Living Cell Microarrays Research Articles

Live-Cell Systems in Real-Time Biomonitoring of Water Pollution: Practical Considerations and Future Perspectives.

Continuous monitoring and early warning of potential water contamination with toxic chemicals is of paramount importance for human health and sustainable food production. During the last few decades there have been noteworthy advances in technologies for the automated sensing of physicochemical parameters of water. These do not translate well into online monitoring of chemical pollutants since most of them are either incapable of real-time detection or unable to detect impacts on biological organisms. As a result, biological early warning systems have been proposed to supplement conventional water quality test strategies. Such systems can continuously evaluate physiological parameters of suitable aquatic species and alert the user to the presence of toxicants. In this regard, single cellular organisms, such as bacteria, cyanobacteria, micro-algae and vertebrate cell lines, offer promising avenues for development of water biosensors. Historically, only a handful of systems utilising single-cell organisms have been deployed as established online water biomonitoring tools. Recent advances in recombinant microorganisms, cell immobilisation techniques, live-cell microarrays and microfluidic Lab-on-a-Chip technologies open new avenues to develop miniaturised systems capable of detecting a broad range of water contaminants. In experimental settings, they have been shown as sensitive and rapid biosensors with capabilities to detect traces of contaminants. In this work, we critically review the recent advances and practical prospects of biological early warning systems based on live-cell biosensors. We demonstrate historical deployment successes, technological innovations, as well as current challenges for the broader deployment of live-cell biosensors in the monitoring of water quality.

Robotic Cell Printing for Constructing Living Yeast Cell Microarrays in Microfluidic Chips

Living cell microarrays in microfluidic chips allow the non-invasive multiplexed molecular analysis of single cells. Here, we developed a simple and affordable perfusion microfluidic chip containing a living yeast cell array composed of a population of cell variants (green fluorescent protein (GFP)-tagged Saccharomyces cerevisiae clones). We combined mechanical patterning in 102 microwells and robotic piezoelectric cell dispensing in the microwells to construct the cell arrays. Robotic yeast cell dispensing of a yeast collection from a multiwell plate to the microfluidic chip microwells was optimized. The developed microfluidic chip and procedure were validated by observing the growth of GFP-tagged yeast clones that are linked to the cell cycle by time-lapse fluorescence microscopy over a few generations. The developed microfluidic technology has the potential to be easily upscaled to a high-density cell array allowing us to perform dynamic proteomics and localizomics experiments.

Primary and Stem Cell Microarrays: Application as Miniaturized Biotesting Systems.

The deposition of living cells on microarray surfaces can be used to create physiologically relevant architecture in vitro. Such living cell microarrays enable the reconstruction of biological processes outside the body in a miniaturized format and have many advantages over traditional cell culture. The present protocol offers an option for the preparation and analysis of living primary and stem cell-based microarrays utilizing the standard microarray equipment (contact-free piezoelectric nanoprinter, microarray scanner), as well as microscopy. To produce living cell microarrays, we applied two kinds of mesenchymal stem cells (MSCs) isolated from umbilical cord and adipose tissue, as well as human umbilical vein endothelial cells (HUVECs) as model cells. We used live imaging microscopy for the online monitoring of cell spots in total size, staining of viable cells with Calcein acetoxymethyl ester (Calcein-AM) and treatment of MSCs with differentiation media to analyze the proliferation, viability, and differentiation potential of printed cells. This way, the general applicability of the established living cell-based microarray production was demonstrated.

Next-Generation Live-Cell Microarray Technologies.

Over the last decades the application of cell-based assays and in vitro cell culture systems has fundamentally transformed our understanding of biological functions on a cellular and organism level. The resulting ubiquitous usage of cell-based assays in today's scientific world has therefore generated a need for advanced in vitro diagnostic systems. This increased demand has further led to the development of miniaturized live-cell microarrays for biomedical applications including high-throughput screening tools and microfluidic systems. The greatest benefit of miniaturized cell analysis systems is the ability to provide quantitative data in real time with high reliability and sensitivity, which are key parameters for any cell-based assay. An additional advantage of live-cell microarrays is their inherent capability for large-scale screening of single cells, multicell populations, as well as spheroids.

Living Cell Microarrays: An Overview of Concepts.

Living cell microarrays are a highly efficient cellular screening system. Due to the low number of cells required per spot, cell microarrays enable the use of primary and stem cells and provide resolution close to the single-cell level. Apart from a variety of conventional static designs, microfluidic microarray systems have also been established. An alternative format is a microarray consisting of three-dimensional cell constructs ranging from cell spheroids to cells encapsulated in hydrogel. These systems provide an in vivo-like microenvironment and are preferably used for the investigation of cellular physiology, cytotoxicity, and drug screening. Thus, many different high-tech microarray platforms are currently available. Disadvantages of many systems include their high cost, the requirement of specialized equipment for their manufacture, and the poor comparability of results between different platforms. In this article, we provide an overview of static, microfluidic, and 3D cell microarrays. In addition, we describe a simple method for the printing of living cell microarrays on modified microscope glass slides using standard DNA microarray equipment available in most laboratories. Applications in research and diagnostics are discussed, e.g., the selective and sensitive detection of biomarkers. Finally, we highlight current limitations and the future prospects of living cell microarrays.

Cell Microarrays for Biomedical Applications.

In this chapter the state of the art of live cell microarrays for high-throughput biological assays are reviewed. The fabrication of novel microarrays with respect to material science and cell patterning methods is included. A main focus of the chapter is on various aspects of the application of cell microarrays by providing selected examples in research fields such as biomaterials, stem cell biology and neuroscience. Additionally, the importance of microfluidic technologies for high-throughput on-chip live-cell microarrays is highlighted for single-cell and multi-cell assays as well as for 3D tissue constructs.

Development of living cell microarrays using non-contact micropipette printing

During the last 30 years cellular screening systems were unidirectional developed towards high throughput applications on single cell level. We developed living cell microarrays, which provide an in vivo-like microenvironment for an advanced method to measure cellular response to external stimuli. To print living cells on glass slides, the classic microarray equipment, which involves printer and scanner, was fully transferred to suspensions of living cells. The microarray production was optimized using a contact-free spotting procedure in order to enhanced cell adhesion and growth rates. The printed model cells, A-549 (lung cancer cell line), were analyzed with conventional cell staining assays like DAPI (cell nuclei staining), calcein acetoxymethyl ester (viable cell staining), and CellTiter-Blue® Cell Viability Assay. After optimization, a reproducible (spot-to-spot variation:±8.6 cells) printing method for small living cell amounts (1200cells and fewer) was established that achieved cell viabilities of up to 88% for ≥0.6μL and good proliferation characteristics. Hence, this method could be advantageous for use in biomedical and diagnostic applications.

Microfluidic Bioreactors for Cellular Microarrays

Living cell microarrays have been combined with microfluidic bioreactors, which provide multiple advantages for multiplex dynamic analyses and high-throughput screening. In the last decade, many developments in this new field have been introduced. The technology has evolved from fixed cell analysis towards living single-cell dynamic systems’ biology and high content analyses. The aim of this review is to provide an updated overview of the developments of living cellular microarrays in microfluidic bioreactors. Cell arrays in microfluidic bioreactors constructed with adherent mammalian cells are compared to non-adherent cells (mainly microbial cells). An overview is given on the design and construction of these microfluidic devices with a particular focus on cell patterning techniques. Cell patterning on adhesive micropatterns using techniques such as microcontact printing, microfluidic patterning, dip-pen nanolithography and polymer pen lithography as well as photo-patterning and laser-patterning strategies are discussed. Additionally, developments in mechanical cell patterning methods and robotic cell printing are reviewed. Two-dimensional (2D) as well as recently developed 3D cell arraying are discussed. Finally, cell array microfluidic setups and operation for single-cell types versus cell population variants are illustrated and compared on the basis of some illustrative examples in the field of drug screening, cytotoxicity evaluation, and basic cellular and microbiology research.

Sol–Gel-Derived Materials for Production of Pin-Printed Reporter Gene Living-Cell Microarrays

We report the fabrication of three-dimensional living-cell microarrays via pin-printing of soft sol-gel-derived silica materials containing bacterial cells. Bacterial cells entrapped in the silica-glycerol microarray spots can express reporter genes and produce strong fluorescence signals. The signals responded to the presence and concentration of inducers or repressors as expected, indicating that the entrapped cells remained metabolically active. Microscopic imaging of individual microarray spots at different culture times suggests that the entrapped cells can grow and divide, phenomena further confirmed by experiments in bulk sol-gel materials that demonstrated the increases of entrapped cell density and fluorescence during incubation in culture media. The cell microarrays can also be printed into 96-well glass bottom microtiter plates in a multiplexed manner, and the fluorescence signals generated were able to quantitatively and selectively respond to the concentration of inducers, thus demonstrating the potential for multitarget biosensing and high-throughput/high-content cell-based screening. The signal levels of bacterial cells in silica were significantly higher than those in alginate arrays, presumably due to viability of the entrapped cells in silica sol-gels. Microarray stability assays proved that the entrapped cells retained their physiological activity after storage for four weeks. Given that a large number of fluorescent and luminescent protein-based cell assays have been developed, the reporter gene living-cell microarrays demonstrated in this paper are expected to be applicable to a wide variety of research areas ranging from bioanalysis and chemical biology to drug discovery and probing of cell-material interactions.

Live-cell Microarray Surface Coatings Supporting Reverse Transduction by Adeno-Associated Viruses

High-throughput live-cell microarray technologies that facilitate combinatorial screening of genes and RNA interference (RNAi) would be invaluable in the identification of key gene expression profiles involved in complex cellular behaviors. Each spot on such a microarray can comprise a unique combination of genes or RNAi packaged into gene delivery vectors. Live target cells seeded on top of the microarrays would express the combination of genetic factors, potentially leading to phenotypic changes within cells. Here, we investigate the feasibility of using adeno-associated virus (AAV) as a gene delivery agent for such live-cell genetic microarrays. A robotic spotter was used to deposit AAV onto gamma-amino propyl silane, amine silane, or nitrocellulose-coated glass slides. Virus deposition and reverse transduction of target cells were found to be surface coating-dependent with nitrocellulose coating yielding the best AAV deposition, while also producing discrete islands of highly transduced cells. Our results demonstrate the feasibility of using nitrocellulose-coated surfaces for the development of AAV-based genetic microarrays.

Simple benchtop patterning of hydrogel grids for living cell microarrays

A living cell microarray consists of an orderly arrangement of cells attached to a solid support such as a glass microscope slide. The chief difficulty of obtaining such arrays is the fabrication of substrates patterned with micro-wells, adhesive spots, or other features to guide orderly cell attachment. Here we report a novel method using woven Nylon mesh to micropattern three-dimensional alginate hydrogel grids on glass slides. The Nylon mesh is both inexpensive and off-the-shelf. By using Nylon mesh we have eliminated any need for lithography, clean room equipment, and microarray printers to generate microarray patterns; thus, this technique can be easily adopted by biological research labs that may lack microfabrication expertise and facilities. We have demonstrated that glass slides micropatterned with hydrogel grids guide the orderly attachment of single fibroblast cells and Schwann cell clusters in microarrays. The fibroblast arrays consisted of 70 microm square compartments at a density of 21,000 compartments per cm(2). The Schwann cell arrays consisted of 100 microm square compartments at a density of 6000 per cm(2). This patterning technique addresses the need for a simple, inexpensive, benchtop method for micro-patterning glass slides and obtaining living cell microarrays.

Living-Cell Microarrays

Living cells are remarkably complex. To unravel this complexity, living-cell assays have been developed that allow delivery of experimental stimuli and measurement of the resulting cellular responses. High-throughput adaptations of these assays, known as living-cell microarrays, which are based on microtiter plates, high-density spotting, microfabrication, and microfluidics technologies, are being developed for two general applications: (a) to screen large-scale chemical and genomic libraries and (b) to systematically investigate the local cellular microenvironment. These emerging experimental platforms offer exciting opportunities to rapidly identify genetic determinants of disease, to discover modulators of cellular function, and to probe the complex and dynamic relationships between cells and their local environment.

Adsorption and Stability of Streptavidin on Cluster-Assembled Nanostructured TiOx Films

The study of the adsorption of proteins on nanostructured surfaces is of fundamental importance to understand and control cell-surface interactions and, notably, cell adhesion and proliferation; it can also play a strategic role in the design and fabrication of nanostructured devices for postgenomic and proteomic applications. We have recently demonstrated that cluster-assembled nanostructured TiO x films produced by supersonic cluster beam deposition possess excellent biocompatibility and that these films can be functionalized with streptavidin, allowing the immobilization of biotinylated retroviral particles and the realization of living-cell microarrays for phenotype screening. Here we present a multitechnique investigation of the adsorption mechanisms of streptavidin on cluster-assembled TiO x films. We show that this nanostructured surface provides an optimal balance between adsorption efficacy and protein functionality. By using low-resolution protein arrays, we demonstrate that a layer of adsorbed streptavidin can be stably maintained on a cluster-assembled TiO x surface under cell culture conditions and that streptavidin retains its biological activity in the adsorbed layer. The adsorption mechanisms are investigated by atomic force microscopy in force spectroscopy mode and by valence-band photoemission spectroscopy, highlighting the potential role of the interaction of the exposed carboxyl groups on streptavidin with the titanium atoms of the nanostructured surface.

Adsorption and Stability of Streptavidin on Cluster-Assembled Nanostructured TiOx Films

The study of the adsorption of proteins on nanostructured surfaces is of fundamental importance to understand and control cell-surface interactions and, notably, cell adhesion and proliferation; it can also play a strategic role in the design and fabrication of nanostructured devices for postgenomic and proteomic applications. We have recently demonstrated that cluster-assembled nanostructured TiO x films produced by supersonic cluster beam deposition possess excellent biocompatibility and that these films can be functionalized with streptavidin, allowing the immobilization of biotinylated retroviral particles and the realization of living-cell microarrays for phenotype screening. Here we present a multitechnique investigation of the adsorption mechanisms of streptavidin on cluster-assembled TiO x films. We show that this nanostructured surface provides an optimal balance between adsorption efficacy and protein functionality. By using low-resolution protein arrays, we demonstrate that a layer of adsorbed streptavidin can be stably maintained on a cluster-assembled TiO x surface under cell culture conditions and that streptavidin retains its biological activity in the adsorbed layer. The adsorption mechanisms are investigated by atomic force microscopy in force spectroscopy mode and by valence-band photoemission spectroscopy, highlighting the potential role of the interaction of the exposed carboxyl groups on streptavidin with the titanium atoms of the nanostructured surface.

Single lymphocyte analysis with a microwell array chip

Following genomics and proteomics, cytomics, a novel method of looking at life, has emerged for analyzing large populations of cells on a single-cell basis with multiple parameters in a quantitative manner. We have developed a highly integrated live-cell microarray system for analyzing the cellular responses of individual cells using a microwell array chip that has 234,000 microwells each of which is just large enough to fit a single cell. Compared with flow cytometry and microscope-based methods, our system can analyze the history of the cellular responses of a large number of cells. We have successfully applied the system to analyze human antigen-specific B-cells and produced human monoclonal antibodies (MoAb) against hepatitis B virus surface antigen. We have also constructed a mouse system to assess hepatitis B virus-neutralization activity and have demonstrated the neutralization activity of our antibodies. Our technology should expand the horizons of cell analysis as well as enable generation of human MoAb for antibody-based therapeutics and diagnosis for infectious diseases such as hepatitis viruses.

Retroviral microarray-based platform on nanostructured TiO 2 for functional genomics and drug discovery

Living-cell microarrays are powerful tools for functional genomics and drug discovery. However, despite several attempts to improve this technology, it is still a challenge to obtain microarrays of cells efficiently overexpressing or downregulating specific genes to address complex phenotypes. Here, we present a cell-based microarray for phenotype screening on primary and cancer cells based on the localized reverse infection by retroviruses. Viral vectors are immobilized on a nanostructured titanium dioxide (ns-TiO 2) film obtained by depositing a supersonic beam of titania clusters on a glass substrate. We validated the retroviral cell array by overexpression of GFP reporter genes in primary and cancer cells, and by RNA interference of p53 in primary cells by analyzing effects in cell growth. We demonstrate that ns-TiO 2 retroviral arrays are an enabling tool for the study of gene function of families of genes for complex phenotypes and for the identification of novel drug targets.

Laser-Guided Assembly of Heterotypic Three-Dimensional Living Cell Microarrays

We have assembled three-dimensional heterotypic networks of living cells in hydrogel without loss of viability using arrays of time-multiplexed, holographic optical traps. The hierarchical control of the cell positions is achieved with, to our knowledge, unprecedented submicron precision, resulting in arrays with an intercell separation <400 nm. In particular, we have assembled networks of Swiss 3T3 fibroblasts surrounded by a ring of bacteria. We have also demonstrated the ability to manipulate hundreds of Pseudomonas aeruginosa simultaneously into two- and three-dimensional arrays with a time-averaged power <2 mW per trap. This is the first time to our knowledge that living cell arrays of such complexity have been synthesized, and it represents a milestone in synthetic biology and tissue engineering.

RNAi living-cell microarrays for loss-of-function screens in Drosophila melanogaster cells

RNA interference (RNAi)-mediated loss-of-function screening in Drosophila melanogaster tissue culture cells is a powerful method for identifying the genes underlying cell biological functions and for annotating the fly genome. Here we describe the development of living-cell microarrays for screening large collections of RNAi-inducing double-stranded RNAs (dsRNAs) in Drosophila cells. The features of the microarrays consist of clusters of cells 200 mum in diameter, each with an RNAi-mediated depletion of a specific gene product. Because of the small size of the features, thousands of distinct dsRNAs can be screened on a single chip. The microarrays are suitable for quantitative and high-content cellular phenotyping and, in combination screens, for the identification of genetic suppressors, enhancers and synthetic lethal interactions. We used a prototype cell microarray with 384 different dsRNAs to identify previously unknown genes that affect cell proliferation and morphology, and, in a combination screen, that regulate dAkt/dPKB phosphorylation in the absence of dPTEN expression.

RNA interference microarrays: high-throughput loss-of-function genetics in mammalian cells.

RNA interference (RNAi) is a biological process in which a double-stranded RNA directs the silencing of target genes in a sequence-specific manner. Exogenously delivered or endogenously encoded double-stranded RNAs can enter the RNAi pathway and guide the suppression of transgenes and cellular genes. This technique has emerged as a powerful tool for reverse genetic studies aimed toward the elucidation of gene function in numerous biological models. Two approaches, the use of small interfering RNAs and short hairpin RNAs (shRNAs), have been developed to permit the application of RNAi technology in mammalian cells. Here we describe the use of a shRNA-based live-cell microarray that allows simple, low-cost, high-throughput screening of phenotypes caused by the silencing of specific endogenous genes. This approach is a variation of "reverse transfection" in which mammalian cells are cultured on a microarray slide spotted with different shRNAs in a transfection carrier. Individual cell clusters become transfected with a defined shRNA that directs the inhibition of a particular gene of interest, potentially producing a specific phenotype. We have validated this approach by targeting genes involved in cytokinesis and proteasome-mediated proteolysis.