Commentary 3015 Deconstructing the third dimension – how 3D culture microenvironments alter cellular cues Brendon M. Baker and Christopher S. Chen* Department of Bioengineering, University of Pennsylvania, 210 South 33rd Street, Philadelphia, PA 19104, USA *Author for correspondence ([email protected]) Journal of Cell Science 125, 3015–3024 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.079509 Summary Much of our understanding of the biological mechanisms that underlie cellular functions, such as migration, differentiation and force- sensing has been garnered from studying cells cultured on two-dimensional (2D) glass or plastic surfaces. However, more recently the cell biology field has come to appreciate the dissimilarity between these flat surfaces and the topographically complex, three- dimensional (3D) extracellular environments in which cells routinely operate in vivo. This has spurred substantial efforts towards the development of in vitro 3D biomimetic environments and has encouraged much cross-disciplinary work among biologists, material scientists and tissue engineers. As we move towards more-physiological culture systems for studying fundamental cellular processes, it is crucial to define exactly which factors are operative in 3D microenvironments. Thus, the focus of this Commentary will be on identifying and describing the fundamental features of 3D cell culture systems that influence cell structure, adhesion, mechanotransduction and signaling in response to soluble factors, which – in turn – regulate overall cellular function in ways that depart dramatically from traditional 2D culture formats. Additionally, we will describe experimental scenarios in which 3D culture is particularly relevant, highlight recent advances in materials engineering for studying cell biology, and discuss examples where studying cells in a 3D context provided insights that would not have been observed in traditional 2D systems. This article is part of a Minifocus on Mechanotransduction. For further reading, please see related articles: ‘Finding the weakest link – exploring integrin-mediated mechanical molecular pathways’ by Pere Roca-Cusachs et al. (J. Cell Sci. 125, 3025-3038). ‘Signalling through mechanical inputs – a coordinated process’ by Huimin Zhang and Michel Labouesse (J. Cell Sci. 125, 3039-3049). ‘United we stand – integrating the actin cytoskeleton and cell–matrix adhesions in cellular mechanotransduction’ by Ulrich S. Schwarz and Margaret L. Gardel (J. Cell Sci. 125, 3051-3060). ‘Mechanosensitive mechanisms in transcriptional regulation’ by Akiko Mammoto et al. (J. Cell Sci. 125, 3061-3073). ‘Molecular force transduction by ion channels – diversity and unifying principles’ by Sergei Sukharev and Frederick Sachs (J. Cell Sci. 125, 3075-3083). Key words: 3D culture models, Cell adhesion, Dimensionality, Mechanotransduction, Microenvironment, Soluble factors Journal of Cell Science Introduction structures and establish a de novo basement membrane (Emerman Our current understanding of many biological processes is based and Pitelka, 1977; Lee et al., 1984; Petersen et al., 1992). largely on studies of homogenous populations of cells cultured on These observations have led to the notion that the dimension in flat, two-dimensional (2D) plastic or glass substrates. However, which cells are cultured is a crucial fate determinant, and to the in vivo, cells primarily exist embedded within a complex and vague impression that culturing cells in monolayer drives information-rich environment that contains multiple extracellular abnormal cell function or dedifferentiation, whereas 3D culture matrix (ECM) components, mixed cell populations that interact elicits a more physiological state. However, we must be wary of heterotypically and a medley of cell-secreted factors. The striking oversimplifying these comparisons into a single difference disparity between traditional monolayer culture and the in vivo between two states, i.e. three-dimensionality versus two- scenario has been a double-edged sword: the simplicity of 2D dimensionality. Presently, dimensionality has become a blanket culture has enabled reductionist approaches to understanding statement for what entails many potential differences between individual cellular phenomena but these findings have come with traditional culture in a 2D monolayer, 3D culture systems and the the caveat that the 2D model might not faithfully capture the physiological setting. Rather than the overall dimensional shape physiological behavior of cells in vivo. of the cell or culture, functional consequences instead originate Indeed, many cell types, when isolated from tissues and placed from the finer features that are inherent to each of these contexts. into planar cell culture, become progressively flatter, divide Thus, rather than simply concluding that a dimensionality factor aberrantly and lose their differentiated phenotype (von der Mark is at play, we must identify and understand the salient features of et al., 1977; Petersen et al., 1992). Interestingly, some of these cell each experimental setting and strive to demystify exactly what types can regain their physiological form and function when 3D culture provides to the cells that differs from more traditional embedded in a three-dimensional (3D) culture environment. For 2D settings. instance, encapsulation of dedifferentiated chondrocytes restores With this goal in mind, this Commentary will examine the their physiological phenotype, including cell shape and the main avenues by which microenvironmental cues are known to expression of cartilaginous markers (Benya and Shaffer, 1982). impact cell function – cell adhesions, mechanical forces and Similarly, mammary epithelial cells embedded in a 3D diffusible factors – and how such cues may be presented in 3D environment halt uncontrolled division, assemble into acinar versus 2D culture. Beyond providing appropriate physiological 3016 Journal of Cell Science 125 (13) cues, 3D culture also facilitates biological responses that might and the migration of cancerous cells through stroma and into not be observable on 2D substrates. For example, the collective lymphatics during metastasis, are all cases of higher-order cell cell migration, force generation and tissue folding that occurs processes that are inherently 3D (Fig. 1). Deconstructing these during gastrulation, the angiogenic sprouting of blood vessels, 3D microenvironments and the associated processes into adhesive, mechanical and chemical components will aid us in understanding the underlying mechanisms that guide A Chondrogenesis these processes. Furthermore, because the technologies for engineering the cellular environment are rapidly evolving, we also examine some of the methods that can be employed for studying these different cues in vitro (see Boxes 1 and 2). This Mechanical cues Commentary is not intended to be an exhaustive compilation of the literature on cell biology in 3D but, rather, seeks to identify Matrix some salient features of 3D experimental systems that should be synthesis and remodeling considered in the questions we pose and the studies we conduct. Interstitial flow Cell adhesion and structure For anchorage-dependent cells, adhesive interactions with the Soluble surrounding ECM and neighboring cells define cell shape and cues Proliferation organization. The organization, composition and number of Adhesive adhesions are among the better understood signals that are cues integrated by the cell in order to regulate many fundamental cellular behaviors, including survival, differentiation, proliferation and migration (Wozniak et al., 2004; Weber et al., 2011; Orr et al., 2006; Geiger et al., 2009). Unavoidably, studying cell biology in B Angiogenesis vitro necessitates stripping cells of these native cell–cell and cell– ECM interactions, and introducing them into a foreign adhesive Repolarization environment that is defined by the culture system. One of the most striking differences observed when comparing Mechanical Migration cues cells in 2D and 3D is the dissimilarity in morphology (Fig. 2). Cells grown in a monolayer are flat, and can adhere and spread Proliferation freely in the horizontal plane but have no support for spreading in the vertical dimension. One consequence of this is that cells that Topographical cues are cultured on 2D surfaces have a forced apical–basal polarity. Soluble and This polarity is arguably relevant for some cell types, such as Journal of Cell Science ECM-bound epithelial cells, but is unnatural for most mesenchymal cells, cues Cell-cell which – when embedded in a 3D ECM – assume a stellate adhesion morphology and only polarize from front to rear during migration Matrix (Mseka et al., 2007). These changes in cell geometry and remodeling organization can directly impact cell function. For instance, Multicellular apical-basal polarity has been shown to modulate the sensitivity reorganization of cells to apoptosis (Weaver et al., 2002). It also has been C Metastasis Fig. 1. 3D cellular phenomena in development, tissue homeostasis and disease are conducted by adhesive, mechanical and chemical cues originating from other cells and the extracellular environment. (A) Chondrocytes (blue) reside within a specialized pericellular ECM, where they are exposed to compressive forces, interstitial fluid flow, adhesive cues Adhesive Polarization and soluble cues in the form of cytokines, which allow the cells to form and cues maintain the surrounding cartilage. (B) In response to soluble