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.     

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.     

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.     

Deformability-based circulating tumor cell separation with conical-shaped microfilters: Concept,optimization, and design criteria

Circulating tumor cells (CTCs) separation technology has made positive impacts on cancer science in many aspects. The ability of detecting and separating CTCs can play a key role in early cancer detection and treatment. In recent years, there has been growing interest in using deformability-based CTC separation microfilters due to their simplicity and low cost. Most of the previous studies in this area are mainly based on experimental work. Although experimental research provides useful insights in designing CTC separation devices, there is still a lack of design guidelines based on fundamental understandings of the cell separation process in the filters. While experimental efforts face challenges, especially microfabrication difficulties, we adopt numerical simulation here to study conical-shaped microfilters using deformability difference between CTCs and blood cells for the separation process. We use the liquid drop model for modeling a CTC passing through such microfilters. The accuracy of the model in predicting the pressure signature of the system is validated by comparing it with previous experiments. Pressure-deformability analysis of the cell going through the channel is then carried out in detail in order to better understand how a CTC behaves throughout the filtration process. Different system design criteria such as system throughput and unclogging of the system are discussed. Specifically, pressure behavior under different system throughput is analyzed. Regarding the unclogging issue, we define pressure ratio as a key parameter representing the ability to overcome clogging in such CTC separation devices and investigate the effect of conical angle on the optimum pressure ratio. Finally, the effect of unclogging applied pressure on the system performance is examined. Our study provides detailed understandings of the cell separation process and its characteristics, which can be used for developing more efficient CTC separation devices.     

Dielectrophoresis has broad applicability to marker-free isolation of tumor cells from blood by microfluidic systems

The number of circulating tumor cells (CTCs) found in blood is known to be a prognostic marker for recurrence of primary tumors, however, most current methods for isolating CTCs rely on cell surface markers that are not universally expressed by CTCs. Dielectrophoresis (DEP) can discriminate and manipulate cancer cells in microfluidic systems and has been proposed as a molecular marker-independent approach for isolating CTCs from blood. To investigate the potential applicability of DEP to different cancer types, the dielectric and density properties of the NCI-60 panel of tumor cell types have been measured by dielectrophoretic field-flow fractionation (DEP-FFF) and compared with like properties of the subpopulations of normal peripheral blood cells. We show that all of the NCI-60 cell types, regardless of tissue of origin, exhibit dielectric properties that facilitate their isolation from blood by DEP. Cell types derived from solid tumors that grew in adherent cultures exhibited dielectric properties that were strikingly different from those of peripheral blood cell subpopulations while leukemia-derived lines that grew in non-adherent cultures exhibited dielectric properties that were closer to those of peripheral blood cell types. Our results suggest that DEP methods have wide applicability for the surface-marker independent isolation of viable CTCs from blood as well as for the concentration of leukemia cells from blood.     

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.     

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.     

Microfluidic cell fragmentation for mechanical phenotyping of cancer cells

Circulating tumor cells (CTCs) shed from the primary tumor undergo significant fragmentation in the microvasculature, and very few escape to instigate metastases. Inspired by this in vivo behavior of CTCs, we report a microfluidic method to phenotype cancer cells based on their ability to arrest and fragment at a micropillar-based bifurcation. We find that in addition to cancer cell size, mechanical properties determine fragmentability. We observe that highly metastatic prostate cancer cells are more resistant to fragmentation than weakly metastatic cells, providing the first indication that metastatic CTCs can escape rupture and potentially initiate secondary tumors. Our method may thus be useful in identifying phenotypes that succumb to or escape mechanical trauma in microcirculation.     

Antibody-independent isolation of circulating tumor cells by continuous-flow dielectrophoresis

Circulating tumor cells (CTCs) are prognostic markers for the recurrence of cancer and may carry molecular information relevant to cancer diagnosis. Dielectrophoresis (DEP) has been proposed as a molecular marker-independent approach for isolating CTCs from blood and has been shown to be broadly applicable to different types of cancers. However, existing batch-mode microfluidic DEP methods have been unable to process 10 ml clinical blood specimens rapidly enough. To achieve the required processing rates of 10⁶ nucleated cells/min, we describe a continuous flow microfluidic processing chamber into which the peripheral blood mononuclear cell fraction of a clinical specimen is slowly injected, deionized by diffusion, and then subjected to a balance of DEP, sedimentation and hydrodynamic lift forces. These forces cause tumor cells to be transported close to the floor of the chamber, while blood cells are carried about three cell diameters above them. The tumor cells are isolated by skimming them from the bottom of the chamber while the blood cells flow to waste. The principles, design, and modeling of the continuous-flow system are presented. To illustrate operation of the technology, we demonstrate the isolation of circulating colon tumor cells from clinical specimens and verify the tumor origin of these cells by molecular analysis.     

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.     

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.     

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.     

High efficiency vortex trapping of circulating tumor cells

Circulating tumor cells (CTCs) are important biomarkers for monitoring tumor dynamics and efficacy of cancer therapy. Several technologies have been demonstrated to isolate CTCs with high efficiency but achieve a low purity from a large background of blood cells. We have previously shown the ability to enrich CTCs with high purity from large volumes of blood through selective capture in microvortices using the Vortex Chip. The device consists of a narrow channel followed by a series of expansion regions called reservoirs. Fast flow in the narrow entry channel gives rise to inertial forces, which direct larger cells into trapping vortices in the reservoirs where they remain circulating in orbits. By studying the entry and stability of particles following entry into reservoirs, we discover that channel cross sectional area plays an important role in controlling the size of trapped particles, not just the orbital trajectories. Using these design modifications, we demonstrate a new device that is able to capture a wider size range of CTCs from clinical samples, uncovering further heterogeneity. This simple biophysical method opens doors for a range of downstream interventions, including genetic analysis, cell culture, and ultimately personalized cancer therapy.     

A microfluidic platform for quantitative analysis of cancer angiogenesis and intravasation

Understanding the mechanism behind cancer metastasis is a major challenge in cancer biology. Several in vitro models have been developed to mimic a cancer microenvironment by engineering cancer–endothelial cell (EC) and cancer-stromal cell interactions. It has been challenging to realistically mimic angiogenesis, intravasation, and extravasation using macro-scale approaches but recent progress in microfluidics technology has begun to yield promising results. We present a metastasis chip that produce microvessels, where EC and stromal cells can be patterned in close proximity to tumor cells. The vessels are formed following a natural morphogenic process and have smooth boundaries with proper cell-cell junctions. The engineered microvessels are perfusable and have well-defined openings toward inlet and outlet channels. The ability to introduce cancer cells into different locations bordering to the microvessel wall allowed generation and maintenance of appropriate spatial gradients of growth factors and attractants. Cancer angiogenesis and its inhibition by anti-vascular endothelial growth factor (bevacizumab) treatment were successfully reproduced in the metastasis chip. Cancer intravasation and its modulation by treatment of tumor necrosis factor-α were also modeled. Compared to other models, the unique design of the metastasis chip that engineers a clear EC-cancer interface allows precise imaging and quantification of angiogenic response as well as tumor cell trans-endothelial migration. The metastasis chip presented here has potential applications in the investigation of fundamental cancer biology as well as in drug screening.     

High-efficiency rare cell identification on a high-density self-assembled cell arrangement chip

Detection of individual target cells among a large amount of blood cells is a major challenge in clinical diagnosis and laboratory protocols. Many researches show that two dimensional cells array technology can be incorporated into routine laboratory procedures for continuously and quantitatively measuring the dynamic behaviours of large number of living cells in parallel, while allowing other manipulations such as staining, rinsing, and even retrieval of targeted cells. In this study, we present a high-density cell self-assembly technology capable of quickly spreading over 300 000 cells to form a dense mono- to triple-layer cell arrangement in 5 min with minimal stacking of cells by the gentle incorporation of gravity and peripheral micro flow. With this self-assembled cell arrangement (SACA) chip technology, common fluorescent microscopy and immunofluorescence can be utilized for detecting and analyzing target cells after immuno-staining. Validated by experiments with real human peripheral blood samples, the SACA chip is suitable for detecting rare cells in blood samples with a ratio lower than 1/100 000. The identified cells can be isolated and further cultured in-situ on a chip for follow-on research and analysis. Furthermore, this technology does not require external mechanical devices, such as pump and valves, which simplifies operation and reduces system complexity and cost. The SACA chip offers a high-efficient, economical, yet simple scheme for identification and analysis of rare cells. Therefore, potentially SACA chip may provide a feasible and economical platform for rare cell detection in the clinic.     

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.     

Selective cell capture and analysis using shallow antibody-coated microchannels

Demand for analysis of rare cells such as circulating tumor cells in blood at the single molecule level has recently grown. For this purpose, several cell separation methods based on antibody-coated micropillars have been developed (e.g., Nagrath et al., Nature 450, 1235–1239 (2007)). However, it is difficult to ensure capture of targeted cells by these methods because capture depends on the probability of cell-micropillar collisions. We developed a new structure that actively exploits cellular flexibility for more efficient capture of a small number of cells in a target area. The depth of the sandwiching channel was slightly smaller than the diameter of the cells to ensure contact with the channel wall. For cell selection, we used anti-epithelial cell adhesion molecule antibodies, which specifically bind epithelial cells. First, we demonstrated cell capture with human promyelocytic leukemia (HL-60) cells, which are relatively homogeneous in size; in situ single molecule analysis was verified by our rolling circle amplification (RCA) method. Then, we used breast cancer cells (SK-BR-3) in blood, and demonstrated selective capture and cancer marker (HER2) detection by RCA. Cell capture by antibody-coated microchannels was greater than with negative control cells (RPMI-1788 lymphocytes) and non-coated microchannels. This system can be used to analyze small numbers of target cells in large quantities of mixed samples.     

On-chip three-dimensional tumor spheroid formation and pump-less perfusion culture using gravity-driven cell aggregation and balanced droplet dispensing

This paper presents a spheroid chip in which three-dimensional (3D) tumor spheroids are not only formed by gravity-driven cell aggregation but also cultured at the perfusion rates controlled by balanced droplet dispensing without fluidic pumps. The previous spheroid chips require additional off-chip processes of spheroid formation and extraction as well as bulky components of fluidic pumps. However, the present spheroid chip, where autonomous medium droplet dispensers are integrated on a well array, achieves the on-chip 3D tumor spheroid formation and perfusion culture using simple structure without bulky fluidic pumps. In the experimental study, we demonstrated that the spheroid chip successfully forms 3D tumor spheroids in the wide diameter range of 220 μm–3.2 mm (uniformity > 90%) using H358, H23, and A549 non-small cell lung cancer cells. At the pump-less perfusion culture (Q = 0.1–0.3 μl/min) of spheroids, the number of H358 cells in the spheroid increased up to 50% from the static culture (Q = 0 μl/min) and the viability of the cultured cells also increased about 10%. Therefore, we experimentally verified that the perfusion environment created by the spheroid chip offers a favourable condition to the spheroids with high increase rate and viability. The present chip achieves on-chip 3D tumor spheroid formation and pump-less perfusion culture with simple structure, thereby exhibiting potential for use in integrated in-vivo-like cell culture systems.     

Quantitative impedimetric monitoring of cell migration under the stimulation of cytokine or anti-cancer drug in a microfluidic chip

Cell migration is a cellular response and results in various biological processes such as cancer metastasis, that is, the primary cause of death for cancer patients. Quantitative investigation of the correlation between cell migration and extracellular stimulation is essential for developing effective therapeutic strategies for controlling invasive cancer cells. The conventional method to determine cell migration rate based on comparison of successive images may not be an objective approach. In this work, a microfluidic chip embedded with measurement electrodes has been developed to quantitatively monitor the cell migration activity based on the impedimetric measurement technique. A no-damage wound was constructed by microfluidic phenomenon and cell migration activity under the stimulation of cytokine and an anti-cancer drug, i.e., interleukin-6 and doxorubicin, were, respectively, investigated. Impedance measurement was concurrently performed during the cell migration process. The impedance change was directly correlated to the cell migration activity; therefore, the migration rate could be calculated. In addition, a good match was found between impedance measurement and conventional imaging analysis. But the impedimetric measurement technique provides an objective and quantitative measurement. Based on our technique, cell migration rates were calculated to be 8.5, 19.1, and 34.9 μm/h under the stimulation of cytokine at concentrations of 0 (control), 5, and 10 ng/ml. This technique has high potential to be developed into a powerful analytical platform for cancer research.