Label-free enrichment of MCF7 breast cancer cells from leukocytes using continuous flow dielectrophoresis

Circulating tumor cells (CTCs) present in the bloodstream are strongly linked to the invasive behavior of cancer; therefore, their detection holds great significance for monitoring disease progression. Currently available CTC isolation tools are often based on tumor-specific antigen or cell size approaches. However, these techniques are limited due to the lack of a unique and universal marker for CTCs, and the overlapping size between CTCs and regular blood cells. Dielectrophoresis (DEP), governed by the intrinsic dielectric properties of the particles, is a promising marker-free, accurate, fast, and low-cost technique that enables the isolation of CTCs from blood cells. This study presents a continuous flow, antibody-free DEP-based microfluidic device to concentrate MCF7 breast cancer cells, a well-established CTC model, in the presence of leukocytes extracted from human blood samples. The enrichment strategy was determined according to the DEP responses of the corresponding cells, obtained in our previously reported DEP spectrum study. It was based on the positive-DEP integrated with hydrodynamic focusing under continuous flow. In the proposed device, the parylene microchannel with two inlets and outlets was built on top of rectangular and equally spaced isolated planar electrodes rotated certain degree relative to the main flow (13°). The recovery of MCF7 cells mixed with leukocytes was 74%–98% at a frequency of 1 MHz and a magnitude of 10–12 V_pp. Overall, the results revealed that the presented system successfully concentrates MCF7 cancer cells from leukocytes, ultimately verifying our DEP spectrum study, in which the enrichment frequency and separation strategy of the microfluidic system were determined.     

Double spiral microchannel for label-free tumor cell separation and enrichment

This work reports on a passive double spiral microfluidic device allowing rapid and label-free tumor cell separation and enrichment from diluted peripheral whole blood, by exploiting the size-dependent hydrodynamic forces. A numerical model is developed to simulate the Dean flow inside the curved geometry and to track the particle/cell trajectories, which is validated against the experimental observations and serves as a theoretical foundation for optimizing the operating conditions. Results from separating tumor cells (MCF-7 and Hela) spiked into whole blood indicate that 92.28% of blood cells and 96.77% of tumor cells are collected at the inner and the middle outlet, respectively, with 88.5% tumor recovery rate at a throughput of 3.33 × 10(7) cells min(-1). We expect that this label-free microfluidic platform, driven by purely hydrodynamic forces, would have an impact on fundamental and clinical studies of circulating tumor cells.     

DNA fragment‐assisted microfluidic chip for capture and release of circulating tumor cells

Circulating tumor cells are specifically referred as cells that detached from the primary tumor and are present in the bloodstream. They could be isolated from blood and used as representative biomarker for predicting cancer prognoses. Here, we developed a microfluidic chip with multiple curved channels, in which DNA fragments and antibody‐based enrichment are exploited to capture circulating tumor cells in blood sample. By introducing DNA fragments as long tentacles, the active antibody could be extended into the microchannel stereoscopically, which could greatly increase the chances of adhesion in a multidirectional way and improve the capture efficacy. Several pivotal factors for cell capturing were optimized to the best state. Compared to conventional chips for planar capturing, the capture efficiency of MCF‐7 cells was greatly increased from 37.17 to 85.10%. For the detection of MCF‐7‐containing artificial blood sample detection, the capture efficiency of tumor cells was about 74.19 ± 2.13%, which was obviously better than the result of flow cytometry (29.67 ± 4.02%). Captured cells were easily released from the surface of microfluidic chip with high cell viability, which could be investigated for the molecular analysis in the field of tumor diagnosis.     

Deformability-based cell classification and enrichment using inertial microfluidics

The ability to detect and isolate rare target cells from heterogeneous samples is in high demand in cell biology research, immunology, tissue engineering and medicine. Techniques allowing label-free cell enrichment or detection are especially important to reduce the complexity and costs towards clinical applications. Single-cell deformability has recently been recognized as a unique label-free biomarker for cell phenotype with implications for assessment of cancer invasiveness. Using a unique combination of fluid dynamic effects in a microfluidic system, we demonstrate high-throughput continuous label-free cell classification and enrichment based on cell size and deformability. The system takes advantage of a balance between deformability-induced and inertial lift forces as cells travel in a microchannel flow. Particles and droplets with varied elasticity and viscosity were found to have separate lateral dynamic equilibrium positions due to this balance of forces. We applied this system to successfully classify various cell types using cell size and deformability as distinguishing markers. Furthermore, using differences in dynamic equilibrium positions, we adapted the system to conduct passive, label-free and continuous cell enrichment based on these markers, enabling off-chip sample collection without significant gene expression changes. The presented method has practical potential for high-throughput deformability measurements and cost-effective cell separation to obtain viable target cells of interest in cancer research, immunology, and regenerative medicine.     

Separation of malignant human breast cancer epithelial cells from healthy epithelial cells using an advanced dielectrophoresis-activated cell sorter (DACS)

In this paper, we successfully separated malignant human breast cancer epithelial cells (MCF 7) from healthy breast cells (MCF 10A) and analyzed the main parameters that influence the separation efficiency with an advanced dielectrophoresis (DEP)-activated cell sorter (DACS). Using the efficient DACS, the malignant cancer cells (MCF 7) were isolated successfully by noninvasive methods from normal cells with similar cell size distributions (MCF 10A), depending on differences between their material properties such as conductivity and permittivity, because our system was able to discern the subtle differences in the properties by generating continuously changed electrical field gradients. In order to evaluate the separation performance without considering size variations, the cells collected from each outlet were divided into size-dependent groups and counted statistically. Following that, the quantitative relative ratio of numbers between MCF 7 and MCF 10A cells in each size-dependent group separated by the DEP were compared according to applied frequencies in the range 48, 51, and 53 MHz with an applied amplitude of 8 V_pp. Finally, under the applied voltage of 48 MHz–8 V_pp and a flow rate of 290 μm/s, MCF 7 and MCF 10A cells were separated with a maximum efficiency of 86.67% and 98.73% respectively. Therefore, our suggested system shows it can be used for detection and separation of cancerous epithelial cells from noncancerous cells in clinical applications.     

Pinched flow coupled shear-modulated inertial microfluidics for high-throughput rare blood cell separation

Blood is a highly complex bio-fluid with cellular components making up >40% of the total volume, thus making its analysis challenging and time-consuming. In this work, we introduce a high-throughput size-based separation method for processing diluted blood using inertial microfluidics. The technique takes advantage of the preferential cell focusing in high aspect-ratio microchannels coupled with pinched flow dynamics for isolating low abundance cells from blood. As an application of the developed technique, we demonstrate the isolation of cancer cells (circulating tumor cells (CTCs)) spiked in blood by exploiting the difference in size between CTCs and hematologic cells. The microchannel dimensions and processing parameters were optimized to enable high throughput and high resolution separation, comparable to existing CTC isolation technologies. Results from experiments conducted with MCF-7 cells spiked into whole blood indicate >80% cell recovery with an impressive 3.25 × 10(5) fold enrichment over red blood cells (RBCs) and 1.2 × 10(4) fold enrichment over peripheral blood leukocytes (PBL). In spite of a 20× sample dilution, the fast operating flow rate allows the processing of ～10(8) cells min(-1) through a single microfluidic device. The device design can be easily customized for isolating other rare cells from blood including peripheral blood leukocytes and fetal nucleated red blood cells by simply varying the 'pinching' width. The advantage of simple label-free separation, combined with the ability to retrieve viable cells post enrichment and minimal sample pre-processing presents numerous applications for use in clinical diagnosis and conducting fundamental studies.     

Examining the lateral displacement of HL60 cells rolling on asymmetric P-selectin patterns

The lateral displacement of cells orthogonal to a flow stream by rolling on asymmetrical receptor patterns presents a new opportunity for the label-free separation and analysis of cells. Understanding the nature of cell rolling trajectories on such substrates is necessary to the engineering of substrates and the design of devices for cell separation and analysis. Here, we investigate the statistical nature of cell rolling and the effect of pattern geometry and flow shear stress on cell rolling trajectories using micrometer-scale patterns of biomolecular receptors with well-defined edges. Leukemic myeloid HL60 cells expressing the PSGL-1 ligand were allowed to flow across a field of patterned lines fabricated using microcontact printing and functionalized with the P-selectin receptor, leveraging both the specific adhesion of this ligand-receptor pair and the asymmetry of the receptor pattern inclination angle with respect to the fluid shear flow direction (α = 5, 10, 15, and 20°). The effects of the fluid shear stress magnitude (τ = 0.5, 1, 1.5, and 2.0 dyn/cm(2)), α, and P-selectin incubation concentration were quantified in terms of the rolling velocity and edge tracking length. Rolling cells tracked along the inclined edges of the patterned lines before detaching and reattaching on another line. The detachment of rolling cells after tracking along the edge was consistent with a Poisson process of history-independent interactions. Increasing the edge inclination angle decreased the edge tracking length in an exponential manner, contrary to the shear stress magnitude and P-selectin incubation concentration, which did not have a significant effect. On the basis of these experimental data, we constructed an empirical model that predicted the occurrence of the maximum lateral displacement at an edge angle of 7.5°. We also used these findings to construct a Monte Carlo simulation for the prediction of rolling trajectories of HL60 cells on P-selectin-patterned substrates with a specified edge inclination angle. The prediction of lateral displacement in the range of 200 μm within a 1 cm separation length supports the feasibility of label-free cell separation via asymmetric receptor patterns in microfluidic devices.     

Tailoring the trajectory of cell rolling with cytotactic surfaces

Cell separation technology is a key tool for biological studies and medical diagnostics that relies primarily on chemical labeling to identify particular phenotypes. An emergent method of sorting cells based on differential rolling on chemically patterned substrates holds potential benefits over existing technologies, but the underlying mechanisms being exploited are not well characterized. In order to better understand cell rolling on complex surfaces, a microfluidic device with chemically patterned stripes of the cell adhesion molecule P-selectin was designed. The behavior of HL-60 cells rolling under flow was analyzed using a high-resolution visual tracking system. This behavior was then correlated to a number of established predictive models. The combination of computational modeling and widely available fabrication techniques described herein represents a crucial step toward the successful development of continuous, label-free methods of cell separation based on rolling adhesion.     

Adenovirus adenine nucleotide translocator-2 shRNA effectively induces apoptosis and enhances chemosensitivity by the down-regulation of ABCG2 in breast cancer stem-like cells

Cancer stem cells (CSCs) are resistant to chemo- and radio-therapy, and can survive to regenerate new tumors. This is an important reason why various anti- cancer therapies often fail to completely control tumors, although they kill and eliminate the bulk of cancer cells. In this study, we determined whether or not adenine nucleotide translocator-2 (ANT2) suppression could also be effective in inducing cell death of breast cancer stem-like cells. A sub-population (SP; CD44+/ CD24-) of breast cancer cells has been reported to have stem/progenitor cell properties. We utilized the adeno- ANT2 shRNA virus to inhibit ANT2 expression and then observed the treatment effect in a SP of breast cancer cell line. In this study, MCF7, MDA-MB-231 cells, and breast epithelial cells (MCF10A) mesenchymally-transdifferentiated through E-cadherin knockdown were used. ANT2 expression was high in both stem-like cells and non-stem-like cells of MCF7 and MDA-MB-231 cells, and was induced and up-regulated by mesenchymal transdifferentiation in MCF10A cells (MCF10A(EMT)). Knockdown of ANT2 by adeno-shRNA virus efficiently induced apoptotic cell death in the stem-like cells of MCF7 and MDA-MB-231 cells, and MCF10A(EMT). Stem-like cells of MCF7 and MDA-MB-231, and MCF10A(EMT) cells exhibited increased drug (doxorubicin) resistance, and expressed a multi-drug resistant related molecule, ABCG2, at a high level. Adeno-ANT2 shRNA virus markedly sensitized the stem-like cells of MCF7 and MDA-MB-231, and the MCF10A(EMT) cells to doxorubicin, which was accompanied by down-regulation of ABCG2. Our results suggest that ANT2 suppression by adeno-shRNA virus is an effective strategy to induce cell death and increase the chemosensitivity of stem-like cells in breast cancer.     

Parallelized continuous flow dielectrophoretic separation of DNA

Numerous microfluidic separation applications have been shown in the past years providing a fast analysis of biological samples like DNA or proteins. Microfluidic separation based on dielectrophoresis (DEP), that is the migration of a polarizable object in an inhomogeneous electric field, provides numerous advantages. However, the main drawback of DEP separation devices is that they are not sufficient for large-scale sample purification due to the lack of high sample throughput. In this work, we present for the first time a microfluidic device with two parallelized dielectrophoretic separations of (biological) samples smaller than 1 µm. The separation is carried out by means of insulator-based DEP, that is an insulating ridge reduced the flow through height and thus created a nanoslit at which the selective DEP forces occur. The device consists of a cross injector, two parallel operation regions and separate harvesting reservoirs where the samples are collected. Each DEP operation region contains an insulating ridge. We successfully demonstrate the separation of 100 and 40 nm beads and 10 and 5 kbp DNA with a separation purity of more than 80%. This states the proof-of-concept for up-scaling of dielectrophoretic separation by parallelization. As the present technique is virtually label-free, it offers a fast purification, for example in the production of gene vaccines.     

Single cell manipulation, analytics, and label-free protein detection in microfluidic devices for systems nanobiology

Single cell analytics for proteomic analysis is considered a key method in the framework of systems nanobiology which allows a novel proteomics without being subjected to ensemble-averaging, cell-cycle, or cell-population effects. We are currently developing a single cell analytical method for protein fingerprinting combining a structured microfluidic device with latest optical laser technology for single cell manipulation (trapping and steering), free-solution electrophoretical protein separation, and (label-free) protein detection. In this paper we report on first results of this novel analytical device focusing on three main issues. First, single biological cells were trapped, injected, steered, and deposited by means of optical tweezers in a poly(dimethylsiloxane) microfluidic device and consecutively lysed with SDS at a predefined position. Second, separation and detection of fluorescent dyes, amino acids, and proteins were achieved with LIF detection in the visible (VIS) (488 nm) as well as in the deep UV (266 nm) spectral range for label-free, native protein detection. Minute concentrations of 100 fM injected fluorescein could be detected in the VIS and a first protein separation and label-free detection could be achieved in the UV spectral range. Third, first analytical experiments with single Sf9 insect cells (Spodoptera frugiperda) in a tailored microfluidic device exhibiting distinct electropherograms of a green fluorescent protein-construct proved the validity of the concept. Thus, the presented microfluidic concept allows novel and fascinating single cell experiments for systems nanobiology in the future.     

In situ detection of live cancer cells by using bioprobes based on Au nanoparticles

We fabricate the high-performance probes based on Au nanoparticles (AuNP) for detection of live cancer cell. AuNP were synthesized with narrow sized distribution (ca. 10 nm) by Au salt reduction method and deposited onto the aminated substrate as a cross-linker and hot spot. Herein, AuNP has enabled the easy and efficient immobilization of the antibody (Cetuximab), which can selectively interact with epidermal growth factor receptor (EGFR) on the surface of epidermal cancer, as detecting moiety onto the AuNP-deposited substrate without nanolithography process. After conjugation of Cetuximab with AuNP-deposited substrate, Cetuximab-conjugated probe as a live cancer cell detector (LCCD) could detect EGFR-highexpressed A431 cells related to epithelial cancer with 54-times larger specificity and sensitivity in comparison with EGFR-deficient MCF7 cells. This implies that AuNP-based probes demonstrate abundant potentials for detection and separation of small biomolecules, cells and other chemicals.     

Tumour cell identification by means of Raman spectroscopy in combination with optical traps and microfluidic environments

Raman spectroscopy has been recognized to be a powerful tool for label-free discrimination of cells. Sampling methods are under development to utilize the unique capabilities to identify cells in body fluids such as saliva, urine or blood. The current study applied optical traps in combination with Raman spectroscopy to acquire spectra of single cells in microfluidic glass channels. Optical traps were realized by two 1070 nm single mode fibre lasers. Microflows were controlled by a syringe pump system. A novel microfluidic glass chip was designed to inject single cells, modify the flow speed, accommodate the laser fibres and sort cells after Raman based identification. Whereas the integrated microchip setup used 514 nm for excitation of Raman spectra, a quartz capillary setup excited spectra with 785 nm laser wavelength. Classification models were trained using linear discriminant analysis to differentiate erythrocytes, leukocytes, acute myeloid leukaemia cells (OCI-AML3), and breast tumour cells BT-20 and MCF-7 with accuracies that are comparable with previous Raman experiments of dried cells and fixed cells in a Petri dish. Implementation into microfluidic environments enables a high degree of automation that is required to improve the throughput of the approach for Raman activated cell sorting.     

Real-time electrical impedance-based measurement to distinguish oral cancer cells and non-cancer oral epithelial cells

In this study, electrical impedance-based measurements were used to distinguish oral cancer cells and non-cancer oral epithelial cells based on their cellular activities on the microelectrodes in a real-time and label-free manner. CAL 27 and Het-1A cell lines were used as the models of oral cancer cells and non-cancer oral epithelial cells, respectively. Various cellular activities, including cell adhesion, spreading, and proliferation were monitored. We found that both the kinetics of cell spreading and the static impedance-based cell index were feasible to distinguish the two cell types. At each given cell number, CAL 27 cell spreading produced a smaller cell index change rate that was 60–70% of those of Het-1A cells. When cells were fully spread, CAL 27 cells generated a cell index more than four times greater than that of Het-1A cells. Since cell spreading and attachment occurs in the first few hours when they were cultured on the microelectrodes, this impedance-based method could be a rapid label-free and non-invasive approach to distinguish oral cancer cells from non-cancer oral epithelial cells. Cell viability analysis was performed along with the impedance-based analysis. Confocal microscopic imaging analysis showed the difference in cell morphology and the thickness of cell monolayers between the two cell types.     

Biomechanical analysis of cancerous and normal cells based on bulge generation in a microfluidic device

This paper presents a new biomechanical analysis method for discrimination between cancerous and normal cells through compression by poly(dimethylsiloxane) (PDMS) membrane deflection in a microfluidic device. When a cell is compressed, cellular membrane will expand and then small bulges will appear on the peripheral cell membrane beyond the allowable strain. It is well known that the amount of F-actin in cancer cells is less than that of normal cells and bulges occur at the sites where cytoskeleton becomes detached from the membrane bilayer. Accordingly, we have demonstrated the difference of the bulge generation between breast cancer cells (MCF7) and normal cells (MCF10A). After excessive deformation, the bulges generated in MCF7 cells were not evenly distributed on the cell periphery. Contrary to this, the bulges of MCF10A cells showed an even distribution. In addition, the morphologies of bulges of MCF7 and MCF10A cells looked swollen protrusion and tubular protrusion, respectively. Peripheral strains at the moment of the bulge generation were also 72% in MCF7 and 46% in MCF10A. The results show that the bulge generation can be correlated with the cytoskeleton quantity inside the cell, providing the first step of a new biomechanical approach.     

Enrichment of putative stem cells from adipose tissue using dielectrophoretic field-flow fractionation

We have applied the microfluidic cell separation method of dielectrophoretic field-flow fractionation (DEP-FFF) to the enrichment of a putative stem cell population from an enzyme-digested adipose tissue derived cell suspension. A DEP-FFF separator device was constructed using a novel microfluidic-microelectronic hybrid flex-circuit fabrication approach that is scaleable and anticipates future low-cost volume manufacturing. We report the separation of a nucleated cell fraction from cell debris and the bulk of the erythrocyte population, with the relatively rare (<2% starting concentration) NG2-positive cell population (pericytes and/or putative progenitor cells) being enriched up to 14-fold. This work demonstrates a potential clinical application for DEP-FFF and further establishes the utility of the method for achieving label-free fractionation of cell subpopulations.     

Dielectrophoretic separation of platelets from diluted whole blood in microfluidic channels

The dielectrophoresis (DEP) phenomenon is used to separate platelets directly from diluted whole blood in microfluidic channels. By exploiting the fact that platelets are the smallest cell type in blood, we utilize the DEP-activated cell sorter (DACS) device to perform size-based fractionation of blood samples and continuously enrich the platelets in a label-free manner. Cytometry analysis revealed that a single pass through the two-stage DACS device yields a high purity of platelets (approximately 95%) at a throughput of approximately 2.2 x 10(4) cells/second/microchannel with minimal platelet activation. This work demonstrates gentle and label-free dielectrophoretic separation of delicate cells from complex samples and such a separation approach may open a path toward continuous screening of blood products by integrated microfluidic devices.     

Continuous separation of breast cancer cells from blood samples using multi-orifice flow fractionation (MOFF) and dielectrophoresis (DEP)

Circulating tumor cells (CTCs) are highly correlated with the invasive behavior of cancer, so their isolations and quantifications are important for biomedical applications such as cancer prognosis and measuring the responses to drug treatments. In this paper, we present the development of a microfluidic device for the separation of CTCs from blood cells based on the physical properties of cells. For use as a CTC model, we successfully separated human breast cancer cells (MCF-7) from a spiked blood cell sample by combining multi-orifice flow fractionation (MOFF) and dielectrophoretic (DEP) cell separation technique. Hydrodynamic separation takes advantage of the massive and high-throughput filtration of blood cells as it can accommodate a very high flow rate. DEP separation plays a role in precise post-processing to enhance the efficiency of the separation. The serial combination of these two different sorting techniques enabled high-speed continuous flow-through separation without labeling. We observed up to a 162-fold increase in MCF-7 cells at a 126 μL min(-1) flow rate. Red and white blood cells were efficiently removed with separation efficiencies of 99.24% and 94.23% respectively. Therefore, we suggest that our system could be used for separation and detection of CTCs from blood cells for biomedical applications.     

Low-cost polymer-film spiral inertial microfluidic device for label-free separation of malignant tumor cells

We developed a low-cost polymer-film spiral inertial microfluidic device for the effective size-dependent separation of malignant tumor cells. The device was fabricated in polymer films by rapid laser cutting and chemical bonding. After fabricating the prototype device, the separation performance of our device was evaluated using particles and cells. The effects of operational flow rate, cell diameter, and cell concentration on the separation performance were explored. Our device successfully separated tumor cells from polydisperse white blood cells according to their different migration modes and lateral positions. Then, the separation of rare cells was carried out using the high-concentration lysed blood spiked with 200 tumor cells. Experimental results showed that 83.90% of the tumor cells could be recovered, while 99.87% of white blood cells could be removed. We successfully employed our device for processing clinical pleural effusion samples from patients with advanced metastatic breast cancer. Malignant tumor cells with an average purity of 2.37% could be effectively enriched, improving downstream diagnostic accuracy. Our device offers the advantages of label-free operation, low cost, and fast fabrication, thus being a potential tool for effective cell separation.     

Hydrogel microfluidic co‐culture device for photothermal therapy and cancer migration

We developed the photo‐crosslinkable hydrogel microfluidic co‐culture device to study photothermal therapy and cancer cell migration. To culture MCF7 human breast carcinoma cells and metastatic U87MG human glioblastoma in the microfluidic device, we used 10 w/v% gelatin methacrylate (GelMA) hydrogels as a semi‐permeable physical barrier. We demonstrated the effect of gold nanorod on photothermal therapy of cancer cells in the microfluidic co‐culture device. Interestingly, we observed that metastatic U87MG human glioblastoma largely migrated toward vascular endothelial growth factor (VEGF)‐treated GelMA hydrogel‐embedding microchannels. The main advantage of this hydrogel microfluidic co‐culture device is to simultaneously analyze the physiological migration behaviors of two cancer cells with different physiochemical motilities and study gold nanorod‐mediated photothermal therapy effect. Therefore, this hydrogel microfluidic co‐culture device could be a potentially powerful tool for photothermal therapy and cancer cell migration applications.