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1.
Vascular function, homeostasis, and pathological development are regulated by the endothelial cells that line blood vessels. Endothelial function is influenced by the integrated effects of multiple factors, including hemodynamic conditions, soluble and insoluble biochemical signals, and interactions with other cell types. Here, we present a membrane microfluidic device that recapitulates key components of the vascular microenvironment, including hemodynamic shear stress, circulating cytokines, extracellular matrix proteins, and multiple interacting cells. The utility of the device was demonstrated by measuring monocyte adhesion to and transmigration through a porcine aortic endothelial cell monolayer. Endothelial cells grown in the membrane microchannels and subjected to 20 dynes∕cm(2) shear stress remained viable, attached, and confluent for several days. Consistent with the data from macroscale systems, 25 ng∕ml tumor necrosis factor (TNF)-α significantly increased RAW264.7 monocyte adhesion. Preconditioning endothelial cells for 24 h under static or 20 dynes∕cm(2) shear stress conditions did not influence TNF-α-induced monocyte attachment. In contrast, simultaneous application of TNF-α and 20 dynes∕cm(2) shear stress caused increased monocyte adhesion compared with endothelial cells treated with TNF-α under static conditions. THP-1 monocytic cells migrated across an activated endothelium, with increased diapedesis in response to monocyte chemoattractant protein (MCP)-1 in the lower channel of the device. This microfluidic platform can be used to study complex cell-matrix and cell-cell interactions in environments that mimic those in native and tissue engineered blood vessels, and offers the potential for parallelization and increased throughput over conventional macroscale systems.  相似文献   

2.
Atherosclerotic lesions occur non-randomly at vascular niches in bends and bifurcations where fluid flow can be characterized as "disturbed" (low shear stress with both forward and retrograde flow). Endothelial cells (ECs) at these locations experience significantly lower average shear stress without change in the levels of pressure or strain, which affects the local balance in mechanical stresses. Common in vitro models of atherosclerosis focus primarily on shear stress without accounting for pressure and strain loading. To overcome this limitation, we used our microfluidic endothelial cell culture model (ECCM) to achieve accurate replication of pressure, strain, and shear stress waveforms associated with both normal flow seen in straight sections of arteries and disturbed flow seen in the abdominal aorta in the infrarenal segment at the wall distal to the inferior mesenteric artery (IMA), which is associated with high incidence of atherosclerotic lesion formation. Human aortic endothelial cells (HAECs) were cultured within the ECCM under both normal and disturbed flow and evaluated for cell shape, cytoskeletal alignment, endothelial barrier function, and inflammation using immunofluorescence microscopy and flow cytometry. Results clearly demonstrate quantifiable differences between cells cultured under disturbed flow conditions, which are cuboidal with short and randomly oriented actin microfilaments and show intermittent expression of β-Catenin and cells cultured under normal flow. However, in the absence of pro-inflammatory stimulation, the levels of expression of activation markers: intra cellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), and vascular endothelial cell growth factor - receptor 2 (VEGF-R2) known to be involved in the initiation of plaque formation were only slightly higher in HAECs cultured under disturbed flow in comparison to cells cultured under normal flow.  相似文献   

3.
Microfluidic platforms enable a variety of physical or chemical stimulation of single or multiple cells to be examined and monitored in real-time. To date, intracellular calcium signalling research is, however, predominantly focused on observing the response of cells to a single mode of stimulation; consequently, the sensitising/desensitising of cell responses under concurrent stimuli is not well studied. In this paper, we provide an extended Discontinuous Dielectrophoresis procedure to investigate the sensitising of chemical stimulation, over an extensive range of shear stress, up to 63 dyn/cm2, which encompasses shear stresses experienced in the arterial and venus systems (10 to 60 dyn/cm2). Furthermore, the TRPV4-selective agonist GSK1016790A, a form of chemical stimulation, did not influence the ability of the cells'' to remain immobilised under high levels of shear stress; thus, enabling us to investigate shear stress stimulation on agonism. Our experiments revealed that shear stress sensitises GSK1016790A-evoked intracellular calcium signalling of cells in a shear-stimulus dependent manner, as observed through a reduction in the cellular response time and an increase in the pharmacological efficacy. Consequently, suggesting that the role of TRPV4 may be underestimated in endothelial cells—which experience high levels of shear stress. This study highlights the importance of conducting studies at high levels of shear stress. Additionally, our approach will be valuable for examining the effect of high levels of shear on different cell types under different conditions, as presented here for agonist activation.  相似文献   

4.
Shear stress is the major mechanical force applied on vascular endothelial cells by blood flow, and is a crucial factor in normal vascular physiology and in the development of some vascular pathologies. The exact mechanisms of cellular mechano-transduction in mammalian cells and tissues have not yet been elucidated, but it is known that mechanically sensitive receptors and ion channels play a crucial role. This paper describes the use of a novel and efficient microfluidic device to study mechanically-sensitive receptors and ion channels in vitro, which has three independent channels from which recordings can be made and has a small surface area such that fewer cells are required than for conventional flow chambers. The contoured channels of the device enabled examination of a range of shear stresses in one field of view, which is not possible with parallel plate flow chambers and other previously used devices, where one level of flow-induced shear stress is produced per fixed flow-rate. We exposed bovine aortic endothelial cells to different levels of shear stress, and measured the resulting change in intracellular calcium levels ([Ca2+]i) using the fluorescent calcium sensitive dye Fluo-4AM. Shear stress caused an elevation of [Ca2+]i that was proportional to the level of shear experienced. The response was temperature dependant such that at lower temperatures more shear stress was required to elicit a given level of calcium signal and the magnitude of influx was reduced. We demonstrated that shear stress-induced elevations in [Ca2+]i are largely due to calcium influx through the transient receptor potential vanilloid type 4 ion channel.  相似文献   

5.
High-throughput size-based rare cell enrichment using microscale vortices   总被引：2，自引：0，他引：2  
Cell isolation in designated regions or from heterogeneous samples is often required for many microfluidic cell-based assays. However, current techniques have either limited throughput or are incapable of viable off-chip collection. We present an innovative approach, allowing high-throughput and label-free cell isolation and enrichment from heterogeneous solution using cell size as a biomarker. The approach utilizes the irreversible migration of particles into microscale vortices, developed in parallel expansion-contraction trapping reservoirs, as the cell isolation mechanism. We empirically determined the critical particle∕cell diameter D(crt) and the operational flow rate above which trapping of cells∕particles in microvortices is initiated. Using this approach we successfully separated larger cancer cells spiked in blood from the smaller blood cells with processing rates as high as 7.5×10(6) cells∕s. Viable long-term culture was established using cells collected off-chip, suggesting that the proposed technique would be useful for clinical and research applications in which in vitro culture is often desired. The presented technology improves on current technology by enriching cells based on size without clogging mechanical filters, employing only a simple single-layered microfluidic device and processing cell solutions at the ml∕min scale.  相似文献   

6.
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.  相似文献   

7.
Various single-cell retention structures (SCRSs) were reported for analysis of single cells within microfluidic devices. Undesirable flow behaviors within micro-environments not only influence single-cell manipulation and retention significantly but also lead to cell damage, biochemical heterogeneity among different individual cells (e.g., different cell signaling pathways induced by shear stress). However, the fundamentals in flow behaviors for single-cell manipulation and shear stress reduction, especially comparison of these behaviors in different microstructures, were not fully investigated in previous reports. Herein, flow distribution and induced shear stress in two different single-cell retention structures (SCRS I and SCRS II) were investigated in detail to study their effects on single-cell trapping using computational fluid dynamics (CFD) methods. The results were successfully verified by experimental results. Comparison between these two SCRS shows that the wasp-waisted configuration of SCRS II has a better performance in trapping and manipulating long cylinder-shaped cardiac myocytes and provides a safer “harbor” for fragile cells to prevent cell damage due to the shear stress induced from strong flows. The simulation results have not only explained flow phenomena observed in experiments but also predict new flow phenomena, providing guidelines for new chip design and optimization, and a better understanding of the cell micro-environment and fundamentals of microfluidic flows in single-cell manipulation and analysis.  相似文献   

8.
A new method is demonstrated to transport particles, cells, and other microorganisms using rectified ac electro-osmotic flows in open microchannels. The rectified flow is obtained by synchronous zeta potential modulation with the driving potential in the microchannel. Experiments were conducted to transport both neutral, charged particles, and microorganisms of various sizes. A maximum speed of 50 μm∕s was obtained for 8 μm polystyrene beads, without any electrolysis, using a symmetrical square waveform driving electric field of 5 V∕mm at 10 Hz and a 360 V gate potential with its polarity synchronized with the driving potential (phase lag=0°).  相似文献   

9.
An electro-osmosis (EOS) diode pumping platform capable of culturing cells in fluidic cellular micro-environments particularly at low volume flow rates has been developed. Diode pumps have been shown to be a viable alternative to mechanically driven pumps. Typically electrokinetic micro-pumps were limited to low-concentration solutions (≤10 mM). In our approach, surface mount diodes were embedded along the sidewalls of a microchannel to rectify externally applied alternating current into pulsed direct current power across the diodes in order to generate EOS flows. This approach has for the first time generated flows at ultra-low flow rates (from 2.0 nl/s to 12.3 nl/s) in aqueous solutions with concentrations greater than 100 mM. The range of flow was generated by changing the electric field strength applied to the diodes from 0.5 Vpp/cm to 10 Vpp/cm. Embedding an additional diode on the upper surface of the enclosed microchannel increased flow rates further. We characterized the diode pump-driven fluidics in terms of intensities and frequencies of electric inputs, pH values of solutions, and solution types. As part of this study, we found that the growth of A549 human lung cancer cells was positively affected in the microfluidic diode pumping system. Though the chemical reaction compromised the fluidic control overtime, the system could be maintained fully functional over a long time if the solution was changed every hour. In conclusion, the advantage of miniature size and ability to accurately control fluids at ultra-low volume flow rates can make this diode pumping system attractive to lab-on-a-chip applications and biomedical engineering in vitro studies.  相似文献   

10.
Fluid shear stress (FSS) plays a critical role in regulating endothelium function and maintaining vascular homeostasis. Current microfluidic devices for studying FSS effects on cells either separate high shear stress zone and low shear stress zone into different culturing chambers, or arranging the zones serially along the flow direction, which complicates subsequent data interpretation. In this paper, we report a diamond shaped microfluidic shear device where the high shear stress zone and the low shear stress zone are arranged in parallel within one culturing chamber. Since the zones with different shear stress magnitudes are aligned normal to the flow direction, the cells in one stress group are not substantially affected by the flow-induced cytokine/chemokine releases by cells in the other group. Cell loading experiments using human umbilical vein endothelial cells show that the device is able to reveal stress magnitude-dependent and loading duration-dependent cell responses. The co-existence of shear stress zones with varied magnitudes within the same culturing chamber not only ensures that all the cells are subject to the identical culturing conditions, but also allows the resemblance of the differential shear stress pattern in natural arterial conditions. The device is expected to provide a new solution for studying the effects of heterogeneous hemodynamic patterns in the onset and progression of various vascular diseases.  相似文献   

11.
Seamless integration of biological components with electrochemical sensors is critical in the development of microdevices for cell analysis. The present paper describes the integration miniature Au electrodes next to immune cells (macrophages) in order to detect cell-secreted hydrogen peroxide (H(2)O(2)). Photopatterning of poly(ethylene glycol) (PEG) hydrogels was used to both immobilize horseradish peroxidase molecules onto electrodes and to define regions for cell attachment in the vicinity of sensing electrodes. Electrodes micropatterned in such a manner were enclosed inside poly(dimethylsiloxane) fluid conduits and incubated with macrophages. The cells attached onto the exposed glass regions in the vicinity of the electrodes and nowhere else on the non-fouling PEG hydrogel surface. A microfluidic device was converted into an electrochemical cell by placing flow-through Ag∕AgCl reference and Pt wire counter electrodes at the outlet and inlet, respectively. This microdevice with integrated H(2)O(2)-sensing electrodes had sensitivity of 27 μA∕cm(2) mM with a limit of detection of 2 μM. Importantly, this microdevice allowed controllable seeding of macrophages next to electrodes, activation of these cells and on-chip monitoring of H(2)O(2) release in real time. In the future, this biosensor platform may be utilized for monitoring of macrophage responses to pathogens or for the study of inflammatory signaling in micropatterned cell cultures.  相似文献   

12.
Microfluidic devices have emerged as important tools for experimental physiology. They allow to study the effects of hydrodynamic flow on physiological and pathophysiological processes, e.g., in the circulatory system of the body. Such dynamic in vitro test systems are essential in order to address fundamental problems in drug delivery and targeted imaging, such as the binding of particles to cells under flow. In the present work an acoustically driven microfluidic platform is presented in which four miniature flow channels can be operated in parallel at distinct flow velocities with only slight inter-experimental variations. The device can accommodate various channel architectures and is fully compatible with cell culture as well as microscopy. Moreover, the flow channels can be readily separated from the surface acoustic wave pumps and subsequently channel-associated luminescence, absorbance, and/or fluorescence can be determined with a standard microplate reader. In order to create artificial blood vessels, different coatings were evaluated for the cultivation of endothelial cells in the microchannels. It was found that 0.01% fibronectin is the most suitable coating for growth of endothelial monolayers. Finally, the microfluidic system was used to study the binding of 1 μm polystyrene microspheres to three different types of endothelial cell monolayers (HUVEC, HUVECtert, HMEC-1) at different average shear rates. It demonstrated that average shear rates between 0.5 s−1 and 2.25 s−1 exert no significant effect on cytoadhesion of particles to all three types of endothelial monolayers. In conclusion, the multichannel microfluidic platform is a promising device to study the impact of hydrodynamic forces on cell physiology and binding of drug carriers to endothelium.  相似文献   

13.
Primary hemostasis and blood clotting is known to be influenced by the red blood cell volume fraction (hematocrit) in blood. Depressed or elevated levels of red blood cells can lead to vascular perfusion problems ranging from bleeding to thrombus formation. The early stage of hemostasis and thus blood clotting in all vessel sections from the arterial to the venous system involves the adhesion of platelets to von Willebrand factor. Here we present experimental and theoretical results showing that the adhesion probability of platelets to von Willebrand factor is strongly and nonlinearly dependent on hematocrit and flow rate. Interestingly, the actual binding forces are not markedly different, which suggest that the origin of such behavior is in the distribution of platelets. Using hydrodynamic simulations of a simple model, we explicitly show that the higher the hematocrit and the flow rate, the larger the amount of platelets residing close to the wall. Our simulation results, which are in excellent agreement with the experimental observations, explain why such phenomena occur. We believe that the nonhomogeneous red blood cell distribution as well as the shear dependent hydrodynamic interaction is key for the accumulation of platelets on the vessel wall. The work we present here is an important step forward from our earlier work on single molecules and extends into the collective cellular behavior of whole blood. It sheds new light on the correlation between hematocrit and the initial steps in hemostasis and thrombosis, and outlines advances for the treatment of vascular diseases associated with high levels of red blood cells. These results are not only highly relevant for the field of hemostasis and the physics of blood clotting but are also of powerful impact in applied science most obviously in drug delivery and colloidal science.  相似文献   

14.
Extravasation of circulating cells is an essential process that governs tissue inflammation and the body''s response to pathogenic infection. To initiate anti-inflammatory and phagocytic functions within tissues, immune cells must cross the vascular endothelial barrier from the vessel lumen to the subluminal extracellular matrix. In this work, we present a microfluidic approach that enables the recreation of a three-dimensional, perfused endothelial vessel formed by human endothelial cells embedded within a collagen-rich matrix. Monocytes are introduced into the vessel perfusate, and we investigate the role of luminal flow and collagen concentration on extravasation. In vessels conditioned with the flow, increased monocyte adhesion to the vascular wall was observed, though fewer monocytes extravasated to the collagen hydrogel. Our results suggest that the lower rates of extravasation are due to the increased vessel integrity and reduced permeability of the endothelial monolayer. We further demonstrate that vascular permeability is a function of collagen hydrogel mass concentration, with increased collagen concentrations leading to elevated vascular permeability and increased extravasation. Collectively, our results demonstrate that extravasation of monocytes is highly regulated by the structural integrity of the endothelial monolayer. The microfluidic approach developed here allows for the dissection of the relative contributions of these cues to further understand the key governing processes that regulate circulating cell extravasation and inflammation.  相似文献   

15.
Studying the effects of pharmacological agents on human endothelium includes the routine use of cell monolayers cultivated in multi-well plates. This configuration fails to recapitulate the complex architecture of vascular networks in vivo and does not capture the relationship between shear stress (i.e. flow) experienced by the cells and dose of the applied pharmacological agents. Microfluidic platforms have been applied extensively to create vascular systems in vitro; however, they rely on bulky external hardware to operate, which hinders the wide application of microfluidic chips by non-microfluidic experts. Here, we have developed a standalone perfusion platform where multiple devices were perfused at a time with a single miniaturized peristaltic pump. Using the platform, multiple micro-vessel networks, that contained three levels of branching structures, were created by culturing endothelial cells within circular micro-channel networks mimicking the geometrical configuration of natural blood vessels. To demonstrate the feasibility of our platform for drug testing and validation assays, a drug induced nitric oxide assay was performed on the engineered micro-vessel network using a panel of vaso-active drugs (acetylcholine, phenylephrine, atorvastatin, and sildenafil), showing both flow and drug dose dependent responses. The interactive effects between flow and drug dose for sildenafil could not be captured by a simple straight rectangular channel coated with endothelial cells, but it was captured in a more physiological branching circular network. A monocyte adhesion assay was also demonstrated with and without stimulation by an inflammatory cytokine, tumor necrosis factor-α.  相似文献   

16.
Optofluidics may hold the key to greater success of photocatalytic water treatment. This is evidenced by our findings in this paper that the planar microfluidic reactor can overcome the limitations of mass transfer and photon transfer in the previous photocatalytic reactors and improve the photoreaction efficiency by more than 100 times. The microreactor has a planar chamber (5 cm×1.8 cm×100 μm) enclosed by two TiO(2)-coated glass slides as the top cover and bottom substrate and a microstructured UV-cured NOA81 layer as the sealant and flow input∕output. In experiment, the microreactor achieves 30% degradation of 3 ml 3×10(-5)M methylene blue within 5 min and shows a reaction rate constant two orders higher than the bulk reactor. Under optimized conditions, a reaction rate of 8% s(-1) is achieved under solar irradiation. The average apparent quantum efficiency is found to be only 0.25%, but the effective apparent quantum efficiency reaches as high as 25%. Optofluidic reactors inherit the merits of microfluidics, such as large surface∕volume ratio, easy flow control, and rapid fabrication and offer a promising prospect for large-volume photocatalytic water treatment.  相似文献   

17.
Microfluidics has become increasingly important for the study of biochemical cues because it enables exquisite spatiotemporal control of the microenvironment. Well-characterized, stable, and reproducible generation of biochemical gradients is critical for understanding the complex behaviors involved in many biological phenomena. Although many microfluidic devices have been developed which achieve these criteria, the ongoing challenge for these platforms is to provide a suitably benign and physiologically relevant environment for cell culture in a user-friendly format. To achieve this paradigm, microfluidic designs must consider the full scope of cell culture from substrate preparation, cell seeding, and long-term maintenance to properly observe gradient sensing behavior. In addition, designs must address the challenges associated with altered culture conditions and shear forces in flow-based devices. With this consideration, we have designed and characterized a microfluidic device based on the principle of stacked flows to achieve highly stable gradients of diffusible molecules over large areas with extremely low shear forces. The device utilizes a benign vacuum sealing strategy for reversible application to pre-established cell cultures. We apply this device to an existing culture of breast cancer cells to demonstrate the negligible effect of its shear flow on migratory behavior. Lastly, we extend the stacked-flow design to demonstrate its scalable architecture with a prototype device for generating an array of combinatorial gradients.  相似文献   

18.
Cell culture in microfluidic systems has primarily been conducted in devices comprised of polydimethylsiloxane (PDMS) or other elastomers. As polystyrene (PS) is the most characterized and commonly used substrate material for cell culture, microfluidic cell culture would ideally be conducted in PS-based microsystems that also enable tight control of perfusion and hydrodynamic conditions, which are especially important for culture of vascular cell types. Here, we report a simple method to prototype perfusable PS microfluidics for endothelial cell culture under flow that can be fabricated using standard lithography and wet laboratory equipment to enable stable perfusion at shear stresses up to 300 dyn/cm2 and pumping pressures up to 26 kPa for at least 100 h. This technique can also be extended to fabricate perfusable hybrid PS-PDMS microfluidics of which one application is for increased efficiency of viral transduction in non-adherent suspension cells by leveraging the high surface area to volume ratio of microfluidics and adhesion molecules that are optimized for PS substrates. These biologically compatible microfluidic devices can be made more accessible to biological-based laboratories through the outsourcing of lithography to various available microfluidic foundries.  相似文献   

19.
Selection of particles or cells of specific shapes from a complex mixture is an essential procedure for various biological and industrial applications, including synchronization of the cell cycle, classification of environmental bacteria, and elimination of aggregates from synthesized particles. Here, we investigate the separation behaviors of nonspherical and spherical particles∕cells in the hydrodynamic filtration (HDF) scheme, which was previously developed for continuous size-dependent particle∕cell separation. Nonspherical particle models were prepared by coating the hemisphere of spherical polymer particles with a thin Au layer and by bonding the Janus particles to form twins and triplets resembling dividing and aggregating cells, respectively. High-speed imaging revealed a difference in the separation behaviors of spherical and nonspherical particles at a branch point; nonspherical particles showed rotation behavior and did not enter the branch channel even when their minor axis was smaller than the virtual width of the flow region entering the branch channel, w(1). The confocal-laser high-speed particle intensity velocimetry system visualized the flow profile inside the HDF microchannel, demonstrating that the steep flow-velocity distribution at the branch point is the main factor causing the rotation behavior of nonspherical particles. As applications, we successfully separated spherical and nonspherical particles with various major∕minor lengths and also demonstrated the selection of budding∕single cells from a yeast cell mixture. We therefore conclude that the HDF scheme can be used for continuous shape-based particle∕cell separation.  相似文献   

20.
The physiology of vascular endothelial cells is strongly affected by fluid shear stress on their surface. In this study, a microfluidic assay was employed to analyze the alignment of actin filaments in endothelial cells in response to shear stress. When cells were cultured in microfluidic channels and subjected to shear stress, the alignment of filaments in the channel direction was significantly higher than in static cultures. By adding inhibitory drugs, the roles of several signaling proteins in the process of alignment were determined. Thus, it is shown how microfluidic technology can be employed to provide a mechanistic insight into cell physiology.  相似文献   

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