Cell Microarray Research Articles

183 Novel OPG Protein Interactions Regulate Survival, Proliferation And Pah-associated Gene Expression in Pulmonary Arterial Smooth Muscle Cells

IntroductionPulmonary arterial hypertension (PAH) is a devastating disease with a high mortality and prognosis worse than many cancers. Pathologically, PAH is characterised by progressive arteriole remodelling driven by pulmonary artery...

High-throughput and combinatorial gene expression on a chip for metabolism-induced toxicology screening.

Differential expression of various drug-metabolizing enzymes in the human liver may cause deviations of pharmacokinetic profiles, resulting in inter-individual variability of drug toxicity and/or efficacy. Here we present the “Transfected Enzyme and Metabolism Chip” (TeamChip), which predicts potential metabolism-induced drug or drug-candidate toxicity. The TeamChip is prepared by delivering genes into miniaturized three-dimensional cellular microarrays on a micropillar chip using recombinant adenoviruses in a complementary microwell chip. The device enables users to manipulate the expression of individual and multiple human metabolizing-enzyme genes (such as CYP3A4, CYP2D6, CYP2C9, CYP1A2, CYP2E1, and UGT1A4) in THLE-2 cell microarrays. To identify specific enzymes involved in drug detoxification, we created 84 combinations of metabolic-gene expressions in a combinatorial fashion on a single microarray. Thus, the TeamChip platform can provide critical information necessary for evaluating metabolism-induced toxicity in a high-throughput manner.

Transcriptional response to vancomycin in a highly vancomycin-resistant Streptomyces coelicolor mutant.

The main objective of this study is to understand the mechanism of vancomycin resistance in a Streptomyces coelicolor disrupted mutant highly resistant to vancomycin. Different techniques have been performed in the study including gene disruption, primer extension, antibiotic susceptibility tests, electron microscopy, confocal microscopy, cell wall analysis and microarrays. During the phenotypical characterization of mutant strains affected in phosphate-regulated genes of unknown function, we found that the S. coelicolor SCO2594 disrupted mutant was highly resistant to vancomycin and had other phenotypic alterations such as antibiotic overproduction, impaired growth and reduction of phosphate cell wall content. Transcriptomic studies with this mutant indicated a relationship between vancomycin resistance and cell wall stress. We identified a S. coelicolor mutant highly resistant to vancomycin in both high and low phosphate media. In addition to Van proteins, others such as WhiB or SigE appear to be involved in this regulatory mechanism.

Facile generation of cell microarrays using vacuum degassing and coverslip sweeping

A simple method to generate cell microarrays with high-percentage well occupancy and well-defined cell confinement is presented. This method uses a synergistic combination of vacuum degassing and coverslip sweeping. The vacuum degassing step dislodges air bubbles from the microwells, which in turn enables the cells to enter the microwells, while the physical sweeping step using a glass coverslip removes the excess cells outside the microwells. This low-cost preparation method provides a simple solution to generating cell microarrays that can be performed in basic research laboratories and point-of-care settings for routine cell-based screening assays.

The submerged printing of cells onto a modified surface using a continuous flow microspotter.

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

Three dimensional cellular microarray platform for human neural stem cell differentiation and toxicology

We developed a three-dimensional (3D) cellular microarray platform for the high-throughput (HT) analysis of human neural stem cell (hNSC) growth and differentiation. The growth of an immortalized hNSC line, ReNcell VM, was evaluated on a miniaturized cell culture chip consisting of 60nl spots of cells encapsulated in alginate, and compared to standard 2D well plate culture conditions. Using a live/dead cell viability assay, we demonstrated that the hNSCs are able to expand on-chip, albeit with lower proliferation rates and viabilities than in conventional 2D culture platforms. Using an in-cell, on-chip immunofluorescence assay, which provides quantitative information on cellular levels of proteins involved in neural fate, we demonstrated that ReNcell VM can preserve its multipotent state during on-chip expansion. Moreover, differentiation of the hNSCs into glial progeny was achieved both off- and on-chip six days after growth factor removal, accompanied by a decrease in the neural progenitor markers. The versatility of the platform was further demonstrated by complementing the cell culture chip with a chamber system that allowed us to screen for differential toxicity of small molecules to hNSCs. Using this approach, we showed differential toxicity when evaluating three neurotoxic compounds and one antiproliferative compound, and the null effect of a non-toxic compound at relevant concentrations. Thus, our 3D high-throughput microarray platform may help predict, in vitro, which compounds pose an increased threat to neural development and should therefore be prioritized for further screening and evaluation.

An improved high‐output cell microarray technology

Cell microarray (CMA) is a high-throughput scientific research tool, which has greatly accelerated many analyses based at the cellular level. However, there are few described methods for constructing CMAs. Here, we introduce a new, simple, high-output CMA method that is applicable to a broad range of cellular samples. In this method, a recipient block (length, 3.6cm; width, 2.7cm; depth, 2cm) with 40 dot markers was moulded using a transparent plastic box. Adenocarcinoma cells were collected from malignant pleural effusions, cell cylinders were moulded with plastic piping and the cylinders were manually arrayed one by one into the corresponding location of the 60°C pre-softened recipient block using the guide holes drilled with a steel needle. We constructed a 40-cylinder CMA to prove this method. The expression of cytokeratin 7 (CK7) in the CMA was examined to confirm antigen preservation and epidermal growth factor receptor (EGFR) gene mutation was screened for in five samples. The CMA prepared by this method had well-defined array configurations, good cellular morphology and well-preserved proteins and DNA. A total of 1000 sections could be easily gained from this CMA block. This simple and low-cost method provides a novel way of preparing a high-output CMA.

Structural proteins of Kaposi’s sarcoma-associated herpesvirus antagonize p53-mediated apoptosis

The tumor suppressor p53 is a central regulatory molecule of apoptosis and is commonly mutated in tumors. Kaposi's sarcoma-associated herpesvirus (KSHV)-related malignancies express wild-type p53. Accordingly, KSHV encodes proteins that counteract the cell death-inducing effects of p53. Here, the effects of all KSHV genes on the p53 signaling pathway were systematically analyzed using the reversely transfected cell microarray technology. With this approach we detected eight KSHV-encoded genes with potent p53 inhibiting activity in addition to the previously described inhibitory effects of KSHV genes ORF50, K10 and K10.5. Interestingly, the three most potent newly identified inhibitors were KSHV structural proteins, namely ORF22 (glycoprotein H), ORF25 (major capsid protein) and ORF64 (tegument protein). Validation of these results with a classical transfection approach showed that these proteins inhibited p53 signaling in a dose-dependent manner and that this effect could be reversed by small interfering RNA-mediated knockdown of the respective viral gene. All three genes inhibited p53-mediated apoptosis in response to Nutlin-3 treatment in non-infected and KSHV-infected cells. Addressing putative mechanisms, we could show that these proteins could also inhibit the transactivation of the promoters of apoptotic mediators of p53 such as BAX and PIG3. Altogether, we demonstrate for the first time that structural proteins of KSHV can counteract p53-induced apoptosis. These proteins are expressed in the late lytic phase of the viral life cycle and are incorporated into the KSHV virion. Accordingly, these genes may inhibit cell death in the productive and in the early entrance phase of KSHV infection.

Differential network analyses of Alzheimer's disease identify early events in Alzheimer's disease pathology.

In late-onset Alzheimer's disease (AD), multiple brain regions are not affected simultaneously. Comparing the gene expression of the affected regions to identify the differences in the biological processes perturbed can lead to greater insight into AD pathogenesis and early characteristics. We identified differentially expressed (DE) genes from single cell microarray data of four AD affected brain regions: entorhinal cortex (EC), hippocampus (HIP), posterior cingulate cortex (PCC), and middle temporal gyrus (MTG). We organized the DE genes in the four brain regions into region-specific gene coexpression networks. Differential neighborhood analyses in the coexpression networks were performed to identify genes with low topological overlap (TO) of their direct neighbors. The low TO genes were used to characterize the biological differences between two regions. Our analyses show that increased oxidative stress, along with alterations in lipid metabolism in neurons, may be some of the very early events occurring in AD pathology. Cellular defense mechanisms try to intervene but fail, finally resulting in AD pathology as the disease progresses. Furthermore, disease annotation of the low TO genes in two independent protein interaction networks has resulted in association between cancer, diabetes, renal diseases, and cardiovascular diseases.

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.

Characterization of Fetal Cells from the Maternal Circulation by Microarray Gene Expression Analysis - Could the Extravillous Trophoblasts Be a Target for Future Cell-Based Non-Invasive Prenatal Diagnosis?

Introduction: Circulating fetal cells in maternal blood provide a tool for risk-free, non-invasive prenatal diagnosis. However, fetal cells in the maternal circulation are scarce, and to effectively isolate enough of them for reliable diagnostics, it is crucial to know which fetal cell type(s) should be targeted. Materials and Methods: Fetal cells were enriched from maternal blood by magnetic-activated cell sorting using the endothelial cell marker CD105 and identified by XY fluorescence in situ hybridization. Expression pattern was compared between fetal cells and maternal blood cells using stem cell microarray analysis. Results: 39 genes were identified as candidates for unique fetal cell markers. More than half of these are genes known to be expressed in the placenta, especially in extravillous trophoblasts (EVTs). Immunohistochemical staining of placental tissue confirmed CD105 staining in EVTs and 76% of fetal cells enriched by CD105 were found to be cytokeratin-positive. Discussion: The unique combination of mesodermal (CD105) and ectodermal (cytokeratin) markers in EVTs could be a potential marker set for cell enrichment of this cell type in maternal blood and could be the basis for future cell-based non-invasive prenatal diagnosis.

Laser Photofabrication of Cell-Containing Hydrogel Constructs

The two-photon polymerization (2PP) of photosensitive gelatin in the presence of living cells is reported. The 2PP technique is based on the localized cross-linking of photopolymers induced by femtosecond laser pulses. The availability of water-soluble photoinitiators (PI) suitable for 2PP is crucial for applying this method to cell-containing materials. Novel PIs developed by our group allow 2PP of formulations with up to 80% cell culture medium. The cytocompatibility of these PIs was evaluated by an MTT assay. The results of cell encapsulation by 2PP show the occurrence of cell damage within the laser-exposed regions. However, some cells located in the immediate vicinity and even within the 2PP-produced structures remain viable and can further proliferate. The control experiments demonstrate that the laser radiation itself does not damage the cells at the parameters used for 2PP. On the basis of these findings and the reports by other groups, we conclude that such localized cell damage is of a chemical origin and can be attributed to reactive species generated during 2PP. The viable cells trapped within the 2PP structures but not exposed to laser radiation continued to proliferate. The live/dead staining after 3 weeks revealed viable cells occupying most of the space available within the 3D hydrogel constructs. While some of the questions raised by this study remain open, the presented results indicate the general practicability of 2PP for 3D processing of cell-containing materials. The potential applications of this highly versatile approach span from precise engineering of 3D tissue models to the fabrication of cellular microarrays.

Expressomal approach for comprehensive analysis and visualization of ligand sensitivities of xenoestrogen responsive genes

Although biological effects of endocrine disrupting chemicals (EDCs) are often observed at unexpectedly low doses with occasional nonmonotonic dose-response characteristics, transcriptome-wide profiles of sensitivities or dose-dependent behaviors of the EDC responsive genes have remained unexplored. Here, we describe expressome analysis for the comprehensive examination of dose-dependent gene responses and its applications to characterize estrogen responsive genes in MCF-7 cells. Transcriptomes of MCF-7 cells exposed to varying concentrations of representative natural and xenobiotic estrogens for 48 h were determined by microarray and used for computational calculation of interpolated approximations of estimated transcriptomes for 300 doses uniformly distributed in log space for each chemical. The entire collection of these estimated transcriptomes, designated as the expressome, has provided unique opportunities to profile chemical-specific distributions of ligand sensitivities for large numbers of estrogen responsive genes, revealing that at low concentrations estrogens generally tended to suppress rather than to activate transcription. Gene ontology analysis demonstrated distinct functional enrichment between high- and low-sensitivity estrogen responsive genes, supporting the notion that a single EDC chemical can cause qualitatively distinct biological responses at different doses. Expressomal heatmap visualization of dose-dependent induction of Bisphenol A inducible genes showed a weak gene activation peak at a very low concentration range (ca. 0.1 nM) in addition to the main, strong gene activation peak at and above 100 nM. Thus, expressome analysis is a powerful approach to understanding the EDC dose-dependent dynamic changes in gene expression at the transcriptomal level, providing important information on the overall profiles of ligand sensitivities and nonmonotonic responses.

Application of cellular micropatterns to miniaturized cell-based biosensor

Because of strong demands for high throughput or high content cell-based assay, significant efforts have focused on the assay miniaturization by fabricating cell microarray using a variety of cell patterning techniques such as spotting, photolithography or soft lithography and by integrating cell microarray into microfluidic devices. Response of cells cultured on microarray can be monitored by using either electrochemical or optical detection methods. Impedancebased detection and potential-based detection have been widely used for electrochemical detection, while optical detection relies mostly on the fluorescence and bioluminescence-based techniques. Resultant cell microarray-based biosensor can be applied for high throughput/content drug screening and detection of pathogens, pollutants and warfare agents. For the successful application of cell-based biosensors to various areas, multi-phenotypic cell microarray should be developed and cells on microarray must be cultured in 3-dimensional environment as they do in real tissue to obtain accurate response of cells against target analytes.

Special issue on tissue engineering

Tissue engineering is an emerging multidisciplinary research field that involves expertise related to cell biology, micro/ nanoscale engineering, and medicine. Engineering artificial tissues are of great benefit for restoring the lost function of diseased or damaged organs. The current research trends of tissue engineering aim to integrate cell-laden biodegradable scaffolds, microvasculatures, biomolecules, and micro/nanoscale engineering platforms. Due to its importance in human health, we select tissue engineering as a theme of this Special Issue of Biomedical Engineering Letters. This Special Issue introduces four review papers covering various state-of-theart technologies, including microengineering technologies, scaffold design and bio-inspired mechanics. The first paper entitled “Application of cellular micropatterns to miniaturized cell-based biosensor” is described by Koh and colleagues. Micropatterning enables the control of cell growth, differentiation, and apoptosis for cell-based sensor applications. Cellular detection on micropatterned substrates is generally performed by electrochemical methods (e.g., impedance-based electrochemical method, potential-based method) and optical methods (e.g., bioluminescence, fluorescence). The cellular micropatterning technique can also be integrated within microfluidics to enable high-throughput drug screening and discovery. Thus, this paper reviews the contemporary micropatterning technologies and their various miniaturized cell-based biosensor applications, such as multiphenotypic cell microarray, drug screening, and pathogen detection. Micropatterning techniques can also be used to fabricate microwells. Park and colleagues present an overview of “Microwell fabrication methods and applications for cellular studies”. This review introduces various fabrication techniques of microwells and mechanisms of cell trapping and handling. Microwells can be fabricated by soft lithography, implementation of physical forces, and micro-injection molding to manipulate the single cell trapping and cell aggregation. By using such microwell platforms, embryonic stem cells can be induced to form uniform-sized embryoid bodies that can be used to generate therapeutic cell types. The third paper entitled “Forming vascular networks within functional cardiac tissue constructs” is described by Bae and colleagues. Hydrogels are of great interest in scaffold fabrication for tissue engineering and regenerative medicine Bong Geun Chung ( ) Department of Mechanical Engineering, Sogang University, Seoul, Republic of Korea Tel : +82-2-705-8823 / Fax : +82-2-712-0799 E-mail : bchung@sogang.ac.kr

Transfer printing of transfected cell microarrays from poly(ethylene glycol)‐oleyl surfaces onto biological hydrogels

We have developed a novel technique for constructing microarrays of transfected mammalian cells on or in extracellular matrix (ECM) hydrogels by transfer printing from patterned poly(ethylene glycol) (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 ink-jet 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 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. The knockdown of a model gene in the transferred cell microarray by the transfected siRNA was successfully confirmed. Transfected cell microarrays were also embedded within three-dimensional ECM hydrogels. In the three-dimensional hydrogel, the inhibition effect of siRNA on cancer cell invasion was evaluated by quantifying the size of cell clusters on the microarrays. These results indicate that transfection of cell microarrays on 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.

The Role of in Vitro Gene Expression Profiling in Particulate Matter Health Research

Exposure to particulate matter (PM) is consistently associated with increased morbidity and mortality rate. The mechanisms for these adverse health effects have been vigorously investigated for many years, but remain uncertain, in part due to the complex interactions between host and exposure. Over the past decade, the use of global gene expression profiling has increased to investigate molecular changes in an attempt to gain more insight into the complex mechanisms that underlie the adverse health effects induced by PM. These experiments have been performed mostly in cell cultures, in part due to the easy availability and maneuverability of different cell types. Whether or not the results obtained from these in vitro experiments are relevant to human exposure is unclear. In this study, cell culture studies were reviewed that used microarray technology to measure global gene expression in response to PM and the findings discussed in the context of global gene expression results obtained from animal and human exposure studies. Ten in vitro studies were identified from PubMed that reported global gene expression results in response to PM exposure. Despite difference in cell types, microarray platforms, incubation time, and PM sources and doses, these experiments showed commonality in the expression of genes and pathways, especially xenobiotic metabolism, oxidative stress, and inflammation. These gene expression profiles were consistent with results from animal and human controlled exposure experiments. The in vitro experiments also uncovered novel biological mechanisms that may be important in PM-induced health effects reported in epidemiological studies. Data indicate that in vitro microarray experiments complement animal and human exposure studies and allow the PM-associated health research to focus on the “toxic” components in PM and novel mechanisms, and may enhance risk assessment beyond the current mass-based standards.

Surface Engineering for Long-Term Culturing of Mesenchymal Stem Cell Microarrays

The cell microarray format can recreate a multitude of cell microenvironments on a single chip using only minimal amounts of reagent. In this study, we describe surface modifications to passivate cell microarrays, aiming to adapt the platform to the study of stem cell behavior over long-term culture periods. Functionalization of glass slides with (3-glycidyloxypropyl) trimethoxysilane enabled covalent anchoring of extracellular matrix proteins on microscale spots printed by a robotic contact printer. Subsequently, the surface was passivated by bovine serum albumin (BSA) or poly(ethylene glycol)bisamine (A-PEG) with molecular weights of 3000, 6000, and 10 000 Da. Cloud-point conditions for A-PEG grafting were attained that were compatible with protein deposition. Passivation strategies were assessed by culturing mesenchymal stem cells on the microarray platform. While both BSA and A-PEG passivation initially blocked cell adhesion between the printed spots, only A-PEG grafting was able to maintain cell pattern integrity over the entire culture period of 3 weeks.

ETS2 Mediated Tumor Suppressive Function and MET Oncogene Inhibition in Human Non–Small Cell Lung Cancer

The ETS2 transcription factor is an evolutionarily conserved gene that is deregulated in cancer. We analyzed the transcriptome of lung adenocarcinomas and normal lung tissue by expression profiling and found that ETS2 was significantly downregulated in adenocarcinomas. In this study, we probed the yet unknown functional role of ETS2 in lung cancer pathogenesis. Lung adenocarcinomas (n = 80) and normal lung tissues (n = 30) were profiled using the Affymetrix Human Gene 1.0 ST platform. Immunohistochemical (IHC) analysis was conducted to determine ETS2 protein expression in non-small cell lung cancer (NSCLC) histologic tissue specimens (n = 201). Patient clinical outcome, based on ETS2 IHC expression, was statistically assessed using the log-rank and Kaplan-Meier tests. RNA interference and overexpression strategies were used to assess the effects of ETS2 expression on the transcriptome and on various malignant phenotypes. ETS2 expression was significantly reduced in lung adenocarcinomas compared with normal lung (P < 0.001). Low ETS2 IHC expression was a significant predictor of shorter time to recurrence in NSCLC (P = 0.009, HR = 1.89) and adenocarcinoma (P = 0.03, HR = 1.86). Moreover, ETS2 was found to significantly inhibit lung cancer cell growth, migration, and invasion (P < 0.05), and microarray and pathways analysis revealed significant (P < 0.001) activation of the HGF pathway following ETS2 knockdown. In addition, ETS2 was found to suppress MET phosphorylation and knockdown of MET expression significantly attenuated (P < 0.05) cell invasion mediated by ETS2-specific siRNA. Furthermore, knockdown of ETS2 augmented HGF-induced MET phosphorylation, cell migration, and invasion. Our findings point to a tumor suppressor role for ETS2 in human NSCLC pathogenesis through inhibition of the MET proto-oncogene.

Bioinspired Superhydrophobic Carbonaceous Hairy Microstructures with Strong Water Adhesion and High Gas Retaining Capability

Various hydrophobic hairy carbonaceous fibers are obtained by a low-temperature CVD process on catalyst-patterned surface patches which are selectively coated with silica to make the surface superhydrophobic and yet allow strong water adhesion for the "Salvinia effect". The versatility of the functional hairy fiber surfaces is demonstrated with a liquid barrier grid for cell microarray, a gas retaining capability under water/liquid for a membrane-free microfluidic chemical process, and functionalized papillae for cell immobilization with green algae.