Single Cell Array Research Articles

Ultrahigh-Throughput Approach for Analyzing Single-Cell Genomic Damage with an Agarose-Based Microfluidic Comet Array

Genomic DNA damage was generally identified with a "comet assay" but limited by low throughput and poor reproducibility. Here we demonstrated an ultrahigh-throughput approach with a microfluidic chip to simultaneously interrogate DNA damage conditions of up to 10,000 individual cells (approximately 100-fold in throughput over the conventional method) with better reproducibility. For experiment, agarose was chosen as the chip fabrication material, which would further act as an electrophoretic sieving matrix for DNA fragments separation. Cancer cells (HeLa or HepG2) were lined up in parallel microchannels by capillary effect to form a dense array of single cells. After treatment with different doses of hydrogen peroxide, individual cells were then lysed for subsequent single-cell gel electrophoresis in the direction vertical to microchannel and fluorescence detection. Through morphological analysis and fluorescent measurement of comet-shaped DNA, the damage conditions of individual cells could be quantified. DNA repair capacity was further evaluated to validate the reliability of this method. It indicated that the agarose-based microfluidic comet array electrophoresis was simple, highly reproducible, and of high throughput, providing a new method for highly efficient single-cell genomic analysis.

Single cell transcriptomic analysis of prostate cancer cells

BackgroundThe ability to interrogate circulating tumor cells (CTC) and disseminated tumor cells (DTC) is restricted by the small number detected and isolated (typically <10). To determine if a commercially available technology could provide a transcriptomic profile of a single prostate cancer (PCa) cell, we clonally selected and cultured a single passage of cell cycle synchronized C4-2B PCa cells. Ten sets of single, 5-, or 10-cells were isolated using a micromanipulator under direct visualization with an inverted microscope. Additionally, two groups of 10 individual DTC, each isolated from bone marrow of 2 patients with metastatic PCa were obtained. RNA was amplified using the WT-Ovation™ One-Direct Amplification System. The amplified material was hybridized on a 44K Whole Human Gene Expression Microarray. A high stringency threshold, a mean Alexa Fluor® 3 signal intensity above 300, was used for gene detection. Relative expression levels were validated for select genes using real-time PCR (RT-qPCR).ResultsUsing this approach, 22,410, 20,423, and 17,009 probes were positive on the arrays from 10-cell pools, 5-cell pools, and single-cells, respectively. The sensitivity and specificity of gene detection on the single-cell analyses were 0.739 and 0.972 respectively when compared to 10-cell pools, and 0.814 and 0.979 respectively when compared to 5-cell pools, demonstrating a low false positive rate. Among 10,000 randomly selected pairs of genes, the Pearson correlation coefficient was 0.875 between the single-cell and 5-cell pools and 0.783 between the single-cell and 10-cell pools. As expected, abundant transcripts in the 5- and 10-cell samples were detected by RT-qPCR in the single-cell isolates, while lower abundance messages were not. Using the same stringency, 16,039 probes were positive on the patient single-cell arrays. Cluster analysis showed that all 10 DTC grouped together within each patient.ConclusionsA transcriptomic profile can be reliably obtained from a single cell using commercially available technology. As expected, fewer amplified genes are detected from a single-cell sample than from pooled-cell samples, however this method can be used to reliably obtain a transcriptomic profile from DTC isolated from the bone marrow of patients with PCa.

Quantification of metal ion induced DNA damage with single cell array based assay

Under physiological and wear conditions, implanted orthopedic devices undergo undesired release of metal ions which cause DNA damage and inflammation of local tissue. However, individuals have personalized responses to identical devices due to varying susceptibility to DNA damage. The current one-size-fits-all approach is therefore not suitable to predict the response of patients to implanted devices. This paper describes a single cell array based method to quantify metal ion induced DNA damage that can potentially be used to predict the response to implanted devices in patients. Ions of several typical metals in implanted devices were used to treat human normal fibroblast cells. After patterning cells on a silicon substrate with cell-catching patches, cells were embedded in hydrogel and treated with alkaline buffer. Damaged DNAs diffuse out of the cell, and are stained to show a characteristic halo. All studied metal ions (Cu(2+), Co(2+), Ni(2+), Cr(2+), Fe(2+), Al(3+)) induce DNA damage and have genotoxicity. Copper ions cause DNA damage at concentrations as low as 1 μM. Cobalt and nickel ions damage DNA at 5 and 10 μM, respectively. Aluminum, iron and chromium ions cause DNA damage at 50 μM. The cytotoxicity assay shows that most ions, except cobalt and copper, are less toxic below 500 μM. The fact that metal ions can cause genotoxicity at lower concentrations than that of cytotoxicity suggests: (1) a single cell based DNA damage assay is more sensitive than a membrane integrity based live/dead assay; and (2) metal ions preferentially induce DNA damage rather than cell membrane damage.

Using surfaces to modulate the morphology and structure of attached cells – a case of cancer cells on chitosan membranes

This paper describes the development of physically and/or chemically modified chitosan membranes to probe cellular behaviors and molecular-level structural responses of NIH-3T3 fibroblasts (normal cells) and Ha-ras-transformed cells (abnormal cells) adhered onto these modified membranes. To prepare chitosan membranes with nanometrically scaled physical features, we have demonstrated an inexpensive and easy-to-handle method that could be easily integrated with IC-based manufacturing processes with mass production potential. These physically or chemically modified chitosan membranes were examined via scanning electron microscopy, atomic force microscopy, X-ray photoelectron spectroscopy, and water contact angle measurement, in order to gain a better understanding of chitosan membrane surface characteristics including surface morphology, stiffness, functional groups, and surface hydrophobicity/hydrophilicity. NIH-3T3 fibroblasts and their Ha-ras-transformed progeny were cultured on these modified chitosan membranes. After 12, 24 and 48 h of culture, these cells were investigated to decipher cellular behaviors. We found that NIH-3T3 fibroblasts and their Ha-ras-transformed progeny exhibited distinct structurally based responses attributable to chitosan membrane surface chemical or physical properties that we demonstrate as possibly applicable, for drug screening applications. Secondarily, but crucially to this study, we developed a chitosan-based micropatterning procedure that allowed us to re-arrange mammalian cells (i.e., HeLa cells in this study, for cancer drug screening) at the desired locations (with a single-cell array format). This procedure was based on cell affinity to different surface topographies of chitosan membranes that we prepared. This cell-based patterning approach has the potential for use in a wide range of applications including use as a promising platform for drug discovery, cytotoxicity studies, functional genomics, and investigations of cellular microenvironment. We believe that this study would provide further understanding of naturally derived biomaterials, lay the foundation for broadening the applications of chitosan, and facilitate the development of new biomedical devices (i.e., artificial stents, implantable artificial tissues, and sustainable implantable biosensors) with unique cell–material interface properties and characteristics, such as in vitro cell culture and diagnostic platforms.

Next-Generation Sequencing of Disseminated Tumor Cells

Disseminated tumor cells (DTCs) detected in the bone marrow have been shown as an independent prognostic factor for women with breast cancer. However, the mechanisms behind the tumor cell dissemination are still unclear and more detailed knowledge is needed to fully understand why some cells remain dormant and others metastasize. Sequencing of single cells has opened for the possibility to dissect the genetic content of subclones of a primary tumor, as well as DTCs. Previous studies of genetic changes in DTCs have employed single-cell array comparative genomic hybridization which provides information about larger aberrations. To date, next-generation sequencing provides the possibility to discover new, smaller, and copy neutral genetic changes. In this study, we performed whole-genome amplification and subsequently next-generation sequencing to analyze DTCs from two breast cancer patients. We compared copy-number profiles of the DTCs and the corresponding primary tumor generated from sequencing and SNP-comparative genomic hybridization (CGH) data, respectively. While one tumor revealed mostly whole-arm gains and losses, the other had more complex alterations, as well as subclonal amplification and deletions. Whole-arm gains or losses in the primary tumor were in general also observed in the corresponding DTC. Both primary tumors showed amplification of chromosome 1q and deletion of parts of chromosome 16q, which was recaptured in the corresponding DTCs. Interestingly, clear differences were also observed, indicating that the DTC underwent further evolution at the copy-number level. This study provides a proof-of-principle for sequencing of DTCs and correlation with primary copy-number profiles. The analyses allow insight into tumor cell dissemination and show ongoing copy-number evolution in DTCs compared to the primary tumors.

Concurrent Transcript and Protein Quantification in a Massive Single Cell Array Enables Population-Wide Observation of Oncogene Escape

The cancer stem cell (CSC) model focuses on the key role these cells can play in drug resistance, since CSCs are believed to reconstitute the cancer after intense chemotherapy treatment. In order to effectively identify and profile CSCs within a heterogeneous tumor population, we are investigating quantitative correlation of messenger RNAs and their translated proteins as a distinctive parameter of the CSC population. However, previous research on mRNA expression and protein abundance has shown the correlations between the two are weak or only “stochastically meaningful” due to the significant level of experimental error originating from ensemble observations. Here, we demonstrate a robust microwell-based method to minimize these errors by monitoring both mRNA expression and the corresponding protein abundance from individual cancer cells. Simultaneous observation of membrane protein expression by immunostaining and detection of mRNA transcripts directly from individual cells using a one-step, reverse transcription polymerase chain reaction have been integrated in a massive single-cell array platform. The proposed experimental scheme was initially tested and validated in three established lung cancer cell lines by correlating mRNA transcript and protein expression levels of individual cells and quantitation of heterogeneity. Responses at the individual cell level to known transcriptional and translational inhibitors, as well as to EGFR-specific inhibitors were evaluated, providing quantitative measures of the heterogeneous response of non-small cell lung cancer cells to the inhibitors. Results showed that drug-treated cell lines displayed oncogene escape due to expunction of drug-sensitive subpopulations in the cell lines. Furthermore, correlation of c-MET mRNA and protein levels revealed unique response patterns in different EGFR-mutated cell lines. Thus, these results demonstrate the potential for molecular profiling at the single cell level to prospectively identify the CSCs subpopulation for effective combinatorial treatments.

Development of a single-cell array for large-scale DNA fluorescence in situ hybridization

DNA fluorescence in situ hybridization (FISH) is a powerful cytogenetic assay, but conventional sample-preparation methods for FISH do not support large-scale high-throughput data acquisition and analysis, which are potentially useful for several biomedical applications. To address this limitation, we have developed a novel FISH sample-preparation method based on generating a centimetre-sized cell array, in which all cells are precisely positioned and separated from their neighbours. This method is simple and capable of patterning nonadherent human cells. We have successfully performed DNA FISH on the single-cell arrays, which facilitates analysis of the FISH results with the FISH-FINDER computer program.

Nanoreplica moulding of polyacrylamide hydrogels

Polyacrylamide (PAM) hydrogels are widely used in bioanalysis and biosensing applications. Scaling down of PAM patterns to micro/nanosize extends PAM applications to lab-on-a-chip, highly sensitive biosensors and cell/tissue analysis. Proposed is a replica moulding technique to pattern the PAM surface down to nanosize. Various patterns on silicon moulds successfully transferred to PAM with a minimum dimension of 60 nm and aspect ratio of up to ∼9. PAM is characterised as a platform for a single cell array and the analysis of cell responses to topographic cues. Moreover, micro/nanopatterned PAM samples were implanted under the back skin of rats to study the topological effects on immuno responses in vivo. Therefore the use of micro/nanoreplica moulding to pattern PAM hydrogel provides opportunities for studying the relationship between soft nanoscaled structures and cell/tissue.

Direct-Write Patterning of Bacterial Cells by Dip-Pen Nanolithography

The ability of dip-pen nanolithography (DPN) to generate nano- or microarrays of soft or hard materials (e.g., small molecules, DNA, proteins, nanoparticles, sols, and polymers) in a direct-write manner has been widely demonstrated. The transporting of large-sized ink materials such as bacteria, however, remains a significant challenge with this technique. The size limitation of the water meniscus formed between the DPN tip and the solid surface becomes a bottleneck in such diffusion-based molecular transport experiments. Herein, we report a straightforward "stamp-on" DPN method that uses a nanostructured poly(2-methyl-2-oxazoline) hydrogel-coated tip and carrier agents to generate patterns of micrometer-sized Escherichia coli JM 109 bacterial cells. We demonstrate that this approach enables the deposition of a single bacterial cell array on a solid surface or arrays of layers of multiple cells by modulating the viscosity of the "ink" solution. Fluorescence microscopy images indicated that the deposited bacterial cells were kept alive on Luria-Bertani-agar layered solid surfaces after DPN patterning.

O2 Altered morphokinetic parameters of embryos identified as aneuploid by single cell array CGH analysis at the 8-cell stage

O2 Altered morphokinetic parameters of embryos identified as aneuploid by single cell array CGH analysis at the 8-cell stage

Deformation measurement of individual cells in large populations using a single-cell microchamber array chip

We analyze the deformability of individual red blood cells (RBCs) using SiCMA technology. Our approach is adequate to quickly measure large numbers of individual cells in heterogeneous populations. Individual cells are trapped in a large-scale array of micro-wells, and dielectrophoretic (DEP) force is applied to deform the cells. The simple structures of micro-wells and DEP electrodes facilitate the analysis of thousands of RBCs in parallel. This unique method allows the correlation of red cell deformation with cell surface and cytosolic characteristics to define the distribution of individual cellular characteristics in heterogeneous populations.

Abrupt and Dynamic Changes in Gene Expression Revealed by Live Cell Arrays

A description of the noise associated with gene expression is presented, based on a simplified form of the combined multistep processes of transcription and translation. These processes are influenced by numerous factors, including the accessibility of promoter regions to the transcriptional machinery, the kinetics of assembly of the transcription complexes, and the synthesis and degradation of both mRNA and proteins, among others. Ultimately, stochasticity in cellular processes results in variation in protein levels. Here we constructed a rationally designed RNA-based transcriptional activator to reduce these variables and provide a cleaner, more detailed portrayal of cellular noise. Functioning at a level comparable to natural transcription activation, this activator is isolated to a lacZ reporter gene in yeast cells to quantitatively describe the efficiency of the combined processes of transcription and translation. By employing single-cell array techniques to monitor individual cells simultaneously and in real time, a statistical approach to investigate noise inherent in gene expression is possible. Live cell arrays enabled cell populations to be characterized temporally at the individual cell level. The array platform allowed for a relative measure of protein production in real time and could characterize protein bursts with variable size and random timing, such that bursts occurred in a temporally indiscriminate fashion. The inherent variability and randomness of these processes is characterized, with almost half (47%) of cells experiencing bursting behavior at least once over the course of the experiment. We demonstrate that cells identified on the upper periphery of activity exhibit behaviors that are substantially different from the majority of the population, and such variable activities within a population will provide a more accurate characterization of the population.

Trapping Yeast Cells on PDMS Micropillar Array

A selectively yeast cell trapping and arraying method was presented, utilizing PDMS micropillar array combining with chemical adhesion. Yeast cells were trapped on the top of micropillar array while swept on the flat area for the pinning of liquid/surface contact line on the micropillars and moving on the flat surface. And the modification of poly-L-lysine on the yeast cells improved the immobilization of cells on the surface of PDMS surface. Both of simulation and experiment results shows that by adjusting the diameter of micropillars, the number of yeast cells on each pillars could be controlled. Single cell array was formed with a 8.3 μm micropillar array, and majority yeast numbers of 3,4,5 was got for the 13.7 μm、18.0 μm and 18.8 μm micropillar array.

Construction of an artificial cell membrane anchor using DARC as a fitting for artificial extracellular functionalities of eukaryotic cells

The need to functionalize cell membranes in a directed way for specific applications as single cell arrays or to force close cell-to-cell contact for artificial intercellular interaction and/or induction concerning stem cell manipulation or in general to have a tool for membrane and cell surface-associated processes, we envisaged a neutral inactive membrane anchor for extracellular entities to facillitate the above mentioned functionalities.The silent Duffy antigen/receptor for chemokines (DARC) is a receptor-like membrane protein of erythrocytes and mediates no cell transduction not at least regarding a missing or truncated G-loop and therefore it seemed to be the candidate for our cell membrane anchor.We isolated the genetic information of DARC from human genomic DNA and cloned it in a mammalian cell line as a fusion protein via a suitable plasmid vector.In this report we demonstrate that the human plasma membrane protein DARC can be used as an artificial anchor molecule in cell surface engineering applications. We constructed the fusion protein SNAP-tag-DARC, consisting of DARC and the self-labeling protein tag SNAP-tag® (Covalys). The SNAP-tag® served as an example for a molecular-technological developed protein that is artificially attached to the extracellular side of the plasma membrane through our DARC-anchor. SnapTag should serve as an example for any extracellular entity and was easy to detect by a commercial detection system. The synthesis of SNAP-tag-DARC, its correct incorporation into the cell membrane and the functionality of the SNAP-tag® were verified by RT-PCR, Western blotting and confocal fluorescence microscopy and showed the desired functionality as an membrane anchor for an extracellular application entity.

High-throughput quantitative imaging of cell spreading dynamics by multi-step microscopy projection photolithography based on a cell-friendly photoresist

A new method for the high-throughput study of cell spreading dynamics is devised by multi-step microscopy projection photolithography based on a cell-friendly photoresist. By releasing a large number of rounded cells in single cell arrays and monitoring their spreading dynamics by interference reflection microscopy, a large number of cell spreading data can be acquired by a single experiment.

Modeling of Arbitrary Power Level PV Array Based on Circuit Equivalent Mechanism

According to the equivalent circuit of single solar cell and the V-I equation of PV cell based on physical mechanism, a simulation model of single solar cell is established. According to this model and structure feature of PV array, the Thevenin circuit equivalent transform theory is used. The control relationship between single solar cell and PV array is given. Then a simple modeling method for arbitrary power level PV array is proposed. To verify the model effectiveness, a Pspice simulation model is established. Output characteristics of the model and its feasibility in PV power generation system are simulated. Simulation results show that the output characteristics of simulation model are in good consistency with actual PV array, and the model can be effectively applied in simulation of PV power generation system. The modeling method is simple and can be used for simulation on some control algorithms of PV power generation system

Shrink-Induced Single-Cell Plastic Microwell Array

The ability to interrogate and track single cells over time in a high-throughput format would provide critical information for fundamental biological understanding of processes and for various applications, including drug screening and toxicology. We have developed an ultrarapid and simple method to create single-cell wells of controllable diameter and depth with commodity shrink-wrap film and tape. Using a programmable CO(2) laser, we cut hole arrays into the tape. The tape then serves as a shadow mask to selectively etch wells into commodity shrink-wrap film by O(2) plasma. When the shrink-wrap film retracts upon briefly heating, high-aspect plastic microwell arrays with diameters down to 20 μm are readily achieved. We calibrated the loading procedure with fluorescent microbeads. Finally, we demonstrate the utility of the wells by loading fluorescently labeled single human embryonic stem cells into the wells.

On the symmetry of siblings: automated single-cell tracking to quantify the behavior of hematopoietic stem cells in a biomimetic setup

The interplay between hematopoietic stem and progenitor cells (HSPC) and their local microenvironment is a key mechanism for the organization of hematopoiesis. To quantitatively study this process, a time-resolved analysis of cellular dynamics at the single-cell level is an essential prerequisite. One way to generate sufficient amounts of appropriate data is automatic single-cell tracking using time-lapse video microscopy. We describe and apply newly developed computational algorithms that allow for an automated generation of high-content data of single-cell characteristics at high temporal and spatial resolution, together with the reconstruction and statistical evaluation of complete genealogical histories. This methodology has been applied to the particular example of purified primary human HSPCs in bioengineered culture conditions. The combination of genealogical information and dynamic profiles of cellular properties identified a marked symmetry between sibling HSPCs regarding cell cycle time, but also migration speed and growth kinetics. Furthermore, we demonstrate that this symmetry of HSPC siblings can be altered by exogenous cues of the local biomimetic microenvironment. Using the example of HSPC growth in biomimetic culture systems, we show that our approach provides a valuable tool for the quantitative analysis of dynamic single-cell features under defined invitro conditions, allowing for integration of functional and genealogical data. The efficiency and accuracy of our approach pave the way for new and intriguing insights into the organizational principles of developmental patterns and the respective influence of exogenous cues not limited to the study of primary HSPCs.

High‐resolution analyses of copy number changes in disseminated tumor cells of patients with breast cancer

The presence of disseminated tumor cells (DTCs) in bone marrow (BM) identifies breast cancer patients with less favorable outcome. Furthermore, molecular characterization is required to investigate the malignant potential of these cells. This study presents a single-cell array comparative genomic hybridization (SCaCGH) method providing molecular analysis of immunomorphologically detected DTCs. The resolution limit of the method was estimated using the cancer cell line SK-BR-3 on 44 and 244k arrays. The technique was further tested on 28 circulating tumor cells and four hematopoietic cells (HCs) from peripheral blood (n = 8 patients). The SCaCGH method was finally applied to 24 DTCs, three immunopositive cells morphologically classified as probable HCs from breast cancer patients and five HC controls from BM (n = 7 patients plus n = 1 healthy donor). The frequency of copy number changes of the DTCs revealed similarities with primary breast tumor samples. Three of the patients had available profiles for DTCs and the corresponding tumor tissue from primary surgery. More than two-third of the analyzed DTCs disclosed equivalent changes, both to each other and to the corresponding primary disease, whereas the rest of the cells showed balanced profiles. The probable HCs revealed either balanced profiles (n = 2) or changes comparable to the tumor tissue and DTCs (n = 1), indicating morphological overlap between HCs and DTCs. Similar aberration patterns were visible in DTCs collected at diagnosis and at 3 years relapse-free follow-up. SCaCGH may be a powerful tool for the molecular characterization of DTCs.

Laser microstructuring of photomodified fluorinated ethylene propylene surface for confined growth of Chinese hamster ovary cells and single cell isolation

We present a method for laser lithography of cell-adhesive arrays on a fluoropolymer surface. The method is based on 172 nm excimer-lamp photomodification in ammonia atmosphere followed by microstructuring by laser ablation. The improved wettability of the polymer is caused by new chemical groups on the surface after the UV treatment that we proved by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy analyses. The cell adhesion properties of micropatterned structures were tested by cultivation of mammalian cells. We show that single elongated cells can grow confined to lines with sharply defined boundaries of the cell-covered areas. In preliminary experiments, we also demonstrate that the described technique allows the production of single-cell arrays with variable cell shape.