Production of reactive oxygen species in endothelial cells under different pulsatile shear stresses and glucose concentrations

A hemodynamic Lab-on-a-chip system was developed in this study. This system has two unique features: (1) it consists of a microfluidic network with an array of endothelial cell seeding sites for testing them under multiple conditions, and (2) the flow rate and the frequency of the culture medium in the microchannel are controlled by a pulsation free pump to mimic the flow profile of the blood in the blood vessel under different physiological conditions. The investigated physiological conditions were: (1) the resting condition in a normal shear stress of 15 dyne cm(-2) with a normal heart rate of 70 bpm, (2) an exhaustive exercise condition with a high shear stress of 30 dyne cm(-2) and a fast heart rate of 140 bpm, and (3) a constant high shear stress of 30 dyne cm(-2). Two chemical conditions were investigated (10 mM and 20 mM glucose) to mimic hyperglycemic conditions in diabetes patients. The effects of various shear stresses either alone or in combination with different glucose concentrations on endothelial cells were examined using the developed hemodynamic Lab-on-a-chip system by assessing two parameters. One is the intracellular level of reactive oxygen species (ROS) determined by a fluorescent probe, H(2)DCFDA. Another is the mitochondrial morphology revealed with a fluorescent dye, MitoTracker Green FM. The results showed that ROS level was elevated nearly 4-fold after 60 min of exhaustive exercise. We found that the pulsatile nature of the fluid was the determination factor for causing ROS generation in the cells as almost no increase of ROS was detected in the constant shear stress condition. Similarly, much higher level of ROS was detected when 10 mM glucose was applied to the cells under normal or high pulsatile shear stresses compared with under a static condition. These results suggest that it is necessary to use pulsatile shear stress to represent the physiological conditions of the blood flow, and demonstrate the advantage of utilizing this newly developed hemodynamic Lab-on-a-chip system over the conventional non-pulsatile system in the future shear stress related studies.     

Endothelin-1 and angiotensin II secretion at different lengths of endothelial cell monolayer in the view of tensile stress accumulation in the upper endothelial cell membrane

Theoretical analysis and experimental observations have shown that tensile stress inside an endothelial cell membrane is capable of growing in the direction opposite to blood flow and can accumulate to a level that is three or more orders of magnitude higher than flow-induced shear stress on the membrane surface. This phenomenon is called cell membrane tension accumulation (CMTA). We hypothesize that correlation may exist between the endothelial cell monolayer length or CMTA and secretory function of endothelial cells. To verify this hypothesis, a paired experimental study was devised to measure the secretion of endothelin (ET-1) and angiotensin II (Ang II) by two monolayers of cultured human glomerular vascular endothelial cell (HGVEC) monolayers subjected an identical steady shear stress. After replicate cultured HGVEC monolayer with two kinds of length of 6 cm and 10 cm were subjected to the same steady laminar shear stress of 0.45 N/m² for 24 h, the average secretion rates of ET-1 and Ang II in 6 cm long increased l.7- and 0.5-fold (n=26, P<0.00l) over 10 cm long, respectively. Over 10 h of exposure to 0.65 N/m², the average secretion rate of both ET-1 and Ang II by HGVEC monolayer of 6 cm in length exceeded 0.5-fold (n=26, P<0.0001) over 10 cm in length. All these demonstrated that the close relationship may exist between length of endothelial cell monolayer and secretion of ET-1 and Ang II by endothelial cells, indicating the possible existence of the cumulative effect of the tensile stress in the upper endothelial cell membrane under the shear flow field.     

Mesoscale constitutive modeling of magnetic dispersions: material functions for shear flows

We recently developed a constitutive model for magnetic dispersions by modeling the magnetic particles as rigid dumbbells dispersed in a solvent. The theory yielded a constitutive equation in which the stress tensor could be expressed as a function of the velocity gradient, an orientational order tensor, S, an average alignment vector, J, and any imposed external magnetic field, H. The constitutive equation is used here to predict material functions for steady shear flow (shear-rate dependent viscosity and first normal stress coefficient) as well as those for unsteady shear flows (stress growth upon inception of steady shear and small-amplitude oscillatory shear). The importance of effects of concentration, equilibrium nematic ordering in the dispersion, and anisotropy in the hydrodynamic drag are emphasized. Comparisons with available experimental data on viscosity for magnetic inks under steady shear flow and inception of steady shear flow show reasonably good agreement.     

A parametric study of human fibroblasts culture in a microchannel bioreactor

The culture of cells in a microbioreactor can be highly beneficial for cell biology studies and tissue engineering applications. The present work provides new insights into the relationship between cell growth, cell morphology, perfusion rate, and design parameters in microchannel bioreactors. We demonstrate the long-term culture of mammalian (human foreskin fibroblasts, HFF) cells in a microbioreactor under constant perfusion in a straightforward simple manner. A perfusion system was used to culture human cells for more than two weeks in a plain microchannel (130 microm x 1 mm x 2 cm). At static conditions and at high flow rates (>0.3 ml h(-1)), the cells did not grow in the microchannel for more than a few days. For low flow rates (<0.2 ml h(-1)), the cells grew well and a confluent layer was obtained. We show that the culture of cells in microchannels under perfusion, even at low rates, affects cell growth kinetics as well as cell morphology. The oxygen level in the microchannel was evaluated using a mass transport model and the maximum cell density measured in the microchannel at steady state. The maximum shear stress, which corresponds to the maximum flow rate used for long term culture, was 20 mPa, which is significantly lower than the shear stress cells may endure under physiological conditions. The effect of channel size and cell type on long term cell culture were also examined and were found to be significant. The presented results demonstrate the importance of understanding the relationship between design parameters and cell behavior in microscale culture system, which vary from physiological and traditional culture conditions.     

Spatially selective reagent delivery into cancer cells using a two-layer microfluidic culture system

In this work, we demonstrate a two-layer microfluidic system capable of spatially selective delivery of drugs and other reagents under low shear stress. Loading occurs by hydrodynamically focusing a reagent stream over a particular region of the cell culture. The system consisted of a cell culture chamber and fluid flow channel, which were located in different layers to reduce shear stress on cells. Cells in the center of the culture chamber were exposed to parallel streams of laminar flow, which allowed fast changes to be made to the cellular environment. The shear force was reduced to 2.7 dyn cm⁻² in the two-layer device (vs. 6.0 dyn cm⁻² in a one-layer device). Cells in the side of the culture chamber were exposed to the side streams of buffer; the shear force was further reduced to a greater extent since the sides of the culture chamber were separated from the main fluid path. The channel shape and flow rate of the multiple streams were optimized for spatially controlled reagent delivery. The boundaries between streams were well controlled at a flow rate of 0.1 mL h⁻¹, which was optimized for all streams. We demonstrated multi-reagent delivery to different regions of the same culture well, as well as selective treatment of cancer cells with a built in control group in the same well. In the case of apoptosis induction using staurosporine, 10% of cells remained viable after 24 h of exposure. Cells in the same chamber, but not exposed to staurosporine, had a viability of 90%. This chip allows dynamic observation of cellular behavior immediately after drug delivery, as well as long-term drug treatment with the benefit of large cell numbers, device simplicity, and low shear stress.     

Rheology and microstructures formation of immiscible model polymer blends under steady state and transient flows

Viscoelastic properties of model immiscible blend were studied here under steady state condition at different initial conditions and transient flow conditions. The flow‐induced microstructure has been studied on these model blends. For this system, the elastic properties of the blend are mainly governed by the interface. Measurement of the dynamic modulus and of the first normal stress difference, both reflecting this enhanced elasticity, have been used to prove the blend morphology. The dynamic moduli after cessation of shear flow, the mean diameter of the disperse phase as generated by the shear flow, have been calculated using the model of Palierne. A procedure based on a direct fitting of the dynamic moduli with the model is compared with the one that uses a weight relaxation spectrum. On the other hand, the steady state normal stress data have been related to the morphology of the blend by means of Doi and Ohta model. The specific interfacial area is found to be inversely proportional to the ratio of interfacial tension over shear stress for the blend. The flow behavior during transient shear flow was also discussed. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 3519–3533, 2005     

A microfluidic system to study cytoadhesion of Plasmodium falciparum infected erythrocytes to primary brain microvascularendothelial cells

The cellular events leading to severe and complicated malaria in some Plasmodium falciparum infections are poorly understood. Additional tools are required to better understand the pathogenesis of this disease. In this technical report, we describe a microfluidic culture system and image processing algorithms that were developed to observe cytoadhesion interactions of P. falciparum parasitized erythrocytes rolling on primary brain microvascularendothelial cells. We isolated and cultured human primary microvascular brain endothelial cells in a closed loop microfluidic culture system where a peristaltic pump and media reservoirs were integrated onto a microscope stage insert. We developed image processing methods to enhance contrast of rolling parasitized erythrocytes on endothelial cells and to estimate the local wall shear stress. The velocity of parasitized erythrocytes rolling on primary brain microvascularendothelial cells was then measured under physiologically relevant wall shear stresses. Finally, we deployed this method successfully at a field site in Blantyre, Malawi. The method is a promising new tool for the investigation of the pathogenesis of severe malaria.     

A study of the static yield stress in a binary Lennard-Jones glass

The stress-strain relations and the yield behavior of a model glass (a 80:20 binary Lennard-Jones mixture) is studied by means of molecular dynamics simulations. In a previous paper it was shown that, at temperatures below the glass transition temperature, Tg, the model exhibits shear banding under imposed shear. It was also suggested that this behavior is closely related to the existence of a (static) yield stress (under applied stress, the system does not flow until the stress sigma exceeds a threshold value sigmay). A thorough analysis of the static yield stress is presented via simulations under imposed stress. Furthermore, using steady shear simulations, the effect of physical aging, shear rate and temperature on the stress-strain relation is investigated. In particular, we find that the stress at the yield point (the "peak"-value of the stress-strain curve) exhibits a logarithmic dependence both on the imposed shear rate and on the "age" of the system in qualitative agreement with experiments on amorphous polymers, and on metallic glasses. In addition to the very observation of the yield stress which is an important feature seen in experiments on complex systems like pastes, dense colloidal suspensions and foams, further links between our model and soft glassy materials are found. An example is the existence of hysteresis loops in the system response to a varying imposed stress. Finally, we measure the static yield stress for our model and study its dependence on temperature. We find that for temperatures far below the mode coupling critical temperature of the model (Tc = 0.435 in Lennard-Jones units), sigmay decreases slowly upon heating followed by a stronger decrease as Tc is approached. We discuss the reliability of results on the static yield stress and give a criterion for its validity in terms of the time scales relevant to the problem.     

Simultaneous optical observation of anisotropic texture for a concentrated solution of poly(γ-benzyl-l-glutamate) under transient stress response in shear flow

Simultaneous optical observation of anisotropic texture was performed under transient stress response in shear flow for a concentrated solution of poly(γ-benzyl-l-glutamate) (PBLG). Transient stress of the PBLG solution immediately after the onset of the shear flow showed a remarkable stress overshoot and damping oscillation behavior, followed by a steady state. On the other hand, simultaneous observation of the polarized optical microscopy showed the remarkable distortion of the banded texture immediately after the onset of the shear flow and the periodical changes in the retardation followed by a steady texture of the PBLG solution. The remarkable stress overshoot and the damping oscillation are closely related to the distortion of the banded texture and to the changes in the orientation of the rod-like molecules of the PBLG, respectively. Received: 15 December 1999/Accepted: 12 July 2000     

Flow mechanisms in the face-centred cubic micellar gel of a diblock copolymer

The mechanisms of flow of a face-centred cubic micellar phase were investigated using small-angle X-ray scattering (SAXS) for samples under either steady or oscillatory shear in two different geometries: Couette cell and planar shear sandwich. The system studied was a gel formed by a poly(oxyethylene)–poly(oxypropylene) diblock copolymer in water. SAXS indicated that under steady shear in a Couette cell, flow occurs via sliding of hexagonal close-packed (hcp) layers with a close-packed [110] direction along the shear direction. Under oscillatory shear in the planar shear sandwich, coexistence between this orientation and one in which the hcp layers are rotated by 30° (and flow is in a [211] direction) was observed; however, when subject to oscillatory shear in the Couette cell, flow only occurred along a [110] direction. This observation of flow in a non-close-packed direction may be due to alignment induced by the walls of the shear sandwich. Received: 24 February 2001 Accepted: 21 March 2001     

Adhesion behaviors of human trophoblast cells by contact with endothelial cells

Although it is still not clear whether migratory trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by a mechanism of directional cell division/proliferation, this process involves the attachment and adhesion of trophoblasts to endothelial cells lining the blood vessel walls. This raises the possibility that the cell–cell contact with endothelial cells may regulate trophoblast cell adhesion behaviors according to the surrounding flow condition. To test this, the adhesion forces of early gestation human trophoblast cells (TCs) cultured on glass slides coated with type I rat collagen or cultured with human umbilical vein endothelial cells (HUVECs) were measured quantitatively using a micropipette aspiration technique. Then, the resistance of TCs co-cultured with HUVECs to flow-induced shear stress was assessed with a flow chamber technique. The results showed that the adhesion force of TCs to glass slides coated with collagen was positively correlated with the concentration of collagen. By contact with endothelial cells, the adhesion force and the resistance to shear stress for the TCs were significantly enhanced. The interdiction of integrin β1 interaction remarkably reduced the adhesion forces of TCs to endothelial cells, hence their resistance to shear stress. The results therefore suggest that the contacts of TCs with endothelial cells enhance the adhesion forces of human TCs, partially by regulating with the integrin β1 according to the flow condition (i.e., the shear stress) in such a way to prevent the TCs from being carried downstream by flowing blood.     

Shear-thickening flow of nanoparticle suspensions flocculated by polymer bridging

The steady-shear viscosity, dynamic viscoelasticity, and stress relaxation behavior were measured for suspensions of silica nanoparticles dispersed in aqueous solutions of poly(ethylene oxide) (PEO). The suspensions of silica with diameters of 8-25 nm show striking shear-thickening profiles in steady shear and highly elastic responses under large strains in oscillatory shear. Since the silica particles are much smaller than the polymer coils, one molecule can extend through several particles by intrachain bridging. Each polymer coil may remain isolated as a floc unit and the silica particles hardly connect two flocs. Therefore, the flow of suspensions is Newtonian with low viscosity at low shear rates. When the polymer coils containing several nanoparticles are subjected to high shear fields, three-dimensional network is developed over the system. The shear-thickening flow may arise from the elastic forces of extended bridges. But, the polymer chain is easily detached from particle surface by thermal energy because of large curvature of particles. As a result, the network structures are reversibly broken down in a quiescent state and the suspensions behaves as viscoelastic fluids with the zero-shear viscosity.     

剪切作用下纳米粘土对LDPE熔体粘弹响应的影响

以LDPE/EVA/纳米粘土复合体系为研究模型,考察了剪切作用下,分散良好的纳米粘土对聚合物基体熔体稳态及瞬态粘弹响应的影响.发现剪切作用下,纳米粘土增加了聚合物熔体粘弹特性对剪切速率、剪切应变及剪切作用史的依赖性,改变了相应的依赖关系.稳态剪切时,纳米粘土的加入使体系第一法向应力差(N1)在低剪切速率区变为负值,而在高剪切速率区N1与粘土的含量无关;同时就瞬态剪切应力及N1的应变依赖关系而言,复合体系明显不同于聚合物基体;预剪切对聚合物基体瞬态粘弹响应几乎没有影响,而当纳米粘土的加入量大于3wt%后,与未经预剪切的样品相比较,经预剪切的复合体系的瞬态剪切应力值、应力过冲程度以及稳态剪切应力值均明显下降,且预剪切前后复合体系达到稳态时其瞬态剪切应力差值随纳米粘土含量的增高而线性增加.此外,纳米粘土的添加对聚合物熔体受剪切作用的非线性粘弹响应存在影响.复合体系熔体呈现特异非线性粘弹响应,其缘由被认为是由于纳米粘土在聚合物基体中剥离分散,或聚合物分子链插层于粘土片层间,形成局部有序结构,受剪切作用而排列取向.     

Microcirculation within grooved substrates regulates cell positioning and cell docking inside microfluidic channels

Immobilization of cells inside microfluidic devices is a promising approach for enabling studies related to drug screening and cell biology. Despite extensive studies in using grooved substrates for immobilizing cells inside channels, a systematic study of the effects of various parameters that influence cell docking and retention within grooved substrates has not been performed. We demonstrate using computational simulations that the fluid dynamic environment within microgrooves significantly varies with groove width, generating microcirculation areas in smaller microgrooves. Wall shear stress simulation predicted that shear stresses were in the opposite direction in smaller grooves (25 and 50 microm wide) in comparison to those in wider grooves (75 and 100 microm wide). To validate the simulations, cells were seeded within microfluidic devices, where microgrooves of different widths were aligned perpendicularly to the direction of the flow. Experimental results showed that, as predicted, the inversion of the local direction of shear stress within the smaller grooves resulted in alignment of cells on two opposite sides of the grooves under the same flow conditions. Also, the amplitude of shear stress within microgrooved channels significantly influenced cell retainment in the channels. Therefore, our studies suggest that microscale shear stresses greatly influence cellular docking, immobilization, and retention in fluidic systems and should be considered for the design of cell-based microdevices.     

Development of an osteoblast-based 3D continuous-perfusion microfluidic system for drug screening

In this work, we demonstrated that biological cells could be cultured in a continuous-perfusion glass microchip system for drug screening. We used mouse Col1a1GFP MC-3T3 E1 osteoblastic cells, which have a marker gene system expressing green fluorescent protein (GFP) under the control of osteoblast-specific promoters. With our microchip-based cell culture system, we realized automated long-term monitoring of cells and sampling of the culture supernatant system for osteoblast differentiation assay using a small number of cells. The system successfully monitored cells for 10 days. Under the 3D microchannel condition, shear stress (0.07 dyne/cm² at a flow rate of 0.2 μL/min) was applied to the cells and it enhanced the GFP expression and differentiation of the osteoblasts. Analysis of alkaline phosphatase (ALP), which is an enzyme marker of osteoblasts, supported the results of GFP expression. In the case of differentiation medium containing bone morphogenetic protein 2, we found that ALP activity in the culture supernatant was enhanced 10 times in the microchannel compared with the static condition in 48-well dishes. A combined system of a microchip and a cell-based sensor might allow us to monitor osteogenic differentiation easily, precisely, and noninvasively. Our system can be applied in high-throughput drug screening assay for discovering osteogenic compounds.     

Steady flow generation in microcirculatory systems

In this paper we explore the mechanical generation of steady-non pulsatile-flow in microfluidic systems. The rationale of the paper is inspired in the example of cardiovascular systems where at the microscale (i.e. capillaries) the flow is steady rather than pulsatile to optimize performance. We present a solution to the generation of steady flow in engineered microfluidic systems either in open or closed loop configurations via the use of disc pumps. The disc pump consists of a flat rotating disc and utilizes both viscous drag and centrifugal force to achieve pumping. Experiments using single loop and double loop microfluidic systems are presented to characterize the disc pump. Continuous flow generated by the disc pumps can be used to separate particles based on size using recirculating loops and for extraction of small particles without disturbing the concentration of bigger particles. The potential impact of this technology includes sample separation and extraction techniques into portable microfluidic labs-on-a-chip, and long term culture systems for cells in suspension.     

聚苯胺类电流变体的稳态剪切流动分析

对聚苯胺、及聚苯胺/聚丙烯酸盐复合粒子的稳态剪切流动行为进行了综合考察.结果表明, 在交流电场下,电流变体的剪切应力和剪切速率的关系符合Bingham流体形为.并对此现象进行了分析,提出用非理想塑性体的新模型来描述交流电场下聚苯胺类电流变体的稳态剪切流动.     

Normal stress and shear stress in a viscoelastic liquid under oscillatory shear flow

Normal stress and shear stress of concentrated polystyrene solutions in a chlorinated diphenyl were measured under steady flow and oscillatory shear flow in a Weissenberg rheogoniometer. The normal stress difference was observed to oscillate at double the frequency of the applied shear strain with amplitude proportional to the square of the applied amplitude, while the shear stress was found to oscillate at the same frequency with amplitude proportional to the applied amplitude. A theoretical relation between the displacement of the oscillatory normal stress difference from zero level and the dynamic modulus derived by Lodge and other investigators was confirmed experimentally, and the theoretical predictions of Coleman and Markovitz concerning the relation among steady-flow normal stress difference and dynamic modulus were also confirmed. However, the theoretical predictions of Lodge, of Spriggs, Huppler and Bird, and of Williams on the relation between the amplitude and phase of oscillatory normal stress and those of oscillatory shear stress did not agree with experimental results.     

Study on Non-Newtonian Behaviors of Lennard-Jones Fluids via Molecular Dynamics Simulations

Using nonequilibrium molecular dynamics simulations, we study the non-Newtonian rheological behaviors of a monoatomic fluid governed by the Lennard-Jones potential. Both steady Couette and oscillatory shear flows are investigated. Shear thinning and normal stress effects are observed in the steady Couette flow simulations. The radial distribution function is calculated at different shear rates to exhibit the change of the microscopic structure of molecules due to shear. We observe that for a larger shear rate the repulsion between molecules is more powerful while the attraction is weaker, and the above phenomena can also be confirmed by the analyses of the potential energy. By applying an oscillatory shear to the system, several findings are worth mentioning here:First, the phase difference between the shear stress and shear rate increases with the frequency. Second, the real part of complex viscosity first increases and then decreases while the imaginary part tends to increase monotonically, which results in the increase of the proportion of the imaginary part to the real part with the increasing frequency. Third, the ratio of the elastic modulus to the viscous modulus also increases with the frequency. These phenomena all indicate the appearance of viscoelasticity and the domination of elasticity over viscosity at high oscillation frequency for Lennard-Jones fluids.     

Transient behavior of entangled polymers at high shear rates

A new model is presented for describing the time-dependent flow of entangled polymer liquids at high shear rates. The results were obtained by extending the Doi and Edwards theory to include the effect of chain stretching. This nonlinear phenomenon is predicted to occur when the product of the shear rate and longitudinal relaxation time of the polymer exceeds one. If a constant-shear-rate flow is started under these conditions, it is shown that the shear stress and the normal stress are considerably larger than that predicted by the original reptation model. We also find that both of these stresses can pass through maxima before reaching a steady state and that the times required to reach these maxima are constants independent of the shear rate. In general the new model requires the numerical solution of coupled partial differential equations. However, at the highest shear rates where reptative relaxation is no longer important, an analytical solution for the stresses is found. The results obtained here are shown to agree well with experimental data and to be an improvement over a simpler model recently proposed.