Inverted open microwells for cell trapping, cell aggregate formation and parallel recovery of live cells

The inverted open microwell is a novel microstructure supporting isolation and trapping of cells, analysis of cell-cell and cell-molecule interactions and functional cell sorting. This work introduces the inverted open microwell concept, demonstrating successful isolation of K562 cells in 75 μm microwells fabricated on a flexible printed circuit board substrate, and recovery of viable cells onto standard microtiter plates after analysis and manipulation. Dielectrophoresis (DEP) was used during the delivery phase to control cell access to the microwell and force the formation of cell aggregates so as to ensure cell-cell contact and interaction. Cells were trapped at the air-fluid interface at the bottom edge of the open microwell. Once trapped, cells were retained on the meniscus even after DEP de-activation and fluid was exchanged to enable perfusion of nutrients and delivery of molecules to the microwell, as demonstrated by a calcein-staining protocol performed in the microsystem. Finally, cell viability was assessed on trapped cells by a calcein release assay and cell proliferation was demonstrated after multiple cells had been recovered in parallel onto standard microtiter plates.     

Microfabrication-based modulation of embryonic stem cell differentiation

Embryonic stem (ES) cells form spontaneous aggregates during differentiation, and cell-cell communication in the aggregates plays an important role in differentiation. The development of a controlled differentiation scheme for ES cells has been hindered by the lack of a reliable method to produce uniform aggregate sizes. Conventional techniques, such as hanging drop and suspension cultures, do not allow precise control over size of ES cell aggregates. To surmount this problem, we microfabricated adhesive stencils to make mouse ES (mES) cell aggregates of specific sizes ranging from 100 microm to 500 microm in diameter. With this technique, we studied the effect of the initial aggregate size on ES cell differentiation. After 20 days of induction of differentiation, we analyzed the stem cell populations using gene and protein expression assays as well as biochemical functions. Notably, we found that germ layer differentiation depends on the initial size of the ES cell aggregate. Among the ES cell aggregate sizes tested, the aggregates with 300 microm diameter showed similar differentiation profiles of three germ layers as embryoid bodies made using the "hanging drop" technique. The smaller (100 microm) aggregates showed the increased expression of ectodermal markers compared to the larger (500 microm) aggregates, while the 500 microm aggregates showed the increased expression of mesodermal and endodermal markers compared to the 100 microm aggregates. These results indicate that the initial size of the aggregate is an important factor for ES cell differentiation, and can affect germ layer selection as well as the extent of differentiation.     

Gelatin methacrylate as a promising hydrogel for 3D microscale organization and proliferation of dielectrophoretically patterned cells

Establishing the 3D microscale organization of cells has numerous practical applications, such as in determining cell fate (e.g., proliferation, migration, differentiation, and apoptosis) and in making functional tissue constructs. One approach to spatially pattern cells is by dielectrophoresis (DEP). DEP has characteristics that are important for cell manipulation, such as high accuracy, speed, scalability, and the ability to handle both adherent and non-adherent cells. However, widespread application of this method is largely restricted because there is a limited number of suitable hydrogels for cell encapsulation. To date, polyethylene glycol-diacrylate (PEG-DA) and agarose have been used extensively for dielectric patterning of cells. In this study, we propose gelatin methacrylate (GelMA) as a promising hydrogel for use in cell dielectropatterning because of its biocompatibility and low viscosity. Compared to PEG hydrogels, GelMA hydrogels showed superior performance when making cell patterns for myoblast (C2C12) and endothelial (HUVEC) cells as well as in maintaining cell viability and growth. We also developed a simple and robust protocol for co-culture of these cells. Combined application of the GelMA hydrogels and the DEP technique is suitable for creating highly complex microscale tissues with important applications in fundamental cell biology and regenerative medicine in a rapid, accurate, and scalable manner.     

A novel ultralow conductivity electromanipulation buffer improves cell viability and enhances dielectrophoretic consistency

Cell separation has become a critical diagnostic, research, and treatment tool for personalized medicine. Despite significant advances in cell separation, most widely used applications require the use of multiple, expensive antibodies to known markers in order to identify subpopulations of cells for separation. Dielectrophoresis (DEP) provides a biophysical separation technique that can target cell subpopulations based on phenotype without labels and return native cells for downstream analysis. One challenge in employing any DEP device is the sample being separated must be transferred into an ultralow conductivity medium, which can be detrimental in retaining cells’ native phenotypes for separation. Here, we measured properties of traditional DEP reagents and determined that after just 1–2 h of exposure and subsequent culture, cells’ viability was significantly reduced below 50%. We developed and tested a novel buffer (Cyto Buffer) that achieved 6 weeks of stable shelf-life and demonstrated significantly improved viability and physiological properties. We then determined the impact of Cyto Buffer on cells’ dielectric properties and morphology and found that cells retained properties more similar to that of their native media. Finally, we vetted Cyto Buffer's usability on a cell separation platform (Cyto R1) to determine combined efficacy for cell separations. Here, more than 80% of cells from different cell lines were recovered and were determined to be >70% viable following exposure to Cyto Buffer, flow stimulation, electromanipulation, and downstream collection and growth. The developed buffer demonstrated improved opportunities for electrical cell manipulation, enrichment, and recovery for next generation cell separations.     

Dielectrophoresis applications in biomedical field and future perspectives in biomedical technology

Dielectrophoresis (DEP) is a technique to manipulate trajectories of polarisable particles in nonuniform electric fields by utilizing unique dielectric properties. The manipulation of a cell using DEP has been demonstrated in various modes, thereby indicating potential applications in the biomedical field. In this review, recent DEP applications in the biomedical field are discussed. This review is intended to highlight research work that shows significant approach related to DEP application in biomedical field reported between 2016 and 2020. First, single-shell model and multiple-shell model of cells are introduced. Current device structures and recently introduced electrode patterns for DEP applications are discussed. Second, the biomedical uses of DEP in liquid biopsies, stem cell-based therapies, and diagnosis of infectious diseases due to bacteria and viruses are presented. Finally, the challenges in DEP research are discussed, and the reported solutions are explained. DEP's potential research directions are mentioned.     

High-throughput dielectrophoretic manipulation of bioparticles within fluids through biocompatible three-dimensional microelectrode array

Dielectrophoresis (DEP) has been deemed as a potential and ideal solution for bioparticle manipulation. A 3-D carbon micro-electro-mechanical system (MEMS) fabricated from the latest developed carbon-MEMS approach has advantages of offering low-cost, biocompatible and high-throughput DEP manipulation for bioparticles. In this paper, a typical process for fabrication of various 3-D microelectrode configurations was demonstrated; accurate numerical analysis was presented on electric field gradient distribution and DEP force based on various microelectrode array configurations. The effects of electrode edge angle, electrode edge-to-edge spacing and electrode height on the electric field distributions were investigated, and optimal design considerations and rules were concluded through analysis of results. The outcomes demonstrate that the sharp edge electrode is more effective in DEP manipulation and both electrode edge-to-edge spacing and electrode height are critical design parameters for seeking optimal DEP manipulation. The gradient magnitude increases exponentially as the electrode spacing is reduced and the electric field extends significantly as the electrode height increases, both of which contribute to a higher throughput for DEP manipulation. These findings are consistent with experimental observations in the literature and will provide critical guidelines for optimal design of DEP devices with 3-D carbon-MEMS.     

Control of surface chemistry, substrate stiffness, and cell function in a novel terpolymer methacrylate library

A focused library of methacrylate terpolymers was synthesized to explore the effects of varying surface chemistry and adhesive peptide ligands on cell function. The chemical diversity of methacrylate monomers enabled construction of a library of polymers in which one can systematically vary the chemical composition to achieve a wide range of contact angle, Young's modulus, and T(g) values. Furthermore, the materials were designed to allow surface immobilization of bioactive peptides. We then examined the effects of these material compositions on protein adsorption and cell attachment, proliferation, and differentiation. We observed that chemical composition of the polymers was an important determinant for NIH 3T3 cell attachment and proliferation, as well as human mesenchymal stem cell differentiation, and correlated directly with the ability of the polymers to adsorb proteins that mediate cell adhesion. Importantly, functionalization of the methacrylate terpolymer library with an adhesive GRGDS peptide normalized cellular responses. RGD-functionalized polymers uniformly exhibited robust attachment, proliferation, and differentiation irrespective of the underlying substrate chemistry. These studies provide a library-based approach to rapidly explore the biological functionality of biomaterials with a wide range of compositions and highlight the importance of cell and protein cell adhesion in predicting their performance.     

Micropatterned dynamically adhesive substrates for cell migration

We present a novel approach to examine cell migration using dynamically adhesive substrates consisting of three spatially and functionally distinct regions: the first is permanently nonadhesive to cells, the second is permanently adhesive, and the final region is electrochemically switched from nonadhesive to adhesive. We applied a double microcontact printing approach to pattern gold surfaces with carboxylic acid-terminated self-assembled monolayers (SAMs) that permit initial cell adhesion, with methyl-terminated SAMs that permit adsorption of a nonadhesive, and with tri(ethylene glycol)-terminated SAMs that can be electrochemically "switched" to permit cell migration from a prespecified pattern onto a new pattern. Using these substrates, we investigated the migration of epithelial cells from monolayers onto narrow, branching tracks of extracellular matrix in order to characterize how lead cells influence the direction of movement of followers. Time-lapse imaging revealed that, on average, five cells consistently chose one branch before other cells entered the second branch, providing evidence to suggest that intercellular communication plays an important role in guiding the cohesive movement of epithelial sheets. This platform may be of use in furthering our understanding of the mechanisms underlying cellular decision-making during migration in both individual and multicellular contexts.     

Metabolic viability of Escherichia coli trapped by dielectrophoresis in microfluidics

The spatial and temporal control of biological species is essential in complex microfluidic biosystems. In addition, if the biological species is a cell, microfluidic handling must ensure that the cell's metabolic viability is maintained. The use of DEP for cell manipulation in microfluidics has many advantages because it is remote and fast, and the voltages required for cell trapping scale well with miniaturization. In this paper, the conditions for bacterial cell (Escherichia coli) trapping using a quadrupole electrode configuration in a PDMS microfluidic channel were developed both for stagnant and for in‐flow fluidic situations. The effect of the electrical conductivity of the fluid, the applied electric field and frequency, and the fluid‐flow velocity were studied. A dynamic exchange between captured and free‐flowing cells during DEP trapping was demonstrated. The metabolic activity of trapped cells was confirmed by using E. coli cells genetically engineered to express green fluorescent protein under the control of an inducible promoter. Noninduced cells trapped by negative DEP and positive DEP were able to express green fluorescent protein minutes after the inducer was inserted in the microchannel system immediately after DEP trapping. Longer times of trapping prior to exposure to the inducer indicated first a degradation of the cell metabolic activity and finally cell death.     

A dielectrophoresis-based platform of cancerous cell capture using aptamer-functionalized gold nanoparticles in a microfluidic channel

In this paper, a microfluidic chip for the manipulation and capture of cancer cells was introduced, in which the combination of dielectrophoresis (DEP) and a binding method based on chemical interactions by using cell-specific aptamers was performed to enhance the capture strength and specificity. The device has been simply constructed from a straight-channel PDMS placed on a glass substrate that has patterned electrode structures and a self-assembled monolayer of gold nanoparticles (AuNPs). The target cells were transported to the manipulation area by flow and attracted down to the region between the electrodes under the influence of positive DEP force. This approach facilitated subsequent selective capture by the modified aptamers on the AuNPs. The distribution of the electric field in the channel has also been simulated to clarify the DEP operation. As a result, the device has been shown to effectively capture target lung cancer cells with a concentration as low as $2\times {10}^4$cells/mL. The capture specificity in a sample of mixed cells is up to 80.4%. This technique has the potential to be applied to detection methods for many types of cancer.     

Simultaneous concentration and separation of microorganisms: insulator-based dielectrophoretic approach

Microanalytical methods offer attractive characteristics for rapid microbial detection and concentration. There is a growing interest in the development of microscale separation techniques. Dielectrophoresis (DEP), a nondestructive electrokinetic transport mechanism, is a technique with great potential for microbe manipulation, since it can achieve concentration and separation in a single step. DEP is the movement of particles due to polarization effects in nonuniform electric fields. The majority of the work on dielectrophoretic manipulation of microbes has employed alternating current fields in arrays of microelectrodes, an approach with some disadvantages. An alternative is to employ insulator-based DEP (iDEP), a dielectrophoretic mode where nonuniform fields are produced by employing arrays of insulating structures. This study presents the concentration and fractionation of a mixture of bacteria and yeast cells employing direct current-iDEP in a microchannel containing an array of cylindrical insulating structures. Negative dielectrophoretic trapping of both types of microorganisms was demonstrated, where yeast cells exhibited a stronger response, opening the possibility for dielectrophoretic differentiation. Simultaneous concentration and fractionation of a mixture of both types of cells was carried out analogous to a chromatographic separation, where a dielectropherogram was obtained in less than 2 min by applying an electric field gradient and achieving concentration factors in the order of 50 and 37 times the inlet concentration for Escherichia coli and Saccharomyces cerevisiae cells, respectively. Encouraging results were also obtained employing a sample of water taken from a pond. The findings demonstrated the great potential of iDEP as a rapid and effective technique for intact microorganism concentration and separation.     

Dielectrophoretic deformation of breast cancer cells for lab on a chip applications

This paper presents the development and experimental analysis of a curved microelectrode platform for the DEP deformation of breast cancer cells (MDA‐MB‐231). The platform is composed of arrays of curved DEP microelectrodes which are patterned onto a glass slide and samples containing MDA‐MB‐231 cells are pipetted onto the platform's surface. Finite element method is utilised to characterise the electric field gradient and DEP field. The performance of the system is assessed with MDA‐MB‐231 cells in a low conductivity 1% DMEM suspending medium. We applied sinusoidal wave AC potential at peak to peak voltages of 2, 5, and 10 V_pp at both 10 kHz and 50 MHz. We observed cell blebbing and cell shrinkage and analyzed the percentage of shrinkage of the cells. The experiments demonstrated higher percentage of cell shrinkage when cells are exposed to higher frequency and peak to peak voltage electric field.     

Development of a new contactless dielectrophoresis system for active particle manipulation using movable liquid electrodes

This study presents a new DEP manipulation technique using a movable liquid electrode, which allows manipulation of particles by actively controlling the locations of electrodes and applying on–off electric input signals. This DEP system consists of mercury as a movable liquid electrode, indium tin oxide (ITO)‐coated glass, SU‐8‐based microchannels for electrode passages, and a PDMS medium chamber. A simple squeezing method was introduced to build a thin PDMS layer at the bottom of the medium chamber to create a contactless DEP system. To determine the operating conditions, the DEP force and the friction force were analytically compared for a single cell. In addition, an appropriate frequency range for effective DEP manipulation was chosen based on an estimation of the Clausius–Mossotti factor and the effective complex permittivity of the yeast cell using the concentric shell model. With this system, we demonstrated the active manipulation of yeast cells, and measured the collection efficiency and the dielectrophoretic velocity of cells for different AC electric field strengths and applied frequencies. The experimental results showed that the maximum collection efficiency reached was approximately 90%, and the dielectrophoretic velocity increased with increasing frequency and attained the maximum value of 10.85 ± 0.95 μm/s at 100 kHz, above which it decreased.     

A correlation of conductivity medium and bioparticle viability on dielectrophoresis-based biomedical applications

Dielectrophoresis (DEP) bioparticle research has progressed from micro to nano levels. It has proven to be a promising and powerful cell manipulation method with an accurate, quick, inexpensive, and label-free technique for therapeutic purposes. DEP, an electrokinetic phenomenon, induces particle movement as a result of polarization effects in a nonuniform electrical field. This review focuses on current research in the biomedical field that demonstrates a practical approach to DEP in terms of cell separation, trapping, discrimination, and enrichment under the influence of the conductive medium in correlation with bioparticle viability. The current review aims to provide readers with an in-depth knowledge of the fundamental theory and principles of the DEP technique, which is influenced by conductive medium and to identify and demonstrate the biomedical application areas. The high conductivity of physiological fluids presents obstacles and opportunities, followed by bioparticle viability in an electric field elaborated in detail. Finally, the drawbacks of DEP-based systems and the outlook for the future are addressed. This article will aid in advancing technology by bridging the gap between bioscience and engineering. We hope the insights presented in this review will improve cell suspension medium and promote DEP-viable bioparticle manipulation for health-care diagnostics and therapeutics.     

Cellular dielectrophoresis: applications to the characterization, manipulation, separation and patterning of cells

Over the past decade, dielectrophoresis (DEP) has evolved into a powerful, robust and flexible method for cellular characterization, manipulation, separation and cell patterning. It is a field with widely varying disciplines, as it is quite common to see DEP integrated with a host of applications including microfluidics, impedance spectroscopy, tissue engineering, real-time PCR, immunoassays, stem-cell characterization, gene transfection and electroporation, just to name a few. The field is finally at the point where analytical and numerical polarization models can be used to adequately describe and characterize the dielectrophoretic behavior of cells, and there is ever increasing evidence demonstrating that electric fields can safely be used to manipulate cells without harm. As such, DEP is slowly making its way into the biological sciences. Today, DEP is being used to manipulate individual cells to specific regions of space for single-cell assays. DEP is able to separate rare cells from a heterogeneous cell suspension, where isolated cells can then be characterized and dynamically studied using nothing more than electric fields. However, there is need for a critical report to integrate the many new features of DEP for cellular applications. Here, a review of the basic theory and current applications of DEP, specifically for cells, is presented.     

A Self‐Digitization Dielectrophoretic (SD‐DEP) Chip for High‐Efficiency Single‐Cell Capture,On‐Demand Compartmentalization,and Downstream Nucleic Acid Analysis

The design and fabrication of a self‐digitization dielectrophoretic (SD‐DEP) chip with simple components for single‐cell manipulation and downstream nucleic acid analysis is presented. The device employed the traditional DEP and insulator DEP to create the local electric field that is tailored to approximately the size of single cells, enabling highly efficient single‐cell capture. The multistep procedures of cell manipulation, compartmentalization, lysis, and analysis were performed in the integrated microdevice, consuming minimal reagents, minimizing contamination, decreasing lysate dilution, and increasing assay sensitivity. The platform developed here could be a promising and powerful tool in single‐cell research for precise medicine.     

A Self‐Digitization Dielectrophoretic (SD‐DEP) Chip for High‐Efficiency Single‐Cell Capture,On‐Demand Compartmentalization,and Downstream Nucleic Acid Analysis

The design and fabrication of a self‐digitization dielectrophoretic (SD‐DEP) chip with simple components for single‐cell manipulation and downstream nucleic acid analysis is presented. The device employed the traditional DEP and insulator DEP to create the local electric field that is tailored to approximately the size of single cells, enabling highly efficient single‐cell capture. The multistep procedures of cell manipulation, compartmentalization, lysis, and analysis were performed in the integrated microdevice, consuming minimal reagents, minimizing contamination, decreasing lysate dilution, and increasing assay sensitivity. The platform developed here could be a promising and powerful tool in single‐cell research for precise medicine.     

Characterization of a hybrid dielectrophoresis and immunocapture microfluidic system for cancer cell capture

The capture of circulating tumor cells (CTCs) from cancer patient blood enables early clinical assessment as well as genetic and pharmacological evaluation of cancer and metastasis. Although there have been many microfluidic immunocapture and electrokinetic techniques developed for isolating rare cancer cells, these techniques are often limited by a capture performance tradeoff between high efficiency and high purity. We present the characterization of shear‐dependent cancer cell capture in a novel hybrid DEP–immunocapture system consisting of interdigitated electrodes fabricated in a Hele‐Shaw flow cell that was functionalized with a monoclonal antibody, J591, which is highly specific to prostate‐specific membrane antigen expressing prostate cancer cells. We measured the positive and negative DEP response of a prostate cancer cell line, LNCaP, as a function of applied electric field frequency, and showed that DEP can control capture performance by promoting or preventing cell interactions with immunocapture surfaces, depending on the sign and magnitude of the applied DEP force, as well as on the local shear stress experienced by cells flowing in the device. This work demonstrates that DEP and immunocapture techniques can work synergistically to improve cell capture performance, and it will aid in the design of future hybrid DEP–immunocapture systems for high‐efficiency CTC capture with enhanced purity.     

Facilitating the culture of mammalian nerve cells with polyelectrolyte multilayers

When neuron-like cells (NLCs) derived from pluripotent embryonal carcinoma cells (P19) are cultured on bare tissue culture substrates, they require a monolayer of fibroblast cells to exhibit normal neurite outgrowth, behavior typical of neuronal cultures. However, substrate treatment with polyelectrolyte multilayers (PEMs) composed of poly(allylamine hydrochloride) (PAH) and poly(styrenesulfonic acid) (PSS) significantly improved these cultures. Cell morphology was more spread, indicative of healthy cells, and direct attachment of neuronal cell bodies to the treated surface was observed. Neuronal outgrowth across the surface was not dependent on an underlying fibroblast monolayer with the PEMs surface treatment. Additionally, the PEMs surface treatment can be used to condition various surfaces, facilitating neuronal cultures on surfaces which are natively hydrophilic (tissue culture polystyrene) or hydrophobic (poly(dimethylsiloxane), PDMS). Microfluidic networks were used to micropattern the PEMs onto PDMS, resulting in confined regions of cellular attachment and directed neuronal outgrowth. The ability of PEMs to encourage NLC attachment without supporting cells to a variety of surfaces and surface geometries greatly simplifies neuronal culture methodology and enables neuronal investigations in new environments.     

Controlling size, shape and homogeneity of embryoid bodies using poly(ethylene glycol) microwells

Directed differentiation of embryonic stem (ES) cells is useful for creating models of human disease and could potentially generate a wide array of functional cell types for therapeutic applications. Methods to differentiate ES cells often involve the formation of cell aggregates called embryoid bodies (EBs), which recapitulate early stages of embryonic development. EBs are typically made from suspension cultures, resulting in heterogeneous structures with a wide range of sizes and shapes, which may influence differentiation. Here, we use microfabricated cell-repellant poly(ethylene glycol) (PEG) wells as templates to initiate the formation of homogenous EBs. ES cell aggregates were formed with controlled sizes and shapes defined by the geometry of the microwells. EBs generated in this manner remained viable and maintained their size and shape within the microwells relative to their suspension counterparts. Intact EBs could be easily retrieved from the microwells with high viability (>95%). These results suggest that the microwell technique could be a useful approach for in vitro studies involving ES cells and, more specifically, for initiating the differentiation of EBs of greater uniformity based on controlled microenvironments.