Microwell Chip Research Articles

Bead-based microarray immunoassay for lung cancer biomarkers using quantum dots as labels

In this study, we developed a multiplex immunoassay system that combines the suspension and planar microarray formats within a single layer of polydimethylsiloxane (PDMS) using soft lithography technology. The suspension format was based on the target proteins forming a sandwich structure between the magnetic beads and the quantum dot (QD) probes through specific antibody–antigen interactions. The planar microarray format was produced by fabricating an array of micro-wells in PDMS. Each micro-well was designed to trap a single microbead and eventually generated a microbead array within the PDMS chamber. The resultant bead-based on-chip assay could be used for simultaneously detecting three lung cancer biomarkers—carcinoembryonic antigen (CEA), fragments of cytokeratin 19 (CYFRA21-1) and neuron-specific enolase (NSE)—in 10μl of human serum, with a wide linear dynamic range (1.03–111ng/mL for CEA and CYFRA21-1; 9.26–1000ng/ml for NSE) and a low detection limit (CEA: 0.19ng/ml; CYFRA21-1: 0.97ng/ml; NSE: 0.37ng/ml; S/N=3). Our micro-well chip does not require complex e-beam lithography or the reactive ion etching process as with existing micro-well systems, which rely on expensive focused ion beam (FIB) milling or optical fiber bundles. Furthermore, the current approach is easy to operate without extra driving equipment such as pumps, and can make parallel detection for multiplexing with rapid binding kinetics, small reagent consumption and low cost. This work has demonstrated the importance of the successful application of on-chip multiplexing sandwich assays for the detection of biomarker proteins.

Full-length single-cell RNA-seq applied to a viral human cancer: applications to HPV expression and splicing analysis in HeLa S3 cells.

BackgroundViral infection causes multiple forms of human cancer, and HPV infection is the primary factor in cervical carcinomas. Recent single-cell RNA-seq studies highlight the tumor heterogeneity present in most cancers, but virally induced tumors have not been studied. HeLa is a well characterized HPV+ cervical cancer cell line.ResultWe developed a new high throughput platform to prepare single-cell RNA on a nanoliter scale based on a customized microwell chip. Using this method, we successfully amplified full-length transcripts of 669 single HeLa S3 cells and 40 of them were randomly selected to perform single-cell RNA sequencing. Based on these data, we obtained a comprehensive understanding of the heterogeneity of HeLa S3 cells in gene expression, alternative splicing and fusions. Furthermore, we identified a high diversity of HPV-18 expression and splicing at the single-cell level. By co-expression analysis we identified 283 E6, E7 co-regulated genes, including CDC25, PCNA, PLK4, BUB1B and IRF1 known to interact with HPV viral proteins.ConclusionOur results reveal the heterogeneity of a virus-infected cell line. It not only provides a transcriptome characterization of HeLa S3 cells at the single cell level, but is a demonstration of the power of single cell RNA-seq analysis of virally infected cells and cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s13742-015-0091-4) contains supplementary material, which is available to authorized users.

Sensitivity-enhanced and noise-reduced surface plasmon resonance sensing with microwell chips

In this paper, we study a surface plasmon resonance (SPR) sensor with a microwell chip that is aimed at improving the sensitivity and reducing the noise. We present both theoretical simulations and experimental results obtained for the proposed structure. In comparison to most SPR sensors that use planar gold films, this proposed well structure enables a sensitivity enhancement of 128%. By confining the surface plasmon wave (SPW) within the well, we are able to reduce the noise by more than twice. The increased sensitivity and lower noise makes our proposed novel microwell chip suitable for small molecule or trace amount detection.

Self-seeding microwell chip for the isolation and characterization of single cells.

A self-seeding microwell chip is introduced for the isolation and interrogation of single cells. A cell suspension is transferred to a microwell chip containing 6400 microwells, each microwell with a single 5 μm pore in the bottom. The fluid enters the microwell and drags a cell onto the pore. After a cell has landed onto the pore, it will stop the fluid flow through this microwell. The remaining fluid and cells will be diverted to the next available microwell. This results in a fast and efficient distribution of single cells in individual microwells. After identification by fluorescence microscopy, the cells of interest are isolated from the microwell by punching the bottom together with the cell. The overall single cell recovery of seeding followed by isolation of the single cell, is >70% with a specificity of 100% as confirmed by the genetic make-up of the isolated cells.

Position detection method for microwell chip on microscope stage by photon multiplier tube

In this paper, we propose a position detection method for a microwell with manageable volume (MMV) chip on the stage of the inverted microscope. When the manipulation robot arm puts an MMV chip on the microscope stage, there is alignment error up to 500 μm. This error affects measurement accuracy of fluorescence from solution filled in a tiny hall of the MMV chip. Usually, CCD image processing is used for detecting a position. However, appending a CCD camera to our microscope increases the complexity of the optical system. Therefore, we used a photon multiplier tube which was already equipped for measuring fluorescence. In the result, the position of the MMV chips was detected by putting a pilot LED directly above the objective lens. The detection error was less than 10 μm. Although this method was devised for our system, we considered that it was available in other micro optics systems.

High throughput micro-well generation of hepatocyte micro-aggregates for tissue engineering.

The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific markers. Moreover we observed that the beneficial effect was dependent on the aggregate dimensions, indicating that aggregate parameters should be carefully considered. In a second part of the study, the selected aggregates were immobilized by encapsulation in methacrylamide-modified gelatin. Phenotype evaluations revealed that a stable hepatocyte phenotype could be maintained during 21 days when encapsulated in the hydrogel. In conclusion we have demonstrated the beneficial use of micro-well chips for hepatocyte aggregation and the size-dependent effects on hepatocyte phenotype. We also pointed out that methacrylamide-modified gelatin is suitable for the encapsulation of these aggregates.

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.

Micropatterned culture of HepG2 spheroids using microwell chip with honeycomb-patterned polymer film

Microwell chip culture is a promising technique for the generation of homogenous spheroids. We investigated the relationship between the structure of the bottom surface of microwell chip and the properties of HepG2 spheroid. We developed a microwell chip, the bottom surface of which consisted of a honeycomb-patterned polymer film (honeycomb film) that had a regular porous structure (HF chip). The chip comprised 270 circular microwells; each microwell was 600μm in diameter and 600μm in depth. At the center of the honeycomb film, an area, 200μm in diameter, was modified with collagen to facilitate cell adhesion. With the exception of the collagen-coated area, the entire microwell was modified with polyethylene glycol to eliminate cell adhesion. HepG2 cells formed uniform spheroids when cultured in the microwells of HF chip. Furthermore, the cells passed through the porous structure of honeycomb film and formed spheroids at its opposite side. The spheroid growth of HepG2 cells cultured in HF chip was greater than that when the cells were culture in a microwell chip, the bottom surface of which was made of poly-methylmethacrylate (PMMA chip). The albumin secretion activity of HepG2 spheroids in HF chip was equal to that in PMMA chip. These results indicate that the microwell bottom with a porous structure enhances the cell growth and maintains well the spheroid function. Thus, HF chip is a promising platform for spheroid cell culture.

Application of the DataChip/MetaChip technology for the evaluation of ajoene toxicity in vitro

The DataChip is a universal platform for three-dimensional (3D) cell cultures on a micropillar chip, which can be applicable to a variety of human cells to simulate organ-specific toxicity. In addition, the MetaChip is developed for various combinations of drug metabolizing enzymes that can be spotted into the microwell chip and incubated with 3D human cells to simulate systematic compound metabolism in the human liver on a microscale format. Ajoenes have been known for various therapeutics activities, including anticancer effects, but there was limited information available in regard to their metabolism and cytotoxicity. In the present work, the metabolism-mediated toxicity of ajoenes was evaluated on a DataChip/MetaChip platform. In detail, we tested cytotoxicity of E- and Z-ajoene on 3D cultured Hep3B human hepatoma cells coupled with mixtures of drug metabolizing enzymes. Metabolic profiles of ajoenes were assessed with 23 representative drug metabolizing enzymes on the MetaChip. As a result, cytotoxicity of E-ajoene was significantly augmented in the presence of cytochrome P450 (CYP) isoforms, such as CYP2E1 and CYP3A5. Both E- and Z-ajoene were drastically detoxified in the presence of Phase II enzymes, including major UGTs, SULTs, NATs, and GSTs. Interestingly, All Mix, an artificial human liver microsome containing representative P450 mixture and phase II enzyme mixture, attenuated P450-induced cytotoxicity of ajoenes. Conclusively, we were able to confirm the metabolism-medicated toxicity of ajoenes on the chip.

Characterization of mouse embryoid bodies cultured on microwell chips with different well sizes

Microwell chip culture is a promising technique for the generation of homogenous embryoid bodies (EBs). In this study, we focused on the relationship between microwell size and mouse EB properties. The basic chip design was 195 microwells in a triangular arrangement on a polymethylmethacrylate plate with a surface modified by polyethylene glycol to render it nonadhesive, and 4 similar chips were fabricated with microwell diameters of 400, 600, 800, and 1000μm. The cell proliferation rate of EBs in larger microwells was higher than that of EBs in smaller microwells. The decrease in the expression levels of undifferentiated marker genes (Oct3/4 and Nanog) in larger microwells was faster than that in smaller microwells. The expression of hepatic (transthyretin and alpha-fetoprotein), cardiac (Nkx2.5 and alpha-myosin heavy chain), and vascular (fetal liver kinase-1; Flk1) markers in larger microwells was higher than that in smaller microwells. The expression levels of differentiation markers except Flk1 in the chip with a diameter of 1000μm were similar to those in hanging drop culture. However, Flk1 expression in microwell chip was markedly lower than that in hanging drop culture, suggesting that microwell chip culture promotes differentiation of hepatic and cardiac lineages. Furthermore, glucose consumption and lactate production were higher in smaller microwells, suggesting that the culture proceeds under anaerobic conditions in smaller microwells. These results indicate that the difference in microwell size affects the proliferation and differentiation of embryonic stem cells, and that microwell culture is a promising technique to control EB properties.

Microwell Chips for Selection of Bio‐macromolecules that Increase the Differentiation Capacities of Mesenchymal Stem Cells

Microwell chips (25 mm × 25 mm) are fabricated to select proper substrates for growing three-dimensional (3D) spheroids from mesenchymal stem cells (MSCs). Different bio-macromolecules and their combinations are immobilized on the chip by air plasma treatment and by polyelectrolyte interaction. Only a small number of MSCs (≈10(5) ) are needed for each chip. The expression level of N-cadherin, a cell-adhesion molecule, is used as an indicator for cell-cell interactions. MSC spheroids expressing the highest N-cadherin level also show the greatest osteogenic potential. The microwell chip may be used as an efficient platform to screen bio-macromolecules that enhance the differentiation potential of MSCs.

High-throughput metabolic genotoxicity screening with a fluidic microwell chip and electrochemiluminescence.

A high throughput electrochemiluminescent (ECL) chip was fabricated and integrated into a fluidic system for screening toxicity-related chemistry of drug and pollutant metabolites. The chip base is conductive pyrolytic graphite onto which are printed 64 microwells capable of holding one-μL droplets. Films combining DNA, metabolic enzymes and an ECL-generating ruthenium metallopolymer (Ru(II)PVP) are fabricated in these microwells. The system runs metabolic enzyme reactions, and subsequently detects DNA damage caused by reactive metabolites. The performance of the chip was tested by measuring DNA damage caused by metabolites of the well-known procarcinogen benzo[a]pyrene (B[a]P). Liver microsomes and cytochrome P450 (cyt P450) enzymes were used with and without epoxide hydrolase (EH), a conjugative enzyme required for multi-enzyme bioactivation of B[a]P. DNA adduct formation was confirmed by determining specific DNA-metabolite adducts using similar films of DNA/enzyme on magnetic bead biocolloid reactors, hydrolyzing the DNA, and analyzing by capillary liquid chromatography-mass spectrometry (CapLC-MS/MS). The fluidic chip was also used to measure IC50-values of inhibitors of cyt P450s. All results show good correlation with reported enzyme activity and inhibition assays.

Openly Accessible Microfluidic Liquid Handlers for Automated High-Throughput Nanoliter Cell Culture

Cell culture is typically performed in Petri dishes, with a few million cells growing together, or in microwell plates with thousands of cells in each compartment. When the throughput of each experiment, especially of screening based assays, is increased, even using microliter solution per well will cost a considerable amount of cells and reagents. We took a rational approach to reduce the volume of each cell culture chamber. We designed and fabricated a poly(dimethylsiloxane) based liquid pipet chip to deliver and transfer nanoliter (50-500 nL) samples and reagents with high accuracy and robustness. A few tens to a few hundreds of cells can be successfully seeded, transferred, passaged, transfected, and stimulated by drugs on a microwell chip using this pipet chip automatically. We have used this system to test the cell growth dynamically, observed the correlation between the culture conditions and cell viabilities, and quantitatively evaluated cell apoptosis induced by cis-diammineplatinum(II) dichloride (cisplatin). This system shows great potential to facilitate large-scale screening and high-throughput cell-array based bioassays with the volume of each individual cell colony at the nanoliter level.

Rehydratable gel for rapid loading of nanoliter solution and its application in protein crystallization

In the work presented, rehydratable polyacrylamide gel is introduced as a medium to uptake and store nanoliter protein solutions in microwells for multiplex bioanalysis. The polyacrylamide gel, produced and stored in the microwells, shrank by 97% upon dehydration and could be reversibly rehydrated to 95% of the initial volume by absorbing aqueous solution. We employed the rehydratable gel to load aqueous solutions of different proteins with molecular weights in the range of 14.7–250 kDa. The protein loading occurred simultaneously with the gel rehydration and reached saturated state in 5 min. The relative protein concentrations in the gel ranged from 92% to 53%, depending on the molecular weight of the proteins. Particularly, the rehydratable gel had a much higher protein loading efficiency than the fresh gel. We applied the protein-carrying gel to the crystallization of four model proteins in the microwells and produced diffraction-quality protein crystals. The rehydratable gel is simple to fabricate, efficient to load with protein, and has good capacity for storing the protein solutions in microwells with minimal dilution effects on the protein solution. The rehydratable gel incorporated microwell chip should be useful in multiplex analysis that requires small sample consumption and high throughput.

Embryoid body culture of mouse embryonic stem cells using microwell and micropatterned chips

The proliferation and differentiation properties of embryoid bodies (EB) from mouse embryonic stem (ES) cells were compared under two microchip conditions: microwell chip and micropatterned chip. The microwell chip contained 270 microwells (diameter, 600 μm; depth, 600 μm) on a polymethylmethacrylate plate and was surface-modified with polyethylene glycol (PEG) to render it non-adhesive. The micropatterned chip contained 270 gelatin spots (diameter, 200 μm) as the cell adhesion area on a glass plate; the region lacking these spots was PEG-modified to render it non-adhesive. The ES cells spontaneously formed the EBs from cell aggregates in each microwell in the chip. In contrast, cells inoculated onto the patterned chip formed a monolayer on the gelatin spots and gradually proliferated to form EBs. The EBs in the patterned chip maintained the high cell growth rate and the expression of endoderm (TTR and AFP) and mesoderm (Nkx2.5, αMHC, Flk1, and PDGFRβ) markers was increased, and these cell properties were similar to the previous methods (hanging drop and round-bottomed 96-well plate cultures). In contrast, the proliferation of ES cells in the microwell chip was lower than in the patterned chip and previous methods, and the EB differentiation proceeded slowly and only formed a small amount of endoderm. These results indicate that the difference of EB generating process in the microchip cultures may affect to the proliferation and differentiation of ES cells, and the existence of microwell structure in the microchip downregulates the cell proliferation and the differentiated progress of ES cells.

Effect of microwell chip structure on cell microsphere production of various animal cells

The formation of three-dimensional cell microspheres such as spheroids, embryoid bodies, and neurospheres has attracted attention as a useful culture technique. In this study, we investigated a technique for effective cell microsphere production by using specially prepared microchip. The basic chip design was a multimicrowell structure in triangular arrangement within a 100-mm(2) region in the center of a polymethylmethacrylate (PMMA) plate (24x24 mm(2)), the surface of which was modified with polyethylene glycol (PEG) to render it nonadhesive to cells. We also designed six similar chips with microwell diameters of 200, 300, 400, 600, 800, and 1000 microm to investigate the effect of the microwell diameter on the cell microsphere diameter. Rat hepatocytes, HepG2 cells, mouse embryonic stem (ES) cells, and mouse neural progenitor/stem (NPS) cells formed hepatocyte spheroids, HepG2 spheroids, embryoid bodies, and neurospheres, respectively, in the microwells within 5 days of culture. For all the cells, a single microsphere was formed in each microwell under all the chip conditions, and such microsphere configurations remained throughout the culture period. Furthermore, the microsphere diameters of each type of cell were strongly positively correlated with the microwell diameters of the chips, suggesting that microsphere diameter can be factitiously controlled by using different chip conditions. Thus, this chip technique is a promising cellular platform for tissue engineering or regenerative medicine research, pharmacological and toxicological studies, and fundamental studies in cell biology.

Nanoporous Titania Coating of Microwell Chips for Stem Cell Culture and Analysis

Stem cell research is today an active and promising field of research. To learn more about the biology of stem cells, technical improvements are needed such as tools to study stem cells in order to characterize them further and to gain insights to the molecular regulations of their maintenance, differentiation and identification. Common procedure when studying stem cells is to coat the surface where the stem cells are to be cultured with organic materials like matri-gel, poly-L-lysine and fibronectin. The resulting coating is usually relatively fragile and it is difficult to know if the coating is evenly distributed. In addition, these forms of coatings cannot be sterilized and re-used, but must be added as an initial, time-consuming step in the daily protocol. A microwell chip with hundreds of 500 nl wells has recently been shown to be a useful tool for stem cell culturing. This platform is here modified to facilitate and improve the coating conditions for adherent cell culture. A robust and highly porous film of TiO2 is deposited in the wells prior cell seeding. TiO2 is known to be biocompatible and provides a surface that is even and well characterized, simple to produce and re-usable. Furthermore it enables the microwell chips to be stored pre-coated for longer periods of time before use. We investigated the growth of rat mesenchymal stem cells on nanoporous titania films and found that they proliferated much faster than on conventional coatings. The combination of the robust TiO2 coating of the microwell chip enables thousands of individually separated single, or clones of, stem cells to be studied simultaneously and opens up the possibility for more user-friendly cell culturing.

A microwell array device with integrated microfluidic components for enhanced single‐cell analysis

Increasing awareness of the importance of cell heterogeneity in many biological and medical contexts is prompting increasing interest in systems that allow single-cell analysis rather than conventional bulk analysis (which provides average values for variables of interest from large numbers of cells). Recently, we presented a microwell chip for long-term, high-throughput single-cell analysis. The chip has proved to be useful for purposes such as screening individual cancer and stem cells for protein/gene markers. However, liquids in the wells can only be added or changed by manually rinsing the chip, or parts of it. This procedure has several well-known drawbacks--including risks of cross-contamination, large dead volumes and laboriousness--but there have been few previous attempts to integrate liquid rinsing/switching channels in "ready-to-use" systems for single-cell analysis. Here we present a microwell system designed (using flow simulations) for single-cell analysis with integrated microfluidic components (microchannels, magnetically driven micropumps and reservoirs) for supplying the cell culture wells with reagents, or rinsing, thus facilitating controlled, directed liquid handling. It can be used totally independently, since tubing is not essential. The practical utility of the integrated system has been demonstrated by culturing endothelial cells in the microwells, and successfully applying live-cell Calcein AM staining. Systems such as this combining high-density microwell chips with microfluidic components have great potential in numerous screening applications, such as exploring the important, but frequently undetected, heterogeneity in drug responses among individual cells.

High-Density Microwell Chip for Culture and Analysis of Stem Cells

With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)-differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. For example, screening of 700,000 substances would take less than two days, using this platform combined with a conventional bio-imaging system. The microwell chip has standard slide format and consists of 672 wells in total. Each well holds 500 nl, a volume small enough to drastically decrease reagent costs but large enough to allow utilization of standard laboratory equipment. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to either maintain the cells as stem/progenitor cells or to study cell differentiation of stem/progenitor cells over time is demonstrated. Clonality is critical for stem cell research, and was accomplished in the microwell chips by isolation and clonal analysis of single mouse embryonic stem cells using flow cytometric cell-sorting. Protocols for practical handling of the microwell chips are presented, describing a rapid and user-friendly method for the simultaneous study of thousands of stem cell cultures in small microwells. This microwell chip has high potential for a wide range of applications, for example directed differentiation assays and screening of reprogramming factors, opening up considerable opportunities in the stem cell field.

A Microwell Array Platform for Picoliter Membrane Protein Assays

A novel microwell chip is developed that can be used to detect protein binding in a liquid environment, together with a liquid handling system that allows the performance of assays with picoliter volumes. A PDMS well structure is cast on a planar optical waveguide, providing reaction containers combined with a high-sensitivity fluorescence readout system. Individual wells of the array can be addressed, filled, and rinsed using a contact-mode pin and ring spotter. This allows for immunoassays in a heavily multiplexed way, as all steps of the assay can be individually chosen per well. An array density of over 1000 wells cm(-2) is used for the current experiments. The wells provide a protected liquid environment in which the handling of proteins in their natural state is possible, thus maintaining their activity. The membrane protein annexin V is chosen as a model protein to demonstrate the current possibilities. Annexin V binds to phosphatidylserine (PS) head groups of lipids in a Ca(2+)-dependent manner and is often chosen as a marker for cell apoptosis. Lipid vesicles with and without PS are spotted in individual wells and spontaneously formed a planar lipid bilayer on the bottom of the buffer-filled wells. Annexin V can be used to distinguish between wells containing PS groups previously incorporated in the membrane patches and reference wells without PS head groups. Also, the dependence on the calcium concentration can be shown. Fluorescence readout of the assays is performed using a highly sensitive system based on a planar optical waveguide.