Migration of connective tissue-derived cells is mediated by ultra-low concentration gradient fields of EGF

The directed migration of cells towards chemical stimuli incorporates simultaneous changes in both the concentration of a chemotactic agent and its concentration gradient, each of which may influence cell migratory response. In this study, we utilized a microfluidic system to examine the interactions between epidermal growth factor (EGF) concentration and EGF gradient in stimulating the chemotaxis of connective tissue-derived fibroblast cells. Cells seeded within microfluidic devices were exposed to concentration gradients established by EGF concentrations that matched or exceeded those required for maximum chemotactic responses seen in transfilter migration assays. The migration of individual cells within the device was measured optically after steady-state gradients had been experimentally established. Results illustrate that motility was maximal at EGF concentration gradients between .01- and 0.1-ng/(mL.mm) for all concentrations used. In contrast, the number of motile cells continually increased with increasing gradient steepness for all concentrations examined. Microfluidics-based experiments exposed cells to minute changes in EGF concentration and gradient that were in line with the acute EGFR phosphorylation measured. Correlation of experimental data with established mathematical models illustrated that the fibroblasts studied exhibit an unreported chemosensitivity to minute changes in EGF concentration, similar to that reported for highly motile cells, such as macrophages. Our results demonstrate that shallow chemotactic gradients, while previously unexplored, are necessary to induce the rate of directed cellular migration and the number of motile cells in the connective tissue-derived cells examined.     

A Portable Chemotaxis Platform for Short and Long Term Analysis

Flow-based microfluidic systems have been widely utilized for cell migration studies given their ability to generate versatile and precisely defined chemical gradients and to permit direct visualization of migrating cells. Nonetheless, the general need for bulky peripherals such as mechanical pumps and tubing and the complicated setup procedures significantly limit the widespread use of these microfluidic systems for cell migration studies. Here we present a simple method to power microfluidic devices for chemotaxis assays using the commercially available ALZET® osmotic pumps. Specifically, we developed a standalone chemotaxis platform that has the same footprint as a multiwell plate and can generate well-defined, stable chemical gradients continuously for up to 7 days. Using this platform, we validated the short-term (24 hours) and long-term (72 hours) concentration dependent PDGF-BB chemotaxis response of human bone marrow derived mesenchymal stem cells.     

Cooperative Roles of SDF-1α and EGF Gradients on Tumor Cell Migration Revealed by a Robust 3D Microfluidic Model

Chemokine-mediated directed tumor cell migration within a three dimensional (3D) matrix, or chemoinvasion, is an important early step in cancer metastasis. Despite its clinical importance, it is largely unknown how cytokine and growth factor gradients within the tumor microenvironment regulate chemoinvasion. We studied tumor cell chemoinvasion in well-defined and stable chemical gradients using a robust 3D microfluidic model. We used CXCL12 (also known as SDF-1α) and epidermal growth factor (EGF), two well-known extracellular signaling molecules that co-exist in the tumor microenvironment (e.g. lymph nodes or intravasation sites), and a malignant breast tumor cell line, MDA-MB-231, embedded in type I collagen. When subjected to SDF-1α gradients alone, MDA-MB-231 cells migrated up the gradient, and the measured chemosensitivity (defined as the average cell velocity along the direction of the gradient) followed the ligand – receptor (SDF-1α – CXCR4) binding kinetics. On the other hand, when subjected to EGF gradients alone, tumor cells increased their overall motility, but without statistically significant chemotactic (directed) migration, in contrast to previous reports using 2D chemotaxis assays. Interestingly, we found that the chemoinvasive behavior to SDF-1α gradients was abrogated or even reversed in the presence of uniform concentrations of EGF; however, the presence of SDF-1α and EGF together modulated tumor cell motility cooperatively. These findings demonstrate the capabilities of our microfluidic model in re-creating complex microenvironments for cells, and the importance of cooperative roles of multiple cytokine and growth factor gradients in regulating cell migration in 3D environments.     

Microfluidic Generated EGF-Gradients Induce Chemokinesis of Transplantable Retinal Progenitor Cells via the JAK/STAT and PI3Kinase Signaling Pathways

A growing number of studies are evaluating retinal progenitor cell (RPC) transplantation as an approach to repair retinal degeneration and restore visual function. To advance cell-replacement strategies for a practical retinal therapy, it is important to define the molecular and biochemical mechanisms guiding RPC motility. We have analyzed RPC expression of the epidermal growth factor receptor (EGFR) and evaluated whether exposure to epidermal growth factor (EGF) can coordinate motogenic activity in vitro. Using Boyden chamber analysis as an initial high-throughput screen, we determined that RPC motility was optimally stimulated by EGF concentrations in the range of 20-400ng/ml, with decreased stimulation at higher concentrations, suggesting concentration-dependence of EGF-induced motility. Using bioinformatics analysis of the EGF ligand in a retina-specific gene network pathway, we predicted a chemotactic function for EGF involving the MAPK and JAK-STAT intracellular signaling pathways. Based on targeted inhibition studies, we show that ligand binding, phosphorylation of EGFR and activation of the intracellular STAT3 and PI3kinase signaling pathways are necessary to drive RPC motility. Using engineered microfluidic devices to generate quantifiable steady-state gradients of EGF coupled with live-cell tracking, we analyzed the dynamics of individual RPC motility. Microfluidic analysis, including center of mass and maximum accumulated distance, revealed that EGF induced motility is chemokinetic with optimal activity observed in response to low concentration gradients. Our combined results show that EGFR expressing RPCs exhibit enhanced chemokinetic motility in the presence of low nanomole levels of EGF. These findings may serve to inform further studies evaluating the extent to which EGFR activity, in response to endogenous ligand, drives motility and migration of RPCs in retinal transplantation paradigms.     

Differential effects of EGF gradient profiles on MDA-MB-231 breast cancer cell chemotaxis

Chemotaxis, directed cell migration in a gradient of chemoattractant, is an important biological phenomenon that plays pivotal roles in cancer metastasis. Newly developed microfluidic chemotaxis chambers (MCC) were used to study chemotaxis of metastatic breast cancer cells, MDA-MB-231, in EGF gradients of well-defined profiles. Migration behaviors of MDA-MB-231 cells in uniform concentrations of EGF (0, 25, 50, and 100 ng/ml) and EGF (0-25, 0-50, and 0-100 ng/ml) with linear and nonlinear polynomial profiles were investigated. MDA-MB-231 cells exhibited increased speed and directionality upon stimulation with uniform concentrations of EGF. The cells were viable and motile for over 24 h, confirming the compatibility of MCC with cancer cells. Linear concentration gradients of different ranges were not effective in inducing chemotactic movement as compared to nonlinear gradients. MDA-MB-231 cells migrating in EGF gradient of 0-50 ng/ml nonlinear polynomial profile exhibited marked directional movement toward higher EGF concentration. This result suggests that MDA-MB-231 cancer cell chemotaxis depends on the shape of gradient profile as well as on the range of EGF concentrations.     

Parallel microfluidic networks for studying cellular response to chemical modulation

A microfluidic chip featuring parallel gradient-generating networks etched on glass plate was designed and fabricated. The dam and weir structures were fabricated to facilitate cell positioning and seeding, respectively. The microchip contains five gradient generators and 30 cell chambers where the resulted concentration gradients of drugs are delivered to stimulate the on-chip cultured cells. This microfluidics exploits the advantage of lab-on-a-chip technology by integrating the generation of drug concentration gradients and a series of cell operations including seeding, culture, stimulation and staining into a chip. Steady parallel concentration gradients were generated by flowing two fluids in each network. The microchip described above was applied in studying the role of reduced glutathione (GSH) in MCF-7 cells' chemotherapy sensitivity. The parental breast cancer cell line, MCF-7 and the derived adriamycin resistant cell line MCF-7(adm) were treated with concentration gradients of arsenic trioxide (ATO) and N-acetyl cysteine (NAC) for GSH modulation, followed by exposure to adriamycin. The intracellular GSH level and cell viability were assessed by fluorescence image analysis. GSH levels of both cell lines were down-regulated upon ATO treatment and up-regulated upon NAC treatment. For both cell lines, suppression of intracellular GSH by treatment with ATO has been shown to increase chemotherapy sensitivity; conversely, elevation of intracellular GSH by treatment with NAC leads to increased drug resistance. The results indicated that high intracellular GSH level has negative effect on chemotherapy sensitivity, while depletion of cellular GSH may serve as an effective way to improve chemotherapy sensitivity. The integrated microfluidic chip is able to perform multiparametric pharmacological profiling with easy operation, thus, holds great potential for extrapolation to the high-content drug screening.     

A hybrid microfluidic-vacuum device for direct interfacing with conventional cell culture methods

Background

Microfluidics is an enabling technology with a number of advantages over traditional tissue culture methods when precise control of cellular microenvironment is required. However, there are a number of practical and technical limitations that impede wider implementation in routine biomedical research. Specialized equipment and protocols required for fabrication and setting up microfluidic experiments present hurdles for routine use by most biology laboratories.

Results

We have developed and validated a novel microfluidic device that can directly interface with conventional tissue culture methods to generate and maintain controlled soluble environments in a Petri dish. It incorporates separate sets of fluidic channels and vacuum networks on a single device that allows reversible application of microfluidic gradients onto wet cell culture surfaces. Stable, precise concentration gradients of soluble factors were generated using simple microfluidic channels that were attached to a perfusion system. We successfully demonstrated real-time optical live/dead cell imaging of neural stem cells exposed to a hydrogen peroxide gradient and chemotaxis of metastatic breast cancer cells in a growth factor gradient.

Conclusion

This paper describes the design and application of a versatile microfluidic device that can directly interface with conventional cell culture methods. This platform provides a simple yet versatile tool for incorporating the advantages of a microfluidic approach to biological assays without changing established tissue culture protocols.     

A microfluidic device to establish concentration gradients using reagent density differences

Microfabrication has become widely utilized to generate controlled microenvironments that establish chemical concentration gradients for a variety of engineering and life science applications. To establish microfluidic flow, the majority of existing devices rely upon additional facilities, equipment, and excessive reagent supplies, which together limit device portability as well as constrain device usage to individuals trained in technological disciplines. The current work presents our laboratory-developed bridged μLane system, which is a stand-alone device that runs via conventional pipette loading and can operate for several days without need of external machinery or additional reagent volumes. The bridged μLane is a two-layer polydimethylsiloxane microfluidic device that is able to establish controlled chemical concentration gradients over time by relying solely upon differences in reagent densities. Fluorescently labeled Dextran was used to validate the design and operation of the bridged μLane by evaluating experimentally measured transport properties within the microsystem in conjunction with numerical simulations and established mathematical transport models. Results demonstrate how the bridged μLane system was used to generate spatial concentration gradients that resulted in an experimentally measured Dextran diffusivity of (0.82 ± 0.01) × 10(-6) cm(2)/s.     

Micrometer-scale oxygen delivery rearranges cells and prevents necrosis in tumor tissue in vitro

Oxygen availability plays a critical role in cancer progression and is correlated with poor prognosis. Despite this connection, the independent effects of oxygen gradients on tumor tissues have not been measured. To address this, we developed an oxygen delivery device that uses microelectrodes to generate oxygen directly underneath three-dimensional tumor cylindroids composed of colon carcinoma cells. The extent of cell death was measured using fluorescence staining. Supplying oxygen for 60 h eliminated the necrotic region typically found in the center of cylindroids despite the continued presence of other nutrient gradients. A mathematical model of cylindroid growth showed that the rate of cell death was more sensitive to oxygen than the growth rate. After oxygenation, a ring of dead cells was observed at the outside edge of cylindroids, and dead cells were observed moving outward from cylindroid centers. This movement suggests that dead cells were pushed by viable cells migrating in response to oxygen gradients, a mechanism that may connect transient oxygen gradients to metastasis formation. These measurements show that oxygen gradients are a primary factor governing cell viability and rearrange cells in tumors.     

Study on Invadopodia Formation for Lung Carcinoma Invasion with a Microfluidic 3D Culture Device

Invadopodia or invasive feet, which are actin-rich membrane protrusions with matrix degradation activity formed by invasive cancer cells, are a key determinant in the malignant invasive progression of tumors and represent an important target for cancer therapies. In this work, we presented a microfluidic 3D culture device with continuous supplement of fresh media via a syringe pump. The device mimicked tumor microenvironment in vivo and could be used to assay invadopodia formation and to study the mechanism of human lung cancer invasion. With this device, we investigated the effects of epidermal growth factor (EGF) and matrix metalloproteinase (MMP) inhibitor, GM6001 on invadopodia formation by human non-small cell lung cancer cell line A549 in 3D matrix model. This device was composed of three units that were capable of achieving the assays on one control group and two experimental groups'' cells, which were simultaneously pretreated with EGF or GM6001 in parallel. Immunofluorescence analysis of invadopodia formation and extracellular matrix degradation was conducted using confocal imaging system. We observed that EGF promoted invadopodia formation by A549 cells in 3D matrix and that GM6001 inhibited the process. These results demonstrated that epidermal growth factor receptor (EGFR) signaling played a significant role in invadopodia formation and related ECM degradation activity. Meanwhile, it was suggested that MMP inhibitor (GM6001) might be a powerful therapeutic agent targeting invadopodia formation in tumor invasion. This work clearly demonstrated that the microfluidic-based 3D culture device provided an applicable platform for elucidating the mechanism of cancer invasion and could be used in testing other anti-invasion agents.     

Modeling of effects of nutrient gradients on cell proliferation in microfluidic bioreactor

Precise control of the microenvironment is highly desirable in cell culture to study the cell biology. Microfluidic based bioreactors provide a promising method for the spatial and temporal control of cell growth and stimuli. A three-dimensional nutrient transport model, incorporating the monolayer cell growth model, has been developed to investigate the influence of gradients of oxygen and epidermal growth factor (EGF) on the cell culture in the continuous-flow microchannel bioreactor. Our results demonstrate that applying inlet concentration gradients of oxygen and EGF can induce variations of cell density in the y-direction. It is further found that compared to the oxygen gradients the EGF concentration gradients are more efficient in regulating the cell growth.     

Activation of endogenous thrombin receptors causes clustering and sensitization of epidermal growth factor receptors of swiss 3T3 cells without transactivation

The G protein-coupled thrombin receptor can induce cellular responses in some systems by transactivating the epidermal growth factor (EGF) receptor. This is in part due to the stimulation of ectoproteases that generate EGF receptor ligands. We show here that this cannot account for the stimulation of proliferation or migration by thrombin of Swiss 3T3 cells. Thrombin has no direct effect on the activation state of the EGF receptor or of its downstream effectors. However, thrombin induces the subcellular clustering of the EGF receptor at filamentous actin-containing structures at the leading edge and actin arcs of migrating cells in association with other signaling molecules, including Shc and phospholipase Cgamma1. In these thrombin-primed cells, the subsequent migratory response to EGF is potentiated. Thrombin did not potentiate the EGF-stimulated EGF receptor phosphorylation. Thus, in Swiss 3T3 cells the G protein-coupled thrombin receptor can potentiate the EGF tyrosine kinase receptor response when activated by EGF, and this appears to be due to the subcellular concentration of the receptor with downstream effectors and not to the overall ability of EGF to induce receptor transphosphorylation. Thus, the EGF receptor subcellular localization which is altered by thrombin appears to be an important determinant of the efficacy of downstream EGF receptor signaling in cell migration.     

Modeling and development of a low frequency contactless dielectrophoresis (cDEP) platform to sort cancer cells from dilute whole blood samples

Contactless dielectrophoresis (cDEP) devices are a new adaptation of dielectrophoresis in which fluid electrodes, isolated from the main microfluidic channel by a thin membrane, provide the electric field gradients necessary to manipulate cells. This work presents a continuous sorting device which is the first cDEP design capable of exploiting the Clausius-Mossotti factor at frequencies where it is both positive and negative for mammalian cells. Experimental devices are fabricated using a cost effective technique which can achieve 50 μm feature sizes and does not require the use of a cleanroom or specialized equipment. An analytical model is developed to evaluate cDEP devices as a network of parallel resistor-capacitor pairs. Two theoretical devices are presented and evaluated using finite element methods to demonstrate the effect of geometry on the development of electric field gradients across a wide frequency spectrum. Finally, we present an experimental device capable of continuously sorting human leukemia cells from dilute blood samples. This is the first cDEP device designed to operate below 100 kHz resulting in successful manipulation of human leukemia cells, while in the background red blood cells are unaffected.     

Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device

Although a wealth of knowledge about chemotaxis has accumulated in the past 40 years, these studies have been hampered by the inability of researchers to generate simple linear gradients instantaneously and to maintain them at steady state. Here we describe a device microfabricated by soft lithography and consisting of a network of microfluidic channels that can generate spatially and temporally controlled gradients of chemotactic factors. When human neutrophils are positioned within a microchannel, their migration in simple and complex interleukin-8 (IL-8) gradients can be tested. The cells exhibit strong directional migration toward increasing concentrations of IL-8 in linear gradients. Neutrophil migration halts abruptly when cells encounter a sudden drop in the chemoattractant concentration to zero ("cliff" gradient). When neutrophils are challenged with a gradual increase and decrease in chemoattractant ("hill" gradient), however, the cells traverse the crest of maximum concentration and migrate further before reversing direction. The technique described in this paper provides a robust method to investigate migratory cells under a variety of conditions not accessible to study by earlier techniques.     

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic microfluidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.     

Single-cell cloning and expansion of human induced pluripotent stem cells by a microfluidic culture device

The microenvironment of cells, which includes basement proteins, shear stress, and extracellular stimuli, should be taken into consideration when examining physiological cell behavior. Although microfluidic devices allow cellular responses to be analyzed with ease at the single-cell level, few have been designed to recover cells. We herein demonstrated that a newly developed microfluidic device helped to improve culture conditions and establish a clonality-validated human pluripotent stem cell line after tracing its growth at the single-cell level. The device will be a helpful tool for capturing various cell types in the human body that have not yet been established in vitro.     

Epidermal growth factor-dependent growth of human KB cells in a defined medium and altered growth factor requirements of KB mutants resistant to EGF-Pseudomonas exotoxin conjugates

A serum-free culture system was established for human KB carcinoma (HeLa) cells that consisted of a chemically defined medium and several growth factors including epidermal growth factor (EGF), insulin, transferrin, hydrocortisone, and ethanolamine. EGF and insulin showed the greatest effects on the growth rate of KB cells. Insulin-like growth factor I (IGF-I) at the same concentration as insulin stimulated cell growth less than insulin. Transferrin, hydrocortisone, or ethanolamine had no growth-stimulatory effects alone but were stimulatory when combined with EGF and/or insulin. Transforming growth factor-beta inhibited growth and triiodothyronine stimulated growth. The growth factor requirements were established for several KB mutants with low EGF receptor levels that had been selected for resistance to a conjugate of EGF with Pseudomonas exotoxin (EGF-PE). Three of five KB mutants did not respond to EGF; two other mutants responded to a lesser extent than the parental KB cells. Four mutants had a reduced response to insulin and responded to T3; one mutant (ET-30) responded to neither. These results indicate that KB cells selected for EGF-PE resistance have lost their growth response to EGF and illustrate the usefulness of serum-free medium for studying the growth factor requirements of mutants with altered receptor levels.     

Flow-Based Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable,Competing Gradients

Chemotaxis is the migration of cells in gradients of chemoeffector molecules. Although multiple, competing gradients must often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to gradients of multiple signals. In this work, we developed a microfluidic device for generating precise and stable gradients of signaling molecules. We used the device to investigate the effects of individual and combined chemoeffector gradients on Escherichia coli chemotaxis. Laminar flow-based diffusive mixing was used to generate gradients, and the chemotactic responses of cells expressing green fluorescent protein were determined using fluorescence microscopy. Quantification of the migration profiles indicated that E. coli was attracted to the quorum-sensing molecule autoinducer-2 (AI-2) but was repelled from the stationary-phase signal indole. Cells also migrated toward higher concentrations of isatin (indole-2,3-dione), an oxidized derivative of indole. Attraction to AI-2 overcame repulsion by indole in equal, competing gradients. Our data suggest that concentration-dependent interactions between attractant and repellent signals may be important determinants of bacterial colonization of the gut.Bacteria sense chemoeffectors using cell surface receptors (13, 29). Cells constantly monitor the concentration of specific molecules, comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement (6, 22). Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Natural habitats for Escherichia coli, such as the gastrointestinal (GI) tract, are typically heterogeneous and contain multiple chemoeffectors with potentially opposing effects. The integrated chemotactic response in such environments is thus likely to be an important factor in bacterial colonization.Conventional approaches for investigating bacterial chemotaxis, such as the swim plate and capillary (1) assays, are not ideal for quantifying bacterial migration. Chemotactic-ring formation in semisolid agar requires metabolizable attractants and is subject to multiple factors, and both it and the traditional capillary assay are poorly designed to investigate repellent taxis. Mao et al. (23) were the first to investigate bacterial taxis in a microfluidic flow cell. In their device, a concentration gradient is formed by the diffusive mixing of two inlet streams. However, the exposure to a fully developed gradient in this device is limited because it takes time for the gradient to develop.Variations of this technique, such as three-channel microfluidic devices (7, 8) in which a linear gradient is generated in the absence of flow or a T-channel device that monitors chemotaxis perpendicular to the direction of fluid flow (18), were developed subsequently. The T-channel system has many of the limitations of the device developed by Mao et al. (23), and nonflow systems, like the capillary assay (1), suffer from a lack of temporal stability of the gradients.Here, we report a flow-based microfluidic chemotaxis device that is coupled to a gradient generator. Bacteria are exposed to precise and temporally stable concentration gradients of chemoeffectors over the length of the microfluidic channel. This device was used to quantify E. coli chemotaxis in response to the canonical chemoeffectors l-aspartate and Ni²⁺. The device was also used to investigate chemotaxis toward cell-cell communication signals such as autoinducer-2 (AI-2), indole, and isatin that are likely to be present in the in vivo microenvironment in which E. coli is present (e.g., the human GI tract). The data obtained reinforce the idea that concentration-dependent interactions between different chemical signals could be important determinants of bacterial colonization in natural environments.     

Visualization of polarized membrane type 1 matrix metalloproteinase activity in live cells by fluorescence resonance energy transfer imaging

Membrane type 1 matrix metalloproteinase (MT1-MMP) plays a critical role in cancer cell biology by proteolytically remodeling the extracellular matrix. Utilizing fluorescence resonance energy transfer (FRET) imaging, we have developed a novel biosensor, with its sensing element anchoring at the extracellular surface of cell membrane, to visualize MT1-MMP activity dynamically in live cells with subcellular resolution. Epidermal growth factor (EGF) induced significant FRET changes in cancer cells expressing MT1-MMP, but not in MT1-MMP-deficient cells. EGF-induced FRET changes in MT1-MMP-deficient cells could be restored after reconstituting with wild-type MT1-MMP, but not MMP-2, MMP-9, or inactive MT1-MMP mutants. Deletion of the transmembrane domain in the biosensor or treatment with tissue inhibitor of metalloproteinase-2, a cell-impermeable MT1-MMP inhibitor, abolished the EGF-induced FRET response, indicating that MT1-MMP acts at the cell surface to generate FRET changes. In response to EGF, active MT1-MMP was directed to the leading edge of migrating cells along micropatterned fibronectin stripes, in tandem with the local accumulation of the EGF receptor, via a process dependent upon an intact cytoskeletal network. Hence, the MT1-MMP biosensor provides a powerful tool for characterizing the molecular processes underlying the spatiotemporal regulation of this critical class of enzymes.     

A microfluidic device with groove patterns for studying cellular behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 microm wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37 degrees C, 5% CO(2)). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.