Increasing label-free stem cell sorting capacity to reach transplantation-scale throughput

Dielectrophoresis (DEP) has proven an invaluable tool for the enrichment of populations of stem and progenitor cells owing to its ability to sort cells in a label-free manner and its biological safety. However, DEP separation devices have suffered from a low throughput preventing researchers from undertaking studies requiring large numbers of cells, such as needed for cell transplantation. We developed a microfluidic device designed for the enrichment of stem and progenitor cell populations that sorts cells at a rate of 150,000 cells/h, corresponding to an improvement in the throughput achieved with our previous device designs by over an order of magnitude. This advancement, coupled with data showing the DEP-sorted cells retain their enrichment and differentiation capacity when expanded in culture for periods of up to 2 weeks, provides sufficient throughput and cell numbers to enable a wider variety of experiments with enriched stem and progenitor cell populations. Furthermore, the sorting devices presented here provide ease of setup and operation, a simple fabrication process, and a low associated cost to use that makes them more amenable for use in common biological research laboratories. To our knowledge, this work represents the first to enrich stem cells and expand them in culture to generate transplantation-scale numbers of differentiation-competent cells using DEP.     

Entry effects of droplet in a micro confinement: Implications for deformation-based circulating tumor cell microfiltration

Deformation-based circulating tumor cell (CTC) microchips are a representative diagnostic device for early cancer detection. This type of device usually involves a process of CTC trapping in a confined microgeometry. Further understanding of the CTC flow regime, as well as the threshold passing-through pressure, is a key to the design of deformation-based CTC filtration devices. In the present numerical study, we investigate the transitional deformation and pressure signature from surface tension dominated flow to viscous shear stress dominated flow using a droplet model. Regarding whether CTC fully blocks the channel inlet, we observe two flow regimes: CTC squeezing and shearing regime. By studying the relation of CTC deformation at the exact critical pressure point for increasing inlet velocity, three different types of cell deformation are observed: (1) hemispherical front, (2) parabolic front, and (3) elongated CTC co-flowing with carrier media. Focusing on the circular channel, we observe a first increasing and then decreasing critical pressure change with increasing flow rate. By pressure analysis, the concept of optimum velocity is proposed to explain the behavior of CTC filtration and design optimization of CTC filter. Similar behavior is also observed in channels with symmetrical cross sections like square and triangular but not in rectangular channels which only results in decreasing critical pressure.     

Label-free isolation of a prostate cancer cell among blood cells and the single-cell measurement of drug accumulation using an integrated microfluidic chip

Circulating tumor cells (CTCs) are found in the blood of patients with cancer. Although these cells are rare, they can provide useful information for chemotherapy. However, isolation of these rare cells from blood is technically challenging because they are small in numbers. An integrated microfluidic chip, dubbed CTC chip, was designed and fabricated for conducting tumor cell isolation. As CTCs usually show multidrug resistance (MDR), the effect of MDR inhibitors on chemotherapeutic drug accumulation in the isolated single tumor cell is measured. As a model of CTC isolation, human prostate cancer cells were mixed with mouse blood cells and the label-free isolation of the tumor cells was conducted based on cell size difference. The major advantages of the CTC chip are the ability for fast cell isolation, followed by multiple rounds of single-cell measurements, suggesting a potential assay for detecting the drug responses based on the liquid biopsy of cancer patients.     

Quick chip assay using locked nucleic acid modified epithelial cell adhesion molecule and nucleolin aptamers for the capture of circulating tumor cells

The role of circulating tumor cells (CTCs) in disease diagnosis, prognosis, monitoring of the therapeutic efficacy, and clinical decision making is immense and has attracted tremendous focus in the last decade. We designed and fabricated simple, flat channel microfluidic devices polydimethylsiloxane (PDMS based) functionalized with locked nucleic acid (LNA) modified aptamers (targeting epithelial cell adhesion molecule (EpCAM) and nucleolin expression) for quick and efficient capture of CTCs and cancer cells. With optimized flow rates (10 μl/min), it was revealed that the aptamer modified devices offered reusability for up to six times while retaining optimal capture efficiency (>90%) and specificity. High capture sensitivity (92%) and specificity (100%) was observed in whole blood samples spiked with Caco-2 cells (10–100 cells/ml). Analysis of blood samples obtained from 25 head and neck cancer patients on the EpCAM LNA aptamer functionalized chip revealed that an average count of 5 ± 3 CTCs/ml of blood were captured from 22/25 samples (88%). EpCAM intracellular domain (EpICD) immunohistochemistry on 9 oral squamous cell carcinomas showed the EpICD positivity in the tumor cells, confirming the EpCAM expression in CTCs from head and neck cancers. These microfluidic devices also maintained viability for in vitro culture and characterization. Use of LNA modified aptamers provided added benefits in terms of cost effectiveness due to increased reusability and sustainability of the devices. Our results present a robust, quick, and efficient CTC capture platform with the use of simple PDMS based devices that are easy to fabricate at low cost and have an immense potential in cancer diagnosis, prognosis, and therapeutic planning.     

Microfluidic impedance cytometry of tumour cells in blood

The dielectric properties of tumour cells are known to differ from normal blood cells, and this difference can be exploited for label-free separation of cells. Conventional measurement techniques are slow and cannot identify rare circulating tumour cells (CTCs) in a realistic timeframe. We use high throughput single cell microfluidic impedance cytometry to measure the dielectric properties of the MCF7 tumour cell line (representative of CTCs), both as pure populations and mixed with whole blood. The data show that the MCF7 cells have a large membrane capacitance and size, enabling clear discrimination from all other leukocytes. Impedance analysis is used to follow changes in cell viability when cells are kept in suspension, a process which can be understood from modelling time-dependent changes in the dielectric properties (predominantly membrane conductivity) of the cells. Impedance cytometry is used to enumerate low numbers of MCF7 cells spiked into whole blood. Chemical lysis is commonly used to remove the abundant erythrocytes, and it is shown that this process does not alter the MCF7 cell count or change their dielectric properties. Combining impedance cytometry with magnetic bead based antibody enrichment enables MCF7 cells to be detected down to 100 MCF7 cells in 1 ml whole blood, a log 3.5 enrichment and a mean recovery of 92%. Microfluidic impedance cytometry could be easily integrated within complex cell separation systems for identification and enumeration of specific cell types, providing a fast in-line single cell characterisation method.     

Efficient capture of circulating tumor cells with a novel immunocytochemical microfluidic device

Ability to perform cytogenetic interrogations on circulating tumor cells (CTCs) from the blood of cancer patients is vital for progressing toward targeted, individualized treatments. CTCs are rare compared to normal (bystander) blood cells, found in ratios as low as 1:10⁹. The most successful isolation techniques have been immunocytochemical technologies that label CTCs for separation based on unique surface antigens that distinguish them from normal bystander cells. The method discussed here utilizes biotin-tagged antibodies that bind selectively to CTCs. The antibodies are introduced into a suspension of blood cells intending that only CTCs will display surface biotin molecules. Next, the cell suspension is passed through a microfluidic channel that contains about 9000 transverse, streptavidin coated posts. A CTC making contact with a post has the opportunity to engage in a biotin-streptavidin reaction that immobilizes the cell. Bystander blood cells remain in suspension and pass through the channel. The goal of the present study is to establish the technical performance of these channels as a function of antigen density and operating conditions, especially flow rate. At 18 μL/min, over 70% of cells are captured at antigen densities greater than 30 000 sites/cell while 50% of cells are captured at antigen densities greater than 10 000. It is found that lower flow rates lead to decreasing cell capture probabilities, indicating that some streamlines develop which are never close enough to a post to allow cell-post contact. Future modeling and streamline studies using computational fluid dynamics software could aid in optimization of channel performance for capture of rare cells.     

Cascaded spiral microfluidic device for deterministic and high purity continuous separation of circulating tumor cells

Inertial microfluidics is an emerging class of technologies developed to separate circulating tumor cells (CTCs). However, defining design parameters and flow conditions for optimal operation remains nondeterministic due to incomplete understanding of the mechanics, which has led to challenges in designing efficient systems. Here, we perform a parametric study of the inertial focusing effects observed in low aspect ratio curvilinear microchannels and utilize the results to demonstrate the isolation of CTCs with high purity. First, we systematically vary parameters including the channel height, width, and radius of curvature over a wide range of flow velocities to analyze its effect on size dependent differential focusing and migration behaviors of binary (10 μm and 20 μm) particles. Second, we use these results to identify optimal flow regimes to achieve maximum separation in various channel configurations and establish design guidelines to readily provide information for developing spiral channels tailored to potentially arbitrary flow conditions that yield a desired equilibrium position for optimal size based CTC separation. Finally, we describe a fully integrated, sheath-less cascaded spiral microfluidic device to continuously isolate CTCs. Human breast cancer epithelial cells were successfully extracted from leukocytes, achieving 86.76% recovery, 97.91% depletion rate, and sustaining high viability upon collection to demonstrate the versatility of the device. Importantly, this device was designed without the cumbersome trail-and-error optimization process that has hindered the development of designing such inertial microfluidic systems.     

Spatially gradated segregation and recovery of circulating tumor cells from peripheral blood of cancer patients

For cancer patients, the enumeration of rare circulating tumor cells (CTCs) in peripheral blood is a strong prognostic indicator of the severity of the cancer; for the general population, the capture of CTCs is needed for use as a clinical tool for cancer screening, early detection, and treatment assessment. Here, we present a fast, high-purity (∼90%) and high-efficiency (>90%) method for the segregation and undamaged recovery of CTCs using a spatially gradated microfluidic chip. Further, by lysing the red blood cells we achieved not only a significant reduction in the overall processing time but also mitigated the blood clogging problem commonly encountered in microfluidic-based CTC isolation systems. To clinically validate the chip, we employed it to detect and capture CTCs from 10 liver cancer patients. Positive CTC enumeration was observed in all the blood samples, and the readings ranged from a low of 1–2 CTCs (1 patient) to a high of >20 CTCs (2 patients) with the balance having 3–20 CTCs per 3-ml blood sample. The work here indicates that our system can be developed for use in cancer screening, metastatic assessment, and chemotherapeutic response and for pharmacological and genetic evaluation of single CTCs.     

Continuous separation of blood cells in spiral microfluidic devices

Blood cell sorting is critical to sample preparation for both clinical diagnosis and therapeutic research. The spiral inertial microfluidic devices can achieve label-free, continuous separation of cell mixtures with high throughput and efficiency. The devices utilize hydrodynamic forces acting on cells within laminar flow, coupled with rotational Dean drag due to curvilinear microchannel geometry. Here, we report on optimized Archimedean spiral devices to achieve cell separation in less than 8 cm of downstream focusing length. These improved devices are small in size (<1 in.²), exhibit high separation efficiency (∼95%), and high throughput with rates up to 1 × 10⁶ cells per minute. These device concepts offer a path towards possible development of a lab-on-chip for point-of-care blood analysis with high efficiency, low cost, and reduced analysis time.     

Rare cell isolation and profiling on a hybrid magnetic/size-sorting chip

We present a hybrid magnetic/size-sorting (HMSS) chip for isolation and molecular analyses of circulating tumor cells (CTCs). The chip employs both negative and positive cell selection in order to provide high throughput, unbiased CTC enrichment. Specifically, the system utilizes a self-assembled magnet to generate high magnetic forces and a weir-style structure for cell sorting. The resulting device thus can perform multiple functions, including magnetic depletion, size-selective cell capture, and on-chip molecular staining. With such capacities, the HMSS device allowed one-step CTC isolation and single cell detection from whole blood, tested with spiked cancer cells. The system further facilitated the study of individual CTCs for heterogeneity in molecular marker expression.Circulating tumor cells (CTCs) have emerged as an important biomarker in clinical practice as well as in fundamental research.^{1, 2} CTCs, shed from primary tumors, have been shown to be an early harbinger of tumor expansion and metastasis³ and have been used to predict disease progression, response to treatment, relapse, and overall survival.^{4, 5, 6} Recent work has shown that CTCs display distinct proteomic and genetic profiles; for example, CTCs in pancreatic cancer, have increased RNA expression of Wnt, implicating this pathway in metastasis.⁷ Proteomic characterization of proliferative markers such as Ki-67, and hormonal markers such as androgen receptor in prostate cancer, also have been shown to be predictive of treatment outcome.^{8, 9}Despite such clinical potential of CTCs, their routine detection and characterization still remains a significant technical challenge.¹⁰ The task requires screening of a large number of cells (e.g., > 10⁷ cells in 10 ml blood) and enrichment of heterogeneous targets against a complex biological background. Two main methods of CTC isolation are typically used: positive and negative selection. In positive selection, CTCs are directly isolated from blood via size-based filtration^{11, 12, 13, 14, 15, 16, 17, 18, 19, 20} or antibody-based capture.^{1, 8, 21} Negative depletion reduces abundant blood cells, often by immunomagnetic separation, for downstream CTC enrichment.²² Both approaches have been used for high throughput CTC isolation from whole blood (SI Table 1).²³ Each method, however, has its own inherent limitations. Positive enrichment could be biased by its selection criteria (e.g., cell size and cell surface markers). Negative selection, albeit unbiased, often requires complex sample processing (e.g., multiple washing steps for CTC isolation) that could result in cell loss.We hypothesized that both positive and negative selection could be combined in a single platform to enable (1) highly efficient and unbiased CTC purification, and (2) in-situ molecular analyses of collected cells. As a proof-of-concept, we herein describe a hybrid magnetic/size-sorting (HMSS) system that integrates magnetic and size-based isolation into a compact microfluidic chip. The HMSS first uses a magnetic filter to deplete leukocytes through immunomagnetic capture. Samples then pass through a size-sorter region that traps individual cells at predefined locations. Since abundant leukocytes are removed by the magnetic filter, the size-sorter could have a low size cut-off (∼5 μm), which allows for the unbiased capture of even small cancer cells. Furthermore, molecular probes can be introduced to perform on-chip, multiplexed analyses at single-cell resolution. We evaluated the utility of the developed system by capturing and profiling tumor cells in whole blood. The HMSS offers the advantages of both negative and positive selection and thereby differs from the recently reported iChip system²⁴ which can operate only in either a negative or a positive selection mode.     

Hydrodynamic mechanisms of cell and particle trapping in microfluidics

Focusing and sorting cells and particles utilizing microfluidic phenomena have been flourishing areas of development in recent years. These processes are largely beneficial in biomedical applications and fundamental studies of cell biology as they provide cost-effective and point-of-care miniaturized diagnostic devices and rare cell enrichment techniques. Due to inherent problems of isolation methods based on the biomarkers and antigens, separation approaches exploiting physical characteristics of cells of interest, such as size, deformability, and electric and magnetic properties, have gained currency in many medical assays. Here, we present an overview of the cell/particle sorting techniques by harnessing intrinsic hydrodynamic effects in microchannels. Our emphasis is on the underlying fluid dynamical mechanisms causing cross stream migration of objects in shear and vortical flows. We also highlight the advantages and drawbacks of each method in terms of throughput, separation efficiency, and cell viability. Finally, we discuss the future research areas for extending the scope of hydrodynamic mechanisms and exploring new physical directions for microfluidic applications.     

ApoStream?, a new dielectrophoretic device for antibody independent isolation and recovery of viable cancer cells from blood

Isolation and enumeration of circulating tumor cells (CTCs) are used to monitor metastatic disease progression and guide cancer therapy. However, currently available technologies are limited to cells expressing specific cell surface markers, such as epithelial cell adhesion molecule (EpCAM) or have limited specificity because they are based on cell size alone. We developed a device, ApoStream^™ that overcomes these limitations by exploiting differences in the biophysical characteristics between cancer cells and normal, healthy blood cells to capture CTCs using dielectrophoretic technology in a microfluidic flow chamber. Further, the system overcomes throughput limitations by operating in continuous mode for efficient isolation and enrichment of CTCs from blood. The performance of the device was optimized using a design of experiment approach for key operating parameters such as frequency, voltage and flow rates, and buffer formulations. Cell spiking studies were conducted using SKOV3 or MDA-MB-231 cell lines that have a high and low expression level of EpCAM, respectively, to demonstrate linearity and precision of recovery independent of EpCAM receptor levels. The average recovery of SKOV3 and MDA-MB-231 cancer cells spiked into approximately 12 × 10⁶ peripheral blood mononuclear cells obtained from 7.5 ml normal human donor blood was 75.4% ± 3.1% (n = 12) and 71.2% ± 1.6% (n = 6), respectively. The intra-day and inter-day precision coefficients of variation of the device were both less than 3%. Linear regression analysis yielded a correlation coefficient (R²) of more than 0.99 for a spiking range of 4–2600 cells. The viability of MDA-MB-231 cancer cells captured with ApoStream was greater than 97.1% and there was no difference in cell growth up to 7 days in culture compared to controls. The ApoStream device demonstrated high precision and linearity of recovery of viable cancer cells independent of their EpCAM expression level. Isolation and enrichment of viable cancer cells from ApoStream enables molecular characterization of CTCs from a wide range of cancer types.     

Microwell perfusion array for high-throughput, long-term imaging of clonal growth

Continuous cell tracking by time-lapse microscopy has led to detailed study of cell differentiation pathways using single cell fate maps. There are a multitude of cell fate outcomes, so hundreds of clonal division histories are required to measure these stochastic branching processes. This study examines the principle of condensing cell imaging information into a relatively small region to maximize live cell imaging throughput. High throughput clonal analysis of non-adherent cells by continuous live cell tracking was possible using a microwell perfusion array with an internal volume of 16 μl and 600 microwells at the base. This study includes examination of biocompatibility of buffer systems, connecting tubing, cell culture substrates, and media degradation. An intermittent perfusion protocol was selected for long-term time-lapse imaging of KG1a cells in the microwell array; 1500 clones were simultaneously cultured and scanned every 3 min at 100 × magnifications for 6 days. The advantages of perfusion microwell culture are continuous long-term cell tracking, higher cell imaging throughput, and greater control over cell microenvironment. Microwell devices facilitate high throughput analysis of cell lineage development and measurement of the probability distribution for cell life events such as mitosis.     

Dielectrophoretic capture of low abundance cell population using thick electrodes

Enrichment of rare cell populations such as Circulating Tumor Cells (CTCs) is a critical step before performing analysis. This paper presents a polymeric microfluidic device with integrated thick Carbon-PolyDimethylSiloxane composite (C-PDMS) electrodes designed to carry out dielectrophoretic (DEP) trapping of low abundance biological cells. Such conductive composite material presents advantages over metallic structures. Indeed, as it combines properties of both the matrix and doping particles, C-PDMS allows the easy and fast integration of conductive microstructures using a soft-lithography approach while preserving O₂ plasma bonding properties of PDMS substrate and avoiding a cumbersome alignment procedure. Here, we first performed numerical simulations to demonstrate the advantage of such thick C-PDMS electrodes over a coplanar electrode configuration. It is well established that dielectrophoretic force (F_DEP) decreases quickly as the distance from the electrode surface increases resulting in coplanar configuration to a low trapping efficiency at high flow rate. Here, we showed quantitatively that by using electrodes as thick as a microchannel height, it is possible to extend the DEP force influence in the whole volume of the channel compared to coplanar electrode configuration and maintaining high trapping efficiency while increasing the throughput. This model was then used to numerically optimize a thick C-PDMS electrode configuration in terms of trapping efficiency. Then, optimized microfluidic configurations were fabricated and tested at various flow rates for the trapping of MDA-MB-231 breast cancer cell line. We reached trapping efficiencies of 97% at 20 μl/h and 78.7% at 80 μl/h, for 100 μm thick electrodes. Finally, we applied our device to the separation and localized trapping of CTCs (MDA-MB-231) from a red blood cells sample (concentration ratio of 1:10).     

A dynamically deformable microfilter for selective separation of specific substances in microfluidics

To study an environmental or biological solution, it is essential to separate its constituents. In this study, a 3D-deformable dynamic microfilter was developed to selectively separate the target substance from a solution. This microfilter is a fine metallic nickel structure fabricated using photolithography and electroplating techniques. It is gold-coated across its entire surface with multiple slits of 10–20 μm in width. Its two-dimensional shape is deformed into a three-dimensional shape when used for fluid separation due to hydrodynamic forces. By adjusting the pressure applied to the microfilter, the size of the gap created by deformation can be changed. To effectively isolate the target substance, the relationship between the solution flow rate and the extent of microfilter deformation was investigated. The filtration experiments demonstrated the microfilter’s ability to isolate the target substance with elastic deformation without undergoing plastic deformation. Additionally, modification of the microfilter surface with nucleic acid aptamers resulted in the selective isolation of the target cell, which further demonstrates the potential application of microfilters in the isolation of specific components of heterogeneous solutions.     

Inertial microfluidics in contraction–expansion microchannels: A review

Inertial microfluidics has brought enormous changes in the conventional cell/particle detection process and now become the main trend of sample pretreatment with outstanding throughput, low cost, and simple control method. However, inertial microfluidics in a straight microchannel is not enough to provide high efficiency and satisfying performance for cell/particle separation. A contraction–expansion microchannel is a widely used and multifunctional channel pattern involving inertial microfluidics, secondary flow, and the vortex in the chamber. The strengthened inertial microfluidics can help us to focus particles with a shorter channel length and less processing time. Both the vortex in the chamber and the secondary flow in the main channel can trap the target particles or separate particles based on their sizes more precisely. The contraction–expansion microchannels are also capable of combining with a curved, spiral, or serpentine channel to further improve the separation performance. Some recent studies have focused on the viscoelastic fluid that utilizes both elastic forces and inertial forces to separate different size particles precisely with a relatively low flow rate for the vulnerable cells. This article comprehensively reviews various contraction–expansion microchannels with Newtonian and viscoelastic fluids for particle focusing, separation, and microfluid mixing and provides particle manipulation performance data analysis for the contraction–expansion microchannel design.     

A microfluidic model for organ-specific extravasation of circulating tumor cells

Circulating tumor cells (CTCs) are the principal vehicle for the spread of non-hematologic cancer disease from a primary tumor, involving extravasation of CTCs across blood vessel walls, to form secondary tumors in remote organs. Herein, a polydimethylsiloxane-based microfluidic system is developed and characterized for in vitro systematic studies of organ-specific extravasation of CTCs. The system recapitulates the two major aspects of the in vivo extravasation microenvironment: local signaling chemokine gradients in a vessel with an endothelial monolayer. The parameters controlling the locally stable chemokine gradients, flow rate, and initial chemokine concentration are investigated experimentally and numerically. The microchannel surface treatment effect on the confluency and adhesion of the endothelial monolayer under applied shear flow has also been characterized experimentally. Further, the conditions for driving a suspension of CTCs through the microfluidic system are discussed while simultaneously maintaining both the local chemokine gradients and the confluent endothelial monolayer. Finally, the microfluidic system is utilized to demonstrate extravasation of MDA-MB-231 cancer cells in the presence of CXCL12 chemokine gradients. Consistent with the hypothesis of organ-specific extravasation, control experiments are presented to substantiate the observation that the MDA-MB-231 cell migration is attributed to chemotaxis rather than a random process.     

The local impact of global climate change: reporting on landscape transformation and threatened identity in the English regional newspaper press

This paper contributes to extant understandings of media representations of climate change by examining the role of the English regional newspaper press in the transformation and dissemination of climate change discourse. Unlike previous accounts, this paper contends that such newspapers shape public understandings of climate change in ways that have yet to be adequately charted. With this in mind, this paper examines the ways in which global climate change is translated into a locally relevant phenomenon. That is, it focuses on its "domestication." Although we acknowledge that there are a number of ways in which this process occurs, specific attention is drawn to stories that highlight the destruction of local landscape features, the transformation of important habitats, and the arrival of "alien" species. The broader significance of such stories is considered in relation to long-standing debates concerning the importance of landscape to notions of national and regional identity.     

Characterization of microfluidic shear-dependent epithelial cell adhesion molecule immunocapture and enrichment of pancreatic cancer cells from blood cells with dielectrophoresis

Current microfluidic techniques for isolating circulating tumor cells (CTCs) from cancer patient blood are limited by low capture purity, and dielectrophoresis (DEP) has the potential to complement existing immunocapture techniques to improve capture performance. We present a hybrid DEP and immunocapture Hele-Shaw flow cell to characterize DEP''s effects on immunocapture of pancreatic cancer cells (Capan-1, PANC-1, and BxPC-3) and peripheral blood mononuclear cells (PBMCs) with an anti-EpCAM (epithelial cell adhesion molecule) antibody. By carefully specifying the applied electric field frequency, we demonstrate that pancreatic cancer cells are attracted to immunocapture surfaces by positive DEP whereas PBMCs are repelled by negative DEP. Using an exponential capture model to interpret our capture data, we show that immunocapture performance is dependent on the applied DEP force sign and magnitude, cell surface EpCAM expression level, and shear stress experienced by cells flowing in the capture device. Our work suggests that DEP can not only repel contaminating blood cells but also enhance capture of cancer cell populations that are less likely to be captured by traditional immunocapture methods. This combination of DEP and immunocapture techniques to potentially increase CTC capture purity can facilitate subsequent biological analyses of captured CTCs and research on cancer metastasis and drug therapies.