Biosensors and Bioelectronics ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Cells on chip for multiplex screening Ophélie I. Berthuy, Loïc J. Blum, Christophe A. Marquette n Institut de Chimie et Biochimie Moléculaires et Supramoléculaires Equipe Génie Enzymatique, Membranes Biomimétiques et Assemblages Supramoléculaires Université Lyon 1-CNRS 5246 ICBMS Bâtiment CPE 43, bd du 11 novembre 1918, 69622 Villeurbanne, Cedex, France

art ic l e i nf o

a b s t r a c t

Article history: Received 23 February 2015 Received in revised form 8 April 2015 Accepted 10 April 2015

Microarray technology was developed in the early 1990s to measure the transcription levels of thousands of genes in parallel. The basic premise of high-density arraying has since been expanded to create cells microarrays. Cells on chip are powerful experimental tools for high-throughput and multiplex screening of samples or cellular functions. Miniaturization increases assay throughput while reducing both reagent consumption and cell population heterogeneity effect, making these systems attractive for a wide range of assays, from drug discovery to toxicology, stem cell research and therapy. One form of cell microarrays, the transfected cell microarray, wherein plasmid DNA or siRNA, spotted on the surface of a substrate, is reverse-transfected locally into adherent cells, has become a standard tool for parallel cell-based analysis. With the advent of technologies, cells can also be directly spotted onto functionalized surfaces using robotic fluid-dispensing devices, or printed directly in bio-ink material. We are providing herein an overview of the last developments in optical cell microarrays allowing high-throughput and high-content analysis. & 2015 Elsevier B.V. All rights reserved.

Keywords: Bioprinting Cell microarrays High-throughput Multiplex Optical detection Reverse transfection

1. Introduction Microarray technology was developed for the analysis of gene expression (Schena et al., 1995), when short nucleotide probes were printed onto the surface of slides for testing their degree of hybridization to the investigated cell-derived cDNA. Since then, many other applications of this technique have been introduced and the types of probes expanded drastically to lead to the most complex ones: living cells. Indeed, since cells are the basic unit of a multicellular organism, understanding the complex effect of the tested molecules may require the use of living, intact cells as detecting agents instead of only one of its purified components. Cell-based array technology permits simultaneous detection of several different activities located at the surfaces or inside cells, allowing the complex characterization of cells with an amount of information that is hardly assessed by any other technique. Furthermore, binding of cells to printed antibodies or ligands may induce their activation, and consequently the outcome of these interactions, such as phosphorylation, gene expression, secretion of various products, differentiation, proliferation and apoptosis of the cells are also measurable on arrays. Moreover, since cells can be transfected with printed vectors, over- or under-expression of selected genes is also achievable simultaneously, creating a nice n

Corresponding author. E-mail address: [email protected] (C.A. Marquette).

tool for assessing the function of a given gene. The high-throughput cell-based microarray technology enables testing the effect of external stimuli on a scale barely thinkable earlier. These microarrays are also of great importance for a variety of applications, including drug testing, toxicology and basic cell biology (Kawasaki, 2004; Rettig and Folch, 2005). Cell-based arrays are built on two approaches. The single-cell microarray technique analyses cells individually, using microchamber array chips with thousands of cells situated in microwells (Taylor and Walt, 2000; Yamamura et al., 2005). The other approach explores the influence of numerous arrayed materials on several cells (Moeller et al., 2008). Cell multiplexing is multiple cell lines on a single assay (or surface), cell arrays are not necessary multiplexed. The challenges in the fabrication of multiplexed cell assays are: (a) keeping cell alive after localization, (b) avoiding cell migration from one location to another, (c) getting a biochemical signal from each cell lines without cross-talking and (d) culture different lines in the same condition. In the present review, we are illustrating a few nonmultiplexed cell microarrays which allow high parallelization of one cell line and one phenotype, then three different possibilities of multiplexed cell microarrays: (a) multiplexing on chip through in-situ transfection which allows multiple phenotypes with a unique cell line transfected, (b) multiplexing on chip through localized cell deposition which allows multiple cell lines and (c) other techniques that allows multiplex cell-based microarrays. This

http://dx.doi.org/10.1016/j.bios.2015.04.024 0956-5663/& 2015 Elsevier B.V. All rights reserved.

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review is focused on optical methods but it is worth to mention that cell-based microarrays could be also developed based on other transduction techniques such as electrochemistry (Kelso et al., 2000; Sen et al., 2012; Takahashi et al., 2009).

2. One cell line, multiple phenotypes To speed up the functional exploration of the human genome, there is a need for high-throughput technologies allowing transfection of thousands of nucleic acids in parallel and the simultaneous analysis of thousands of resulting phenotypes. In 2001, Ziauddin and Sabatini succeeded in scaling down high-throughput gene function analysis to the microarray level. Different cDNA expression plasmids were spotted onto slides using a microarray spotter. The dried slides were exposed to a transfection reagent, placed in a culture dish and covered with adherent mammalian cells in medium. This operation created microarrays of simultaneously transfected cell clusters with different plasmids in distinct and defined areas of a lawn of cells (Ziauddin and Sabatini, 2001). With this cell chip, they pave the way to the transfected cell microarray systems. Based on the reverse transfection format, Baghdoyan et al. developed a cell microarray to simultaneously transfect thousands of different nucleic acid molecules, to analyse and quantify phenotypes resulting from either gain or loss gene function. Using a regular DNA arrayer, they were able to print and simultaneously transfect high numbers of nucleic acid. As the cell microarrays are also printed by the same robots as regular DNA arrays, massively parallel transfection of up to 5000 cell clusters per slide could be feasible for effective siRNA (Kumar et al., 2003) or shRNA probes for inhibition of target gene expression (Mousses et al., 2003). The applications of current microarray-based transfection techniques were limited to cells that are easy to grow and transfect, such as HEK293T and COS-7 (Wu et al., 2002). These cells, while typically used as models, have limited relevance to physiological systems in the biomedical field, whereas relevant primary cells, such as hMSCs, are notoriously difficult to handle and transfect (Michiels et al., 2002). That's why Yoshikawa et al. realized a transfection microarray for hMSCs by using the property of fibronectin to increase the transfection efficiency of DNA/polymer complexes. The robustness and versatility of the developed technology was demonstrated through its use in the achievement of on-chip RNAi gene knockdown as well as generalization of the technique to a multitude of mammalian cells (HEK293, hMSC, HeLa, HepG2, NIH3T3) (Yoshikawa et al., 2004). However, since existing methods of measuring transcription provide discrete measurements of a transcriptional response obtained from large populations of cells, they suffer from two major drawbacks. First, quantifying transcription dynamics using microarrays at multiple time-points is expensive when long processes are under study. Second, despite improvements in assay sensitivity, these approaches typically involve pooling mRNA from thousands of cells. The averaged response measured in this way is adequate for classifying different cell or tissue type, but it is not well-suited for studying processes with cell-to-cell variation, such as cell division, differentiation, or drug responsiveness. Recent developments in cell-based assays combined with advances in reporter technology allow to address these limitations, since expression levels can be repeatedly assayed in single-cells. Rajan et al. described a high-throughput platform for measuring transcriptional changes in real time in single mammalian cells. They used reverse transfection microarrays to be able to transfect fluorescent reporter plasmids into 600 independent clusters of cells plated on a single microscope slide and imaged these clusters every 20 min. They used a fast-maturing, destabilized and

nuclear-localized reporter (Venus-NLS-PEST fluorescent reporter (Nagoshi et al., 2004)) that is suitable for automated segmentation to accurately measure promoter activity in single-cells. They tested this platform with synthetic drug-inducible promoters that showed robust induction over 24 h. Automated segmentation and tracking of over 11 million cell images during this period revealed that cells display substantial heterogeneity in their responses to applied treatment, including a large proportion of transfected cells that do not respond at all (Rajan et al., 2011). Though the cell microarrays have slowly evolved to become a more widely accepted screening technology, in many publications, the individual arrays have contained only a modest number of samples, and data from multiple small arrays have been combined for large-scale coverage due to technological limitations of the methods (Doil et al., 2009; Neumann et al., 2010). Rantala et al. described the optimization of a cell spot microarray (CSMA) method which provides a patterned array platform with spatially confined cell spots that allow simple production of cell microarrays with significantly increased sample coverage in microplatesized array plates readily compatible with standard imaging instruments. To allow rapid adaptation of the technique, they optimized an application protocol of the CSMA for 85 cell types and applied the platform to functional genetics profiling of G-protein coupled receptor coding genes in cultured prostate cancer cells and non-malignant epithelial prostate cells, demonstrating the potential of the CSMA for context specific target discovery (Fig. 1A) (Rantala et al. 2011). It is usually necessary to maintain a distance of 500 mm or more between micro-spots on a transfected cell microarrays (Erfle et al., 2007). For higher-density micro-spots, methodological breakthroughs are required to prevent the migration of cells and to limit the diffusion of spotted materials among the micro-spots in the array. The micropatterning of a glass substrate via generation of a hydrophobic or hydrophilic surface is very effective for the regulation of cell adhesion and prevention of the migration of cells among spots (Hook et al., 2009). Fujita et al. described the development of a super-dense transfected cell microarray. To create this microarray, they used an inkjet printer to spot a mixture of plasmid, extracellular matrix (ECM) protein, and other reagents for induction of reverse transfection on a glass substrate that had been previously grafted with polyethylene glycol (PEG). The microspots containing ECM were separated from one another by a hydrophobic barrier generated by PEG, which has proven to be extremely effective in preventing the migration of cells and the cross contamination of reagents among adjacent spots. The densest transfected cell microarray that they prepared had arrays with spots of 50 mm in diameter and 150 mm in pitch (Fig. 1B) (Fujita et al., 2013). To improve reverse transcription efficiency and to prevent spot-to-spot diffusion, the distance between the different transfection clusters was adjusted and various glass material coatings have been used (Hodges et al., 2005; Peterbauer et al., 2006). However, these methods still have as limitation high cost coating material and complex experimental steps. In their study, Oh et al. used a polyethylene glycol diacrylate (PEGA) microwell to generate a spatially separated cell adhesion area and applied it to cell culture and reverse transfection platform for cell-based highthroughput screening. For the first time, olfactory receptors were expressed on the microwell platform using reverse transfection technique. The various olfactory receptors can be expressed simultaneously using this technique and the microwell spotted with olfactory receptors genes can be used as a high-throughput screening platform. The odorant response was detected via fluorescence analysis on the microwell using a cAMP response element (CRE) reporter assay (Oh et al., 2014). Recently, Yamaguchi et al. developed a novel technique for

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constructing microarrays of transfected mammalian cells on or in ECM hydrogels by transfer printing from patterned PEG-oleyl surfaces. A mixed solution of small interfering RNA (siRNA) and a transfection reagent was spotted on PEG-oleyl-coated glass slides using an inkjet printer and the cells were then transiently immobilized on the patterned transfection mixtures. After overlaying an ECM hydrogel sheet onto the immobilized cells, the cells sandwiched between the glass slide and the hydrogel sheet were

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incubated at 37 °C for simultaneous transfection of siRNA into cells and adhesion of cells to the hydrogel sheet. Transfer of the adhered, transfected cells was completed by peeling off the hydrogel sheet (Fig. 1C) (Yamaguchi et al., 2013). These results indicate that transfection of cell microarrays or in a biological matrix is a promising technique for high-throughput screening of disease-related genes by direct observation of cellular phenomena in a physiologically relevant environment.

Fig. 1. Some examples of reverse transfection cell chip. (A) (1) Principle of the cell spot microarray (CSMA) method developed by Rantala et al. (2) Microscope images of 200 mm diameter array displaying the parallel staining of multiple cell types using CSMA method (Rantala et al., 2011). (B) (1) Schematic illustration of the construction of the super-dense TCM developed by Fujita et al. (2) Merged phase-contrast and fluorescence images of HeLa cells on TCM (Fujita et al., 2013). (C) Schematic illustration of the transfer printing of a transfected cell microarray (Yamaguchi et al., 2013).

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3. Multiple cell lines With the advent of robotic spotting technology and microfabrication, it is now possible to distribute nanoliter volumes of different chemicals, biomolecules and cells in a spatially addressable footprint (Falsey et al., 2001; Yamazoe and Iwata, 2005). Therefore, cell-based microarrays are especially well-suited for high-throughput screening of large numbers of very small samples (Howbrook et al., 2003; Nicholson et al., 2007). Soft lithography using elastomeric materials has also been used to generate microbioreactor arrays for high-throughput experiments using human embryonic stem cells (Figallo et al., 2007) and patterned surfaces for the growth of neural stem cells (Ruiz et al., 2008). Polydimethylsiloxane (PDMS) has been used to generate substrates for the culture of single-cells in microwell arrays with 3d cell shape control (Ochsner et al., 2007), and micropatterning of hydrogels, such as hyaluronic acid or agarose, can also serve as an alternative means of generating arrays of cells for high-throughput screening applications (Khademhosseini et al., 2005). Cells can also be directly spotted onto functionalized glass surfaces using robotic fluid-dispensing devices. With this strategy it is possible to generate patterns of cells encapsulated in 3D hydrogel matrices (e.g. collagen or alginate), which support cell growth at the micro-scale. One demonstration reported by Lee and colleagues involves the use of glass slides that are spin-coated with a polymeric material (poly[stryrene-co-maleic anhydride], PS-MA) to increase hydrophobicity of the surface while providing reactive functional groups to attach a hydrogel matrix. A mixture of poly-L-lysine (PLL) and barium chloride (BaCl2) is first spotted onto glass slides coated with PS-MA. The positively charged PLL serves as a substrate to bind Ba² þ ions and assist the attachment of the negatively charged alginate. The Ba² þ ions also cause immediate alginate gelation to give rise to 3D cell-containing matrix spots possessing volumes as low as 20 nL (Lee et al., 2008). Bioprinting is an emerging technology that highlights a growing trend in the fusion of biology and engineering. The ability to design and fabricate complex structures by printing living cells, biomaterials and other biological molecules is crucial to the success of tissue engineering (Calvert, 2007; Guillotin and Guillemot, 2011), and enables new possibilities in drug screening and toxicology (Castel et al., 2006). Cell sorting and separation techniques are essentials tools for biology research and for many diagnostic and therapeutic applications. For many of these applications, it is imperative that heterogeneous populations of cells are segregated according to their cell type and that individual cells can be isolated and analysed. A novel technique to isolate single-cells encapsulated in picolitre sized droplets that are then deposited by inkjet-like printing at defined locations for downstream genomic analysis was presented by Yusof et al. The single-cell manipulation (SCM) developed for this purpose consists of a dispenser chip used to print cells contained in a free flying droplet. A computer vision system is used to detect single-cell inside the dispenser chip prior to printing, and appropriate automation equipment used to print single-cells containing droplet onto defined locations on a substrate. This technique is spatially dynamic, enabling cell printing on a wide range of commonly used substrates such as microscope slides, membranes and microtiter plates (Fig. 2A) (Yusof et al., 2011). Recently, Wu et al. used this technique to construct a cell microarray containing 32 publicly available immortalized breast cell lines with the goal of creating a method to rapidly screen for antigens of interest in breast cancer research in a relatively easy, rapid and cost-effective manner (Wu et al., 2014). Piezoelectric inkjet print-heads with multiple nozzles are the current standard for high-end printing applications (Xu et al., 2005), and could allow for higher throughput and fabrication of

larger cellular constructs (Roth et al., 2004). Rather than developing bio-inks that are suitable for use in these systems, bio-ink design has focused on two-component fast-gelling reactive schemes. Cells have been mixed with alginate and printed into cross-linking Ca2 þ solutions (Arai et al., 2011), or mixed with Ca2 þ and printed into either alginate or alginate/collagen solutions (Xu et al., 2008). Similar approaches have utilized the fibrin/thrombin reaction (Cui and Boland, 2009) or photopolymer inks (Cui et al., 2012). However, these printed environments are not suitable for all cell types and applications. Ferris et al. reported a bio-ink based on a novel microgel suspension in a surfactant-containing tissue culture medium that can be used to reproducibly print several different cell types, from two different commercially available drop-on-demand printing systems (microvalve deposition system (Deerac™, Labcyte Inc.) and many-nozzle piezoelectric inkjet print-heads (Xaar-126, Xaars)), over long printing period (Fig. 2B) (Ferris et al., 2013). In this context, we have described a fully automated fabrication process for the production of polystyrene microwells on gold surface and their filling with adherent cells. We have also demonstrated the high adhesion contrast of the microstructured surface and the ability of the encapsulation spotting technique to generate multiplex arrays of living adherent cells (Fig. 2C) (Berthuy et al., 2014). As high-throughput single-cell measurements of cellular responses are of great importance for a variety of applications, we have recently developed a new encapsulating process permitting to have one cell per drop. In this purpose, a thin film of CaCl2 was spread onto a glass slide and one drop of a 1% alginate solution containing 2.5  106 cells/mL HeLa cells was spotted on this film using a piezoelectric spotter (sciFLEXARRAYER S1, Scienion, Germany). This method allowed us to produce hundred spots composed of one of alginate bead encapsulating a single-cell (Fig. 2D). A new approach to prepare arrays of sessile droplets of living single-cell cultures using a liquid hydrophobic barrier was proposed to prevent sample dehydration, and to allow spatially addressable arrays for statistical quantitative single-cell studies. By carefully moving a thin layer of mineral oil on the substrate over the droplets during the printing, dehydration of the droplets can be prevented, and the vitality of the cells can be maintained. The net result of this confluence of submerged cell culturing and inkjet printing is facile access to spatially addressable arrays of isolated single-cells on surfaces (Liberski et al., 2011). More recently, Salehi-Reyhani et al. reported the use of oil-encapsulated droplet microarrays employed to perform a sandwich antibody assay for single-cell protein analysis of the tumor-suppressor protein p53. A simple apparatus is used to dispense cell-containing droplets on top of an antibody spot printed using a microcontact arrayer. The addressability of the droplet array is demonstrated by the loading of single human cancer cells into droplets and their individual lysis (Salehi-Reyhani et al., 2014). The on-demand printing of living cells using inkjet technologies has recently been demonstrated and allows for the controlled deposition of cells in microarrays. Ellis et al. showed that such arrays can be interrogated directly by robot-control liquid micro-extraction coupled with chip-based nano-electrospray mass spectrometry. Such automated analyses generate profiles of abundant membrane lipids that are characteristic of cell type. Significantly, the spatial control in both deposition and extraction steps combined with the sensitivity of the mass spectrometric detection allows for robust molecular profiling of individual cells (Ellis et al., 2012). More recently, Zhou et al. reported an easy but versatile method for patterning different cells on a single substrate by using a microfluidic approach that allows not only spatial and temporal control of multiple microenvironments but also retrieval of specific treated cells to profile their expressed genetic information at around 10-cell resolution. By taking advantages of increased surface area of gold nanoparticles on a PDMS coated substrate, cell

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Fig. 2. Some examples of techniques allowing handling of multiple cell lines on chip. (A) Single-cell manipulator (SCM) system for printing single-cells consists of (1) dispenser chip mounted to the aluminum case that hosts the piezo-stack actuator, (2) target for single-cell printing (e.g. 96-well plate) mounted on motorized linear stage, (3) external illumination, (4) objective of a CCD camera for image recognition and cell detection, (5) reservoir (Yusof et al., 2011), (B) Patterning of two cell types printed simultaneously from separate inkjet print-heads onto collagen substrates, C2C12 (red) and PC12 (green) cells were pre-stained with CellTracker™ dyes and printed in various patterns (the scale bars represent 500 mm for the images on the left and 200 mm for the images on the right) (Ferris et al., 2013), (C) Optical (top) and fluorescent (bottom) microscopy images of HeLa [þ ] cell (1) and HeLa-eGP cell spots (2) 72 h of co-culture after spotting with alginate in polystyrene microwells (the scale bars represent 100 mm) (Berthuy et al., 2014), (D) One drop, one cell, (1) Principle of the deposition method, (2) Microscope images of three alginate beads with one cell inside each bead (the scale bar represents 50 mm), (E) Schematic of “Ip-Do assay” method for cell patterning and gene analysis (Zhou et al., 2015) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

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Fig. 3. (A) Patterning cellular co-culture (Rodriguez et al., 2014). (B) Schematic of the in-cell, on chip Western technique (Fernandes et al., 2008). (C) Schematic of the DataChip platform for direct testing of compound toxicity or coupling with the MetaChip for evaluating toxicity of P450-generated metabolites (Lee et al., 2008). (D) General scheme of the cell capture–release strategy developed by Leroy et al. (2014).

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adhesive-promotive protein, human fibronectin (hFN), can be significantly accumulated on designed regions where cells which can recognize the protein spread out. Microfluidic chip reversibly bond with a PDMS-coated glass substrate and was used as an “Ip-Do Assay” device. To synthesize the gold nanofilm on PDMS as a strippattern, gold(III) chloride solution was delivered into the chip and incubated to conduct the reduction reaction, followed by human fibronectin incubation and blow-drying. Cells were introduced into channels which have been orthogonally aligned with the nanogold strips after the first layer of the chip was peeled off. Cells attached and formed patterns on hFN modified gold-islands in channels after overnight incubation. On the basis of imaging analysis, the desired pellet of patterned cells can be further retrieved by using a mouthpipet to perform consequent biochemical reactions in the tube and qPCR (Fig. 2E) (Zhou et al., 2015). 3.1. Other multiplex cell-based microarrays The ability to control the spatial localization and geometry of cells via surface engineering has contributed greatly to our understanding of how cell adhesion regulates a wide variety of cellular functions. Microcontact printing of adhesive proteins, a surface patterning tool based on soft lithography techniques, developed by Whitesides and colleagues, restricts cell adhesion to specific regions (Kane et al., 1999; Singhvi et al., 1994) and has enabled numerous studies, illuminating mechanisms by which cell adhesion and adhesion region shape impact cell survival, apoptosis proliferation, differentiation and migration (Chen et al., 1997; Thery et al., 2005). However, micropatterned surfaces generated via conventional microcontact printing are binary: one region permanently permits cell adhesion, and the remaining region permanently prevents cell adhesion. Thus, conventional microcontact printing is not well-suited to pattern more than two regions and does not allow for the patterning of multiple cell types. To overcome this limitation, subsequent patterning techniques were proposed for the fabrication of multicolor substrates. These techniques were sequential stamping with multiple proteins (Rogers et al., 1998), multimask photolithography (Hui and Bhatia, 2007), photoresist barriers and aminosilane-linked biomolecules (Bhatia et al., 1997), multilevel stamps (Tien et al., 2002) and stampoff (Desai et al., 2011). These multicolor substrates were composed of more than one type of adhesion region and have been used to spatially segregate different cell types or subcellular components by exploiting the preferential attachment of certain cell types or receptors to specific adhesive ligands. More recently, Rodriguez et al. developed a technique that combines microcontact printing with a simple dynamic attachment chemistry to achieve multicolor patterns with three distinct functional regions: adhesive (microcontact printed fibronectin), non-adhesive (Pluronics F127), and a initially nonadhesive region (microcontact printed neutravidin) that can be induced to become adhesive by the capture of biotinylated fibronectin (Fig. 3A) (Rodriguez et al., 2014). Elitas et al. presented a microchip platform, which was built upon their previous high-throughput single-cell secretomic microchip. They demonstrated the measurement of 16 secreted proteins in a large array of subnanoliter microchambers containing individual glioma cells, individual macrophage cells, or various combinations of both on the same device. This simple device, which has 5000 þ microchambers, does not require precise control of cell trapping, but allows for creating hundreds of individual tumor–macrophage pairs simply through a random-loading method (Elitas et al., 2014). Fernandes et al. developed an immunofluorescence-based assay for high-throughput analysis of target proteins on a three-dimensional cellular microarray platform. This process integrates the use of three-dimensional cellular microarrays, which should better mimic the cellular microenvironment, with sensitive

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immunofluorescence detection and provides quantitative information on cell function. To demonstrate this assay platform, they examined the accumulation of the α subunit of the hypoxiainducible factor (HIF-1α) after chemical stimulation of human pancreatic tumor cells encapsulated in 3D alginate spots in volumes as low as 60 nL. They also tested the effect of the known dysregulator of HIF-1α, 2-methoxyestradiol (2ME2), on the level of HIF-1α using a dual microarray stamping technique. This chipbased in-situ Western immunoassay protocol was able to provide quantitative information on cell function, namely, the cellular response to hypoxia, mimicking conditions and the reduction of HIF1α levels after cell treatment with 2ME2. This system is the first to enable high-content screening of cellular protein levels on a 3D human cell microarray platform (Fig. 3B) (Fernandes et al., 2008). Another example of miniaturized 3D cell array is the Data Analysis Toxicology Assay Chip (DataChip) developed by Lee et al. for high-throughput toxicity screening of drug candidates and their cytochrome P450-generated metabolites. The DataChip consists of human cells encapsulated in collagen or alginate gels (as small as 20 nL) arrayed on a functionalized glass slide for spatially addressable screening against multiple compounds. A single DataChip containing 1080 individual cell cultures, used in conjunction with the complementary human P450-containing microarray (the Metabolizing Enzyme Toxicoly Assay Chip or MetaChip), simultaneously provided IC50 values for nine compounds and their metabolites from CYP1A2, CYP2D6, and CYP3A4 and a mixture of the three P450 designed to emulate the human liver. Similar responses were obtained with the DataChip and conventional 96-well plate assays, demonstrating that the near 2000-fold miniaturization does not influence the cytotoxicity response detection (Fig. 3C) (Lee et al., 2008). More recently, Leroy et al. proposed a miniaturized system able to perform multiple cell capture followed by cell-type selective release from a biochip surface. Unlabeled lymphocytes were first specifically captured onto a DNA array by antibody-DNA conjugates. The immobilized cells were subsequently released under spatiotemporal control using local heating generated by intense surface plasmon resonance (SPR) produced by laser illumination. Therefore, this approach constitutes an example of a non-invasive and gentle method for multiplexed cell sorting, performed under precise control and without consumption of additional reagents. The biochip could be designed to sort and release more cell types and also to enable assays of crude biological samples such as blood tests (Fig. 3D) (Leroy et al., 2014). Otherwise, a novel miniaturized 3D cell culture chip platform that can maintain specific biochemical and morphological features of human cancer cells similar to their corresponding tumors with a high-throughput manner had been developed by Lee and colleagues. This chip platform is ideally suited for encapsulating primary cancer cells in nanoscale spots of hydrogels, generating efficacy data of various drugs. As a proof of concept, authors encapsulated a U251 brain cancer cell line and three primary brain cancer cells from patients (448T, 464T and 775T) in 30 nL droplets of alginate and tested therapeutic efficacy of 24 anticancer drugs by measuring their dose response variations (Lee et al., 2014). Li et al. developed a ready-to-use micro-scaffold array chip suitable for performing high-throughput cell-based assays in 3D at the benchtop. The sponge-like micro-scaffolds function both as absorbents to realize parallel auto-loading of cells or drugs and as barriers to prevent cell loss during medium exchange via centrifugation. An array of isolated and miniaturized reaction chambers could be created by ‘sandwiching’ an additional drug-laden PEG microsponge array chip with the cell-laden micro-scaffold array chip in an addressable way. Increased drug resistance of cancer cells was observed using this platform when compared to cancer cells cultured on the planar substrate, which could be

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partially attributed to the enhanced cell–matrix interactions and malignancy of cancer cells in 3D scaffold culture. In addition, high drug resistance of the 3D cultured cells was independent of cell density as compared to the density dependent drug responses of 2D cultured cells. The benchtop 3D culture-based platform offers powerful tools for rapid and inexpensive cell-based drug screening accessible to common laboratories with only basic cell culture setups. They anticipate broad applications of the current platform for cell biology, tissue engineering, biochemical detection and drug discovery (Li et al., 2014). Quantitative cell motility studies are necessary for understanding biophysical processes, developing models for cell locomotion and for drug discovery. Such studies are typically performed by controlling environmental conditions around a lens-based microscope, requiring costly instruments while still remaining limited in field-of-view. Pushkarsky et al. presented a compact cell monitoring platform utilizing a wide-field (24 mm2) lensless holographic microscope enabling automated single-cell tracking of large populations, compatible with a standard laboratory incubator. This platform was used to track NIH 3T3 cells on polyacrylamide gels over 20 h. The increased throughput associated with lensfree onchip imaging enables higher statistical significance in observed cell behavior and may facilitate rapid screening of drugs and genes that affect cell motility (Pushkarsky et al., 2014). Microfluidic devices that enable parallel analysis in a highthroughput manner have also been produced using soft lithography techniques (Kang et al., 2008). An increasing demand for fully automated and quantitative cell culture technology has driven the development of microfluidic chip-based arrays (Hung et al., 2005). For example, the continuous perfusion of medium inside parallel culture chambers allowed the evaluation of how a range of transient stimulation schedules influenced proliferation, differentiation and motility of human mesenchymal stem cells (Gomez-Sjoeberg et al., 2007). Microfluidic cell culture arrays, therefore, offer an attractive platform with a wide range of applications in highthroughput cell-based screening and quantitative cell biology. Finally, Zhou et al. reported on a laser scanning cytometry approach exploiting a bench-top microarray scanner as an end-point reader to perform rapid and automated fluorescence imaging of cells cultured on a chip. Using high-content imaging analysis algorithms, they demonstrated multiplexed measurements of morphometric and proteomic parameters from multiple single-cells. Their approach shows the improvement of both sensitivity and dynamic range by two orders of magnitude as compared to conventional epifluorescence microscopy. They applied this technology to highthroughput analysis of mesenchymal stem cells on an extracellular matrix protein array and characterization of heterotypic cell populations. This work establishes an alternative approach to perform high-content single-cell analysis and this approach can be readily applied to a wide range of lab-on-a-chip systems (Zhou et al., 2012).

4. Summary and conclusions We have presented an overview of the last developments on cell microarrays, particularly optical microarrays with multiplexing abilities allowing high-content and high-throughput analysis. We have shown that, among the many cell microarrays, only few of them allow multiplexed cell culture. Whereas, cell-based arrays allowing reverse transfection permit to study several phenotypes with only one cell line on the same chip, the novel technologies such as bioprinting are essential tools to be able to address different cell lines on the same substrate. The miniaturization of cell-based assays promises then to have a profound impact on high-throughput screening of compounds by minimizing the consumption of reagents and cells.

5. Future perspectives To achieve routine adoption of high-throughput cellular microscale platforms, future development will most certainly need to focus on automated, high-throughput and multiplex methods for the study of cellular microenvironments and growth conditions in 3D environment (Jongpaiboonkit et al., 2008). In this regard, arraybased formats have been developed that have proved their usefulness as enhanced-throughput platforms for 3D culture of various cell types (Gottwald et al., 2007), and much effort is now being directed towards cell culture models that better reflect in vivo function. In this context, organs-on-chip have emerged to provide new tools for better evaluating the effects of various chemicals on human tissues (Huh et al., 2011; Polini et al., 2014; Van der Meer and Van den Berg, 2012).

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