scholar 

  55. 55

    Wang, D. W., Fermor, B., Gimble, J. M., Awad, H. A. & Guilak, F. Influence of oxygen on the proliferation and metabolism of adipose derived adult stem cells. J. Cell Physiol. 204, 184?191 (2005).

    Article  CAS  Google Scholar 

  56. 56

    Zhao, F. et al. Effects of oxygen transport on 3-D human mesenchymal stem cell metabolic activity in perfusion and static cultures: experiments and mathematical model. Biotechnol. Prog. 21, 1269?1280 (2005).

    Article  CAS  Google Scholar 

  57. 57

    Chow, D. C., Wenning, L. A., Miller, W. M. & Papoutsakis, E. T. Modeling pO2 distributions in the bone marrow hematopoietic compartment. II. Modified Kroghian models. Biophys. J. 81, 685?696 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. 58

    Radisky, D. C. et al. Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability. Nature 436, 123?127 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. 59

    Wojciak-Stothard, B., Tsang, L. Y. & Haworth, S. G. Rac and Rho play opposing roles in the regulation of hypoxia/reoxygenation-induced permeability changes in pulmonary artery endothelial cells. Am. J. Physiol. Lung Cell Mol. Physiol. 288, L749?L760 (2005).

    Article  CAS  Google Scholar 

  60. 60

    Morin, J. P., Preterre, D., Keravec, V. & Thuillez, C. Rotating wall vessel as a new in vitro shear stress generation system: application to rat coronary endothelial cell cultures. Cell Biol. Toxicol. 19, 227?242 (2003).

    Article  CAS  Google Scholar 

  61. 61

    MacDonald, J. M., Wolfe, S. P., Roy-Chowdhury, I., Kubota, H. & Reid, L. M. Effect of flow configuration and membrane characteristics on membrane fouling in a novel multicoaxial hollow-fiber bioartificial liver. Ann. NY Acad. Sci. 944, 334?343 (2001).

    Article  CAS  Google Scholar 

  62. 62

    Zeilinger, K. et al. Time course of primary liver cell reorganization in three-dimensional high-density bioreactors for extracorporeal liver support: an immunohistochemical and ultrastructural study. Tissue Eng. 10, 1113?1124 (2004).

    Article  CAS  Google Scholar 

  63. 63

    Reddy, C. C., Niyogi, S. K., Wells, A., Wiley, H. S. & Lauffenburger, D. A. Engineering EGF for enhanced mitogenic potency. Nature Biotechnol. 14, 1696?1699 (1996).

    Article  CAS  Google Scholar 

  64. 64

    Janowska-Wieczorek, A., Majka, M., Ratajczak, J. & Ratajczak, M. Z. Autocrine/paracrine mechanisms in human hematopoiesis. Stem Cells 19, 99?107 (2001).

    Article  CAS  Google Scholar 

  65. 65

    Prabhu, S. D. Cytokine-induced modulation of cardiac function. Circ. Res. 95, 1140?1153 (2004).

    Article  CAS  Google Scholar 

  66. 66

    Singh, A. B. & Harris, R. C. Autocrine, paracrine and juxtacrine signaling by EGFR ligands. Cell. Signal. 17, 1183?1193 (2005).

    Article  CAS  Google Scholar 

  67. 67

    Janes, K. A. et al. A systems model of signaling identifies a molecular basis set for cytokine-induced apoptosis. Science 310, 1646?1653 (2005).

    Article  CAS  Google Scholar 

  68. 68

    DeWitt, A. et al. Affinity regulates spatial range of EGF receptor autocrine ligand binding. Dev. Biol. 250, 305?316 (2002). Determined quantitative properties that govern tissue distribution of secreted growth factors.

    Article  CAS  Google Scholar 

  69. 69

    Wiley, H. S., Shvartsman, S. Y. & Lauffenburger, D. A. Computational modeling of the EGF-receptor system: a paradigm for systems biology. Trends Cell Biol. 13, 43?50 (2003).

    Article  CAS  Google Scholar 

  70. 70

    Cartmell, S. H., Porter, B. D., Garcia, A. J. & Guldberg, R. E. Effects of medium perfusion rate on cell-seeded three-dimensional bone constructs in vitro. Tissue Eng. 9, 1197?1203 (2003).

    Article  CAS  Google Scholar 

  71. 71

    Chary, S. R. & Jain, R. K. Direct measurement of interstitial convection and diffusion of albumin in normal and neoplastic tissues by fluorescence photobleaching. Proc. Natl Acad. Sci. USA 86, 5385?5389 (1989).

    Article  CAS  Google Scholar 

  72. 72

    Dafni, H., Israely, T., Bhujwalla, Z. M., Benjamin, L. E. & Neeman, M. Overexpression of vascular endothelial growth factor 165 drives peritumor interstitial convection and induces lymphatic drain: magnetic resonance imaging, confocal microscopy, and histological tracking of triple-labeled albumin. Cancer Res. 62, 6731?6739 (2002).

    CAS  PubMed  Google Scholar 

  73. 73

    Gurdon, J. B. & Bourillot, P. Y. Morphogen gradient interpretation. Nature 413, 797?803 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. 74

    Quinn, T. M., Grodzinsky, A. J., Buschmann, M. D., Kim, Y. J. & Hunziker, E. B. Mechanical compression alters proteoglycan deposition and matrix deformation around individual cells in cartilage explants. J. Cell Sci. 111, 573?583 (1998).

    CAS  PubMed  Google Scholar 

  75. 75

    Garcia, A. M., Lark, M. W., Trippel, S. B. & Grodzinsky, A. J. Transport of tissue inhibitor of metalloproteinases-1 through cartilage: Contributions of fluid flow and electrical migration. J. Orthop. Res. 16, 734?742 (1998).

    Article  CAS  Google Scholar 

  76. 76

    Semino, C. E., Kamm, R. D. & Lauffenburger, D. A. Autocrine EGF receptor activation mediates endothelial cell migration and vascular morphogenesis induced by VEGF under interstitial flow. Exp. Cell Res. 312, 289?298 (2006).

    CAS  PubMed  Google Scholar 

  77. 77

    Swartz, M. A., Tschumperlin, D. J., Kamm, R. D. & Drazen, J. M. Mechanical stress is communicated between cell types to elicit matrix remodeling. Proc. Natl Acad. Sci. USA 98, 6180?6185 (2001).

    Article  CAS  Google Scholar 

  78. 78

    Choe, M. M., Sporn, P. H. S. & Swartz, M. A. An in vitro airway wall model of remodeling. Am. J. Physiol. Lung Cell Physiol. 285, L427?L433 (2003).

    Article  CAS  Google Scholar 

  79. 79

    Popel, A. S. & Johnson, P. C. Microcirculation and microrheology. Ann. Rev. Fluid Mech. 37, 43?69 (2005).

    Article  Google Scholar 

  80. 80

    Schmid-Schonbein, G. W. Biomechanics of microcirculatory blood perfusion. Annu. Rev. Biomed. Eng. 1, 73?102 (1999).

    Article  CAS  Google Scholar 

  81. 81

    Boardman, K. C. & Swartz, M. A. Interstitial flow as a guide for lymphangiogenesis. Circ. Res. 92, 801?808 (2003).

    Article  CAS  Google Scholar 

  82. 82

    Ng, C. P., Helm, C. L. & Swartz, M. A. Interstitial flow differentially stimulates blood and lymphatic endothelial cell morphogenesis in vitro. Microvasc. Res. 68, 258?264 (2004).

    Article  Google Scholar 

  83. 83

    LeCouter, J. et al. Angiogenesis-independent endothelial protection of liver: role of VEGFR-1. Science 299, 890?893 (2003).

    Article  CAS  Google Scholar 

  84. 84

    Shin, V., Zebboudj, A. F. & Bostrom, K. Endothelial cells modulate osteogenesis in calcifying vascular cells. J. Vasc. Res. 41, 193?201 (2004).

    Article  Google Scholar 

  85. 85

    Matsumoto, K., Yoshitomi, H., Roussant, J. & Zaret, K. Liver organogenesis promoted by endothelial cells prior to vascular function. Science 294, 559?563 (2001).

    Article  CAS  Google Scholar 

  86. 86

    Cao, Y. Emerging mechanisms of tumour lymphangiogenesis and lymphatic metastasis. Nature Rev. Cancer 5, 735?743 (2005).

    Article  CAS  Google Scholar 

  87. 87

    Swartz, M. A. & Skobe, M. Lymphatic function, lymphangiogenesis, and cancer metastasis. Microsc. Res. Tech. 55, 92?99 (2001).

    Article  CAS  Google Scholar 

  88. 88

    Saharinen, P., Tammela, T., Karkkainen, M. J. & Alitalo, K. Lymphatic vasculature: development, molecular regulation and role in tumor metastasis and inflammation. Trends Immunol. 25, 387?395 (2004).

    Article  CAS  Google Scholar 

  89. 89

    Van Trappen, P. O. & Pepper, M. S. Lymphatic dissemination of tumour cells and the formation of micrometastases. Lancet Oncol. 3, 44?52 (2002).

    Article  CAS  Google Scholar 

  90. 90

    Balkwill, F. Cancer and the chemokine network. Nature Rev. Cancer 4, 540?550 (2004).

    Article  CAS  Google Scholar 

  91. 91

    Mougel, L. et al. Three-dimensional culture and multidrug resistance: effects on immune reactivity of MCF-7 cells by monocytes. Anticancer Res. 24, 935?941 (2004).

    CAS  PubMed  Google Scholar 

  92. 92

    Elgert, K. D., Alleva, D. G. & Mullins, D. W. Tumor-induced immune dysfunction: The macrophage connection. J. Leukoc. Biol. 64, 275?290 (1998).

    Article  CAS  Google Scholar 

  93. 93

    Wiley, H. E., Gonzalez, E. B., Maki, W., Wu, M. T. & Hwang, S. T. Expression of CC chemokine receptor-7 and regional lymph node metastasis of B16 murine melanoma. J. Natl Cancer Inst. 93, 1638?1643 (2001).

    Article  CAS  Google Scholar 

  94. 94

    Patel, D. D. et al. Chemokines have diverse abilities to form solid phase gradients. Clin. Immunol. 99, 43?52 (2001).

    Article  CAS  Google Scholar 

  95. 95

    Goswami, S. et al. Macrophages promote the invasion of breast carcinoma cells via a colony-stimulating factor-1/epidermal growth factor paracrine loop. Cancer Res. 65, 5278?5283 (2005). Compelling evidence of a cell-generated chemotactic gradient that operates in 3D on a heterotypic cell type.

    Article  CAS  Google Scholar 

  96. 96

    Muschler, G. F., Nakamoto, C. & Griffith, L. G. Engineering principles of clinical cell-based tissue engineering. J. Bone Joint Surg. Am. 86-A, 1541?1558 (2004).

    Article  Google Scholar 

  97. 97

    Stern, R., McPherson, M. & Longaker, M. T. Histologic study of artificial skin used in the treatment of full-thickness thermal injury. J. Burn Care Rehabil. 11, 7?13 (1990).

    Article  CAS  Google Scholar 

  98. 98

    Mansbridge, J., Liu, K., Patch, R., Symons, K. & Pinney, E. Three-dimensional fibroblast culture implant for the treatment of diabetic foot ulcers: metabolic activity and therapeutic range. Tissue Eng. 4, 403?414 (1998).

    Article  CAS  Google Scholar 

  99. 99

    Yannas, I. V. Synthesis of tissues and organs. ChemBioChem 5, 26?39 (2004).

    Article  CAS  Google Scholar 

  100. 100

    Stock, U. A. & Vacanti, J. P. Tissue engineering: current state and prospects. Annu. Rev. Med. 52, 443?451 (2001).

    Article  CAS  Google Scholar 

  101. 101

    Yannas, I. V., Lee, E., Orgill, D. P., Skrabut, E. M. & Murphy, G. F. Synthesis and characterization of a model extracellular matrix that induces partial regeneration of adult mammalian skin. Proc. Natl Acad. Sci. USA 86, 933?937 (1989). Pioneering demonstration of design principles that are applied to the development of synthetic scaffolds for tissue regeneration.

    Article  CAS  Google Scholar 

  102. 102

    Jakab, K., Neagu, A., Mironov, V., Markwald, R. R. & Forgacs, G. Engineering biological structures of prescribed shape using self-assembling multicellular systems. Proc. Natl Acad. Sci. USA 101, 2864?2869 (2004).

    Article  CAS  Google Scholar 

  103. 103

    Tremblay, P. L., Hudon, V., Berthod, F., Germain, L. & Auger, F. A. Inosculation of tissue-engineered capillaries with the host's vasculature in a reconstructed skin transplanted on mice. Am. J. Transplant. 5, 1002?1010 (2005).

    Article  Google Scholar 

  104. 104

    Levenberg, S. et al. Engineering vascularized skeletal muscle tissue. Nature Biotechnol. 23, 879?884 (2005).

    Article  CAS  Google Scholar 

  105. 105

    Tlsty, T. D. & Hein, P. W. Know thy neighbor: stromal cells can contribute oncogenic signals. Curr. Opin. Genet. Dev. 11, 54?59 (2001).

    Article  CAS  Google Scholar 

  106. 106

    Jasmund, I. & Bader, A. Bioreactor developments for tissue engineering applications by the example of the bioartificial liver. Adv. Biochem. Eng. Biotechnol. 74, 99?109 (2002).

    CAS  PubMed  Google Scholar 

  107. 107

    Poznansky, M. C. et al. Efficient generation of human T cells from a tissue-engineered thymic organoid. Nature Biotechnol. 18, 729?734 (2000).

    Article  CAS  Google Scholar 

  108. 108

    Stachowiak, A. N., Bershteyn, A. & Irvine, D. J. Bioactive hydrogels with an ordered cellular structure combine interconnected macroporosity and robust mechanical properties. Adv. Mater. 17, 399?403 (2005). Demonstration of an innovative approach in creating synthetic scaffolds for the recreation of complex tissue behaviours in vitro.

    Article  CAS  Google Scholar 

  109. 109

    Whitesides, G. M., Ostuni, E., Takayama, S., Jiang, X. & Ingber, D. E. Soft lithography in biology and biochemistry. Annu. Rev. Biomed. Eng. 3, 335?373 (2001).

    Article  CAS  Google Scholar 

  110. 110

    Klebe, R. J. Cytoscribing: a method for micropositioning cells and the construction of two- and three-dimensional synthetic tissues. Exp. Cell Res. 179, 362?373 (1988).

    Article  CAS  Google Scholar 

  111. 111

    Tsuda, Y. et al. The use of patterned dual thermoresponsive surfaces for the collective recovery as co-cultured cell sheets. Biomaterials 26, 1885?1893 (2005).

    Article  CAS  Google Scholar 

  112. 112

    Andersson, H. & van den Berg, A. Microfabrication and microfluidics for tissue engineering: state of the art and future opportunities. Lab Chip 4, 98?103 (2004).

    Article  CAS  Google Scholar 

  113. 113

    Tan, W. & Desai, T. A. Microscale multilayer cocultures for biomimetic blood vessels. J. Biomed. Mater. Res. A 72, 146?160 (2005).

    Article  CAS  Google Scholar 

  114. 114

    Shin, M. et al. Endothelialized networks with a vascular geometry in microfabricated poly(dimethyl siloxane). Biomed. Microdevices 6, 269?278 (2004).

    Article  CAS  Google Scholar 

  115. 115

    Weibel, D. B., Garstecki, P. & Whitesides, G. M. Combining microscience and neurobiology. Curr. Opin. Neurobiol. 15, 560?567 (2005).

    Article  CAS  Google Scholar 

  116. 116

    Hansen, C. & Quake, S. R. Microfluidics in structural biology: smaller, faster em leader better. Curr. Opin. Struct. Biol. 13, 538?544 (2003).

    Article  CAS  Google Scholar 

  117. 117

    Lin, F. et al. Neutrophil migration in opposing chemoattractant gradients using microfluidic chemotaxis devices. Ann. Biomed. Eng. 33, 475?482 (2005).

    Article  Google Scholar 

  118. 118

    Lu, H. et al. Microfluidic shear devices for quantitative analysis of cell adhesion. Anal. Chem. 76, 5257?5264 (2004).

    Article  CAS  Google Scholar 

  119. 119

    Song, J. W. et al. Computer-controlled microcirculatory support system for endothelial cell culture and shearing. Anal. Chem. 77, 3993?3999 (2005).

    Article  CAS  Google Scholar 

  120. 120

    Sin, A. et al. The design and fabrication of three-chamber microscale cell culture analog devices with integrated dissolved oxygen sensors. Biotechnol. Prog. 20, 338?345 (2004). Shows the principles of multi-compartment tissue models 'on a chip', incorporating many principles of microfabrication and microfluidics.

    Article  CAS  Google Scholar 

  121. 121

    Yaakov, N., Schwartz, R. E., Hu, W.-S., Verfaillie, C. & Odde, D. J. Endothelium-mediated hepatocyte recruitment in the establishment of liver-like tissue in vitro. Tissue Eng. (in the press).

  122. 122

    DeLeve, L. D., Wang, X., Hu, L., McCuskey, M. K. & McCuskey, R. S. Rat liver sinusoidal endothelial cell phenotype is maintained by paracrine and autocrine regulation. Am. J. Physiol. Gastrointest. Liver Physiol. 287, G757?G763 (2004).

    Article  CAS  Google Scholar 

  123. 123

    Powers, M. J. et al. Functional behavior of primary rat liver cells in a three-dimensional perfused microarray bioreactor. Tissue Eng. 8, 499?513 (2002).

    Article  Google Scholar 

  124. 124

    Powers, M. J. et al. A microfabricated array bioreactor for perfused 3D liver culture. Biotechnol. Bioeng. 78, 257?269 (2002).

    Article  CAS  Google Scholar 

  125. 125

    Lin, C. Y., Kikuchi, N. & Hollister, S. J. A novel method for biomaterial scaffold internal architecture design to match bone elastic properties with desired porosity. J. Biomech. 37, 623?636 (2004).

    Article  Google Scholar 

  126. 126

    Yeong, W. Y., Chua, C. K., Leong, K. F. & Chandrasekaran, M. Rapid prototyping in tissue engineering: challenges and potential. Trends Biotechnol. 22, 643?652 (2004).

    Article  CAS  Google Scholar 

  127. 127

    Sherwood, J. K. et al. A three-dimensional osteochondral composite scaffold for articular cartilage repair. Biomaterials 23, 4739?4751 (2002).

    Article  CAS  Google Scholar 

  128. 128

    Bornstein, P. & Sage, E. H. Matricellular proteins: extracellular modulators of cell function. Curr. Opin. Cell Biol. 14, 608?616 (2002).

    Article  CAS  Google Scholar 

  129. 129

    Sigal, S. H., Brill, S., Fiorino, A. S. & Reid, L. M. The liver as a stem cell and lineage system. Am. J. Physiol. 263, G139?G148 (1992).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank A. Hwa, A. Wells, D. Stolz, S. Watkins, C. Yates, G. Papworth and P.T. So for the use of unpublished photos. We thank D. Lauffenburger, F. Gertler and V. Weaver for critical review of the manuscript.

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Correspondence to Linda G. Griffith.

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Glossary

Feature

An architectural or compositional component of a scaffold that delineates a distinct, defined region. For example, features in a scaffold with a honeycomb architecture would include: the walls, the hexagonal channels and the overall shape.

Acinar

Pertaining to a sac-like tissue structure in the shape of an acinus, which is a polarized epithelial layer surrounding a small lumen that contains secretions (such as milk) from epithelial cells.

Proteoglycans

Extracellular matrix components that consist of a protein core and glycosamino side chains. They are huge molecules (>100 MDa) with a high fixed-charge density and are crucial to maintaining the fluid balance and storing growth factors, cytokines and other morphogens in the matrix.

Matrigel

Commercially available extract of the basement membrane-like ECM that is secreted by the murine Engelbreth-Holm-Swarm (EHS) tumour and that is rich in laminin, type IV collagen, heparan sulphate proteoglycans and growth factors. It supports the in vitro formation of tubes from endothelial cells, as well as the in vitro differentiation of many epithelial cell types.

Convection

Transport by fluid flow (as opposed to diffusion); can refer to the transport of fluid or of solute that is dissolved in the fluid and carried by the flow.

Autocrine loop

Mode of growth-factor signalling in which a cell that expresses a particular growth-factor receptor also synthesizes and releases the corresponding ligand, by which the receptor is activated.

Interstitial flow

Flow through or within the 3D extracellular matrix (as opposed to across a surface or within a vessel).

Starling force

A force that drives fluid movement, including gradients or differences in hydrostatic pressure (which drives fluid flow from higher to lower pressures) and osmotic pressure (which drives fluid flow from less concentrated to more concentrated areas).

Fluid shear stress

Mechanical stress on a surface (for example, of a cell or ECM fibre) caused by fluid flow across that surface.

Angiogenesis

The growth of new blood vessels by sprouting from existing vessels in a process that involves endothelial-cell migration and proliferation.

Glycosaminoglycans

Polysaccharide chains of ECM proteoglycans, comprising disaccharide-repeat units with one amino sugar and one negatively charged (carboxylated or sulphated) sugar.

Resolution

The smallest dimensions over which the placement or size of a feature can be controlled during the fabrication of a device or scaffold. There are three measures of resolution: positive feature size (the minimum width of a wall that can be created); negative feature size (the minimum possible width of a channel or hole) and feature placement (how reproducible the spacing is between features).

Capillary bed

A region of tissue that contains a local network of blood microvessels, where the intimate exchange of fluid and molecular components between blood and tissues occurs.

Sinusoidal capillary

A discontinuous capillary that consists of endothelial cells with unusually wide gaps between them, and (partially) lacking a basement membrane. Sinusoidal capillaries can be found in liver, spleen and bone marrow.

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Griffith, L., Swartz, M. Capturing complex 3D tissue physiology in vitro. Nat Rev Mol Cell Biol 7, 211–224 (2006). https://doi.org/10.1038/nrm1858

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