Cells on chips Jamil El-Ali1, Peter K. Sorger2 & Klavs F. Jensen1 Microsystems create new opportunities for the spatial and temporal control of cell growth and stimuli by combining surfaces that mimic complex biochemistries and geometries of the extracellular matrix with microfluidic channels that regulate transport of fluids and soluble factors. Further integration with bioanalytic microsystems results in multifunctional platforms for basic biological insights into cells and tissues, as well as for cell-based sensors with biochemical, biomedical and environmental functions. Highly integrated microdevices show great promise for basic biomedical and pharmaceutical research, and robust and portable point-of-care devices could be used in clinical settings, in both the developed and the developing world. In their normal environment, cells are subject to multiple cues that vary in time and space, including gradients of cytokines and secreted proteins from neighbouring cells, biochemical and mechanical interactions with the extracellular matrix (ECM), and direct cell���cell contacts (Box 1). Microfabricated systems can present cells with these cues in a control- lable and reproducible fashion that cannot easily be achieved by standard tissue culture, and can be used to link cell culture with integrated analy- tical devices that can probe the biochemical processes that govern cell behaviour. Some cell-based microsystems simply represent miniaturized versions of conventional laboratory techniques, whereas others exploit the advantages of small length scales and low Reynolds numbers1, such as favourable scaling of electrical fields and the ability to create well-control- led laminar flows. In this Review, we discuss the application of microtech- nology to cell biology and describe methods for cell culture, regulation of extracellular cues, cell fractionations and biochemical analysis on a micrometre scale (Fig. 1). Emphasis is placed on microsystems aimed at gaining biological insight, as well as on efforts to realize increasing cell- handling integration and biochemical analysis levels on chips. We believe these devices will become increasingly implemented in applied and basic biomedical research, mainly because soft lithography2 has put microfluidics within the reach of biology-focused academic laboratories. Elastomeric materials used in soft lithography, typically 1Department of Chemical Engineering, and 2Department of Biology and Biological Engineering, Center for Cell Decision Processes, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. poly(dimethylsiloxane) (PDMS), are relatively easy to fabricate, and are compatible with most biological assays. Devices that are based on micro- fabrication of silicon and glass require access to advanced cleanroom facilities similar to those used for microelectronics. This typically involves higher cost, but has unique advantages for specialized applications, such as electrophoresis in glass devices. Much cell-based microsystem research takes place under a ���lab-on- a-chip��� or ���micro-total-analysis-system��� (��TAS) framework that seeks to create microsystems incorporating several steps of an assay into a single system3���5. Integrated microfluidic devices perform rapid and reproduc- ible measurements on small sample volumes while eliminating the need for labour-intensive and potentially error-prone laboratory manipu- lations. Thus, microfluidics allows experiments to be carried out that cannot be performed simply by miniaturizing and mechanizing conven- tional laboratory procedures using robotics and microplates. For exam- ple, in cell-based studies, the transition from 384- to 1,536-well plates is proving challenging, largely because edge effects and uncontrolled evaporation from very small wells result in poorly defined culture condi- tions. Conventional handling of very small fluidic volumes is difficult, and subject to both variability and high fixed losses. The fabrication of many copies of an analytic device, small reagent volumes, and dimin- ished labour associated with use of automated microfabricated devices 403 INSIGHT REVIEW NATURE|Vol 442|27 July 2006|doi:10.1038/nature05063 Soluble signalling molecules include hormones, cytokines and growth factors produced by local or distant cells (giving rise to paracrine and endocrine signals, respectively), and even by the receiving cell itself (autocrine signals). Insoluble signalling molecules include components of the ECM and membrane-bound proteins on neighbouring cells. Cells sense most extracellular signals (including proteins, peptides and carbohydrates) via transmembrane receptors that activate complex biochemical cascades of kinases, proteases, adaptor proteins, transcription factors and so on, which together act to regulate cell physiology. At the same time, cells alter their surrounding environment by making or destroying ECM or soluble factors and by exerting mechanical force99,100. In animals, cells typically reside in environments with very specific three-dimensional (3D) features. Cells are sensitive to the presence of neighbouring cells of similar or different type and often make long-lasting mechanical and biochemical connections to them. In columnar epithelia, for example, identical cells lined up side by side assemble junctions with neighbours to form continuous impermeable sheets. These sheets are polarized such that cells interact with their surroundings in very different ways on the lumenal and basolateral surfaces. In addition, epithelia usually establish a close relationship with specific types of immune cell. In these epithelia, both homotypic and heterotypic cell���cell interactions are essential to maintain cell and tissue function. Most in vitro experiments with adherent human cells are performed in two-dimensional (2D) cultures in which cells are plated onto plastic surfaces treated to stimulate cell binding. Depending on their type, cells either grow directly on the plastic, secrete ECM components that coat the plastic to facilitate cell adhesion, or require pre-coating of the plastic with ECM. Standard 2D culture conditions are poor mimics of the cellular environment in an animal: soluble growth factors are present at abnormally high concentrations, 3D cues are largely absent, oxygen tension is too high and cell���cell interactions are rarely organized. Attempts have been made to overcome these problems using organ culture and various laboratory-scale bioreactors, but microsystems provide a much more effective means of controlling cell environment in vitro. Particularly promising are various artificial organ systems in which multiple cell types are grown together under conditions that mimic normal 3D environmental and circulatory cues. Box 1 | Cell physiology, phenotype and fate are regulated by cell-autonomous processes and extracellular signalling molecules Nature Publishing Group ��2006

should make them highly cost effective. Moreover, the small footprint and low power consumption of integrated systems creates opportunities for portable, point-of-care devices that can perform analyses hitherto possible only in the research or clinical laboratory. Devices such as these with sophisticated diagnostic capabilities are likely to become important in the personalization of medical care. Many of the promises of ��TAS have yet to be realized: integration and packaging of several functionalities into a single system is proving to be a complex task (Fig. 1), and many cell-based microsystems avail- able today are still in the proof-of-concept phase. Typical unit opera- tions (for example, growth, treatment, selection, lysis, separation and analysis) have been demonstrated (Fig. 2), but robust approaches to fabrication, integration and packaging (such as communication with the macroenvironment) remain major areas of research. Microfabricated cell cultures Culturing cells in vitro is one of the cornerstones of modern biology. Nevertheless, even for intensively studied tissues, many of the factors that induce or stabilize differentiated phenotypes are poorly under- stood and difficult to mimic in vitro6. One approach to increase control over cell���cell and soluble cues typical of in vivo cell environments is to combine microfabrication of 3D ECM structures and microfluidic networks that transport soluble factors such as nutrients and oxygen. Microfluidics has the additional advantage of being capable of creating mechanical strain, through shear, in the physiological range. Cells and the extracellular matrix Microfabrication integrating micropatterning techniques with advanced surface chemistry makes it possible to reproducibly engineer cell micro- environment at cellular resolution. A large variety of surface-patterning techniques are available, including standard photolithography liftoff techniques, photoreactive chemistry and, increasingly, techniques based on soft lithography (microcontact printing and fluidic patterning)7. Surface patterning of micrometre-sized features allows micrometre- scale control over cell���ECM interactions and can be used to generate ensembles of cells with defined geometry. Lamination, moulding and photo-polymerization techniques all allow fabrication of 3D scaffolds with feature sizes in the lower micrometre range, including microstruc- tured scaffolds made of biodegradable materials8. The precise control of the cellular environment that has been made pos- sible by microtechnology provides new opportunities for understanding biochemical and mechanical processes responsible for changes in behav- iour such as the effects of cell shape on the anchorage-dependence of cell growth9,10. For example, by altering the spacing of a grid of cell-adhesive islands it is possible to control the extent of cell spreading, while keeping the cell���ECM contact area constant10 (Fig. 3a). Human capillary endothe- lial cells confined to closely spaced islands undergo apoptosis, whereas cells that can spread freely survive and proliferate normally10. Adhesive ECM patches can also be designed so that the locations of focal adhesions (integrin-mediated links between the ECM and actin cytoskeleton) result in the same overall cell shape, but with a different underlying cytoskeletal organization (Fig. 3b). By allowing cells to spread and proliferate on these adhesive patches the orientation of the cell division axis can be controlled11. Similar regulation of the division axis by the ECM is likely to be important for tissue morphogenesis and other developmental processes. The force exerted on the ECM by cells can be measured in several ways. A particularly powerful method involves measuring the deflec- tion of arrays of micrometre-sized vertical elastomer posts (Fig. 3c). When tested with smooth muscle cells, forces acting in the plane of the substrate are in the range of 100 nN, and appear to scale with the area covered by focal adhesions12. Compared with conventional meth- ods that rely on substrate distortion, the elastomer-post technique has Growth media Stimulation Cell lysis Tissue organization Cell selection Cell imaging Biochemical analysis Figure 1 | Tissue organization, culture and analysis in microsystems. Advanced tissue organization and culture can be performed in microsystems by integrating homogeneous and heterogeneous cell ensembles, 3D scaffolds to guide cell growth, and microfluidic systems for transport of nutrients and other soluble factors. Soluble factors ��� for example, cytokines for cell stimulation ��� can be presented to the cells in precisely defined spatial and temporal patterns using integrated microfluidic systems. Microsystems technology can also fractionate heterogeneous cell populations into homogeneous populations, including single-cell selection, so different cell types can be analysed separately. Microsystems can incorporate numerous techniques for the analysis of the biochemical reactions in cells, including image-based analysis and techniques for gene and protein analysis of cell lysates. This makes microtechnology an excellent tool in cell-based applications and in the fundamental study of cell biology. As indicated by the yellow arrows, the different microfluidic components can be connected with each other to form an integrated system, realizing multiple functionalities on a single chip. However, this integration is challenging with respect to fluidic and sample matching between the different components, not least because of the difficulty in simultaneously packaging fluidic, optical, electronic and biological components into a single system. 404 NATURE|Vol 442|27 July 2006 INSIGHT REVIEW Nature Publishing Group ��2006