Microfluidic devices modulate tumor cell line susceptibility to NK cell recognition

This study aims to adoptively reduce the major histocompatibility complex class I (MHC-I) molecule surface expression of cancer cells by exposure to microfluid shear stress and a monoclonal antibody. A microfluidic system is developed and tumor cells are injected at different flow rates. The bottom surface of the microfluidic system is biofunctionalized with antibodies (W6/32) specific for the MHC-I molecules with a simple method based on microfluidic protocols. The antibodies promote binding between the bottom surface and the MHC-I molecules on the tumor cell membrane. The cells are injected at an optimized flow rate, then roll on the bottom surface and are subjected to shear stress. The stress is localized and enhanced on the part of the membrane where MHC-I proteins are expressed, since they stick to the antibodies of the system. The localized stress allows a stripping effect and consequent reduction of the MHC-I expression. It is shown that it is possible to specifically treat and recover eukaryotic cells without damaging the biological samples. MHC-I molecule expression on treated and control cell surfaces is measured on tumor and healthy cells. After the cell rolling treatment a clear reduction of MHC-I levels on the tumor cell membrane is observed, whereas no changes are observed on healthy cells (monocytes). The MHC-I reduction is investigated and the possibility that the developed system could induce a loss of these molecules from the tumor cell surface is addressed. The percentage of living tumor cells (viability) that remain after the treatment is measured. The changes induced by the microfluidic system are analyzed by fluorescence-activated cell sorting and confocal microscopy. Cytotoxicity tests show a relevant increased susceptibility of natural killer (NK) cells on microchip-treated tumor cells.     

Shear-induced force transmission in a multicomponent,multicell model of the endothelium

Haemodynamic forces applied at the apical surface of vascular endothelial cells (ECs) provide the mechanical signals at intracellular organelles and through the inter-connected cellular network. The objective of this study is to quantify the intracellular and intercellular stresses in a confluent vascular EC monolayer. A novel three-dimensional, multiscale and multicomponent model of focally adhered ECs is developed to account for the role of potential mechanosensors (glycocalyx layer, actin cortical layer, nucleus, cytoskeleton, focal adhesions (FAs) and adherens junctions (ADJs)) in mechanotransmission and EC deformation. The overriding issue addressed is the stress amplification in these regions, which may play a role in subcellular localization of mechanotransmission. The model predicts that the stresses are amplified 250–600-fold over apical values at ADJs and 175–200-fold at FAs for ECs exposed to a mean shear stress of 10 dyne cm⁻². Estimates of forces per molecule in the cell attachment points to the external cellular matrix and cell–cell adhesion points are of the order of 8 pN at FAs and as high as 3 pN at ADJs, suggesting that direct force-induced mechanotransmission by single molecules is possible in both. The maximum deformation of an EC in the monolayer is calculated as 400 nm in response to a mean shear stress of 1 Pa applied over the EC surface which is in accord with measurements. The model also predicts that the magnitude of the cell–cell junction inclination angle is independent of the cytoskeleton and glycocalyx. The inclination angle of the cell–cell junction is calculated to be 6.6° in an EC monolayer, which is somewhat below the measured value (9.9°) reported previously for ECs subjected to 1.6 Pa shear stress for 30 min. The present model is able, for the first time, to cross the boundaries between different length scales in order to provide a global view of potential locations of mechanotransmission.     

Fibrin-mediated endothelial cell adhesion to vascular biomaterials resists shear stress due to flow

In vitro endothelial cell (EC) seeding onto biomaterials for blood-contacting applications can improve the blood compatibility of materials. Adhesive proteins adsorbed from serum that is supplemented with the culture medium intercede the initial cell adhesion and subsequent spreading on material surface during culture. Nevertheless, physical and chemical properties of vascular biomaterial surface fluctuate widely between materials resulting in dissimilarity in protein adsorption characteristics. Thus, a variation is expected in cell adhesion, growth and the ability of cell to resist shear stress when tissue engineering on to vascular biomaterials is attempted. This study was carried out with an objective to determine the significance of a matrix coating on cell adhesion and shear stress resistance when cells are cultured on materials such as polytetrafluoroethylene (PTFE, Teflon) and polyethyleneterephthalate (Dacron), ultra high molecular weight polyethylene (UHMWPE) and titanium (Ti), that are used for prosthetic devices. The study illustrates the distinction of EC attachment and proliferation between uncoated and matrix-coated surfaces. The cell attachment and proliferation on uncoated UHMWPE and titanium surfaces were not significantly different from matrix-coated surfaces. However, shear stress resistance of the cells grown on composite coated surfaces appeared superior compared to the cells grown on uncoated surface. On uncoated vascular graft materials, the cell adhesion was not supported by serum alone and proliferation was scanty as compared to matrix-coated surface. Therefore, coating of implant devices with a composite of adhesive proteins and growth factors can improve EC attachment and resistance of the cells to the forces of flow.     

Integrating Flexible Electrochemical Sensor into Microfluidic Chip for Simulating and Monitoring Vascular Mechanotransduction

As an interface between the blood flow and vessel wall, endothelial cells (ECs) are exposed to hemodynamic forces, and the biochemical molecules released from ECs–blood flow interaction are important determinants of vascular homeostasis. Versatile microfluidic chips have been designed to simulate the biological and physiological parameters of the human vascular system, but in situ and real‐time monitoring of the mechanical force–triggered signals during vascular mechanotransduction still remains a significant challenge. Here, such challenge is fulfilled for the first time, by preparation of a flexible and stretchable electrochemical sensor and its incorporation into a microfluidic vascular chip. This allows simulating of in vivo physiological and biomechanical parameters of blood vessels, and simultaneously monitoring the mechanically induced biochemical signals in real time. Specifically, the cyclic circumferential stretch that is actually exerted on endothelium but is hard to reproduce in vitro is successfully recapitulated, and nitric oxide signals under normal blood pressure, as well as reactive oxygen species signals under hypertensive states, are well documented. Here, the first integration of a flexible electrochemical sensor into a microfluidic chip is reported, therefore paving a way to evaluate in vitro organs by built‐in flexible sensors.     

Experimental studies on the suitability of human mesothelial cells for seeding vascular prostheses: shear stress resistance in vitro

This investigation forms part of a study on the suitability of human omentum mesothelial cells (HOMES) as an alternative to endothelial cells (EC) for seeding vascular grafts. Isolated HOMES were grown in primary culture and characterized by their morphology (light microscopy and scanning electron microscopy (SEM)), as well as by fluorescence-activated cell sorting (FACS) and immunocytochemistry. The latter two methods showed cells which were positive for smooth muscle-type actin and cytokeratin, but negative for factor VIII-related antigen. HOMES were grown to confluence on glass with or without a fibronectin coating. Controlled shear stress was applied for up to 30 min using a plate and cone rheometer at 20 dynes/cm². These dynamic culture conditions led to loss of only occasional cells. The most marked alterations seen on SEM were some cell elongation, marked raising of the nucleus and loss of luminal cytoplasmic microvilli. Time-lapse video microscopy revealed that shear stress also increased the spreading capacity of some cells. Similar experiments with venous endothelial cells gave a shearing off of a confluent monolayer. This investigation shows the marked shear-stress resistance of HOMES, a pre-requisite for their use to seed vascular prostheses.     

Study of powder flow patterns in a Couette cell with axial flow using tracers and solid fraction measurements

We present different aspects of dense granular flows in a Couette geometry using a variety of particulate materials with shape and size distributions. Tracer studies point to an apparent coupling of particle size with flow and stress field gradients. While there is a clear industrial motivation to use “real” materials as a means to expand basic physical and engineering research in granular dynamics, the current study suggests additional academic motivations. Indeed, particles with distributed characteristics uncover rich interactions between flow and stress fields that might otherwise go un-noticed with model materials such as spherical glass beads. Distribution of size and shape play a strong role in how stress is transmitted in granular media (Kheiripour Langroudi et al. in Powder Technol 203:23–32, 2010) and how particle pattern arrangements evolve. Direct solid fraction measurements, using a capacitance probe, show that dense particle flows exhibit significant variations in solid fraction in both sheared and stagnant layers. Furthermore, these measurements also show different dependence of the solid fraction on shearing rate: solid fraction decreases in sheared layers and increases in stagnant layers as the shear rate is increased. From these results the thickness of the shear band could be estimated and was found to vary as a function of particle shape and the roughness of the container walls. The main result is that shear stress (or torque) (see also Kheiripour Langroudi et al. in Powder Technol 197:91–101, 2010) and solid fraction profiles depend on particle shape and whether or not an extra degree of freedom in their movement is provided so that the system can dilate under various shear states in the Couette cell. This extra degree of freedom is assured in the present experimental work by allowing a slight axial outflow from the Couette device while the driven shear fields are in the radial and tangential directions.     

Endothelial cell culture model for replication of physiological profiles of pressure, flow, stretch, and shear stress in vitro

The phenotype and function of vascular cells in vivo are influenced by complex mechanical signals generated by pulsatile hemodynamic loading. Physiologically relevant in vitro studies of vascular cells therefore require realistic environments where in vivo mechanical loading conditions can be accurately reproduced. To accomplish a realistic in vivo-like loading environment, we designed and fabricated an Endothelial Cell Culture Model (ECCM) to generate physiological pressure, stretch, and shear stress profiles associated with normal and pathological cardiac flow states. Cells within this system were cultured on a stretchable, thin (～500 μm) planar membrane within a rectangular flow channel and subject to constant fluid flow. Under pressure, the thin planar membrane assumed a concave shape, representing a segment of the blood vessel wall. Pulsatility was introduced using a programmable pneumatically controlled collapsible chamber. Human aortic endothelial cells (HAECs) were cultured within this system under normal conditions and compared to HAECs cultured under static and "flow only" (13 dyn/cm(2)) control conditions using microscopy. Cells cultured within the ECCM were larger than both controls and assumed an ellipsoidal shape. In contrast to static control control cells, ECCM-cultured cells exhibited alignment of cytoskeletal actin filaments and high and continuous expression levels of β-catenin indicating an in vivo-like phenotype. In conclusion, design, fabrication, testing, and validation of the ECCM for culture of ECs under realistic pressure, flow, strain, and shear loading seen in normal and pathological conditions was accomplished. The ECCM therefore is an enabling technology that allows for study of ECs under physiologically relevant biomechanical loading conditions in vitro.     

Double-shearing theory applied to instability and strain localization in granular materials

An account is given of the non-dilatant double-shearing theory of plane flow of granular materials, and it is shown that the theory may be formulated as a special form of hypoplasticity theory. It is shown that according to this theory, simple shearing flows may be supported by a time-independent stress field, but that this solution is unstable. An alternative solution in which the stress in time-dependent is also derived, and shear flow takes place under decreasing shear stress. The strain localization theory of Rudnicki and Rice is applied in conjunction with the double-shearing theory, and it is shown that the theory admits bifurcations in which shear bands form on planes that coincide with the shear plane. Similarly, in pure shear, there exists an unstable solution with time-independent stress, and a solution with time-dependent stress in which the compressive load falls as the deformation increases, and shear bands may form at surfaces on which, according to the Coulomb criterion, the critical shear stress is mobilized. The double-shearing theory for axially symmetric flow is summarized, and applied to compression of a circular cylinder. Again there is an unstable constant stress solution, a time-dependent stress solution in which the axial pressure decreases as the compression of the cylinder increases, and conical shear bands may form on conical surfaces on which the critical shear stress is mobilized.     

Influence of hydrodynamic conditions on quantitative cellular assays in microfluidic systems

This study demonstrates the importance of the hydrodynamic environment in microfluidic systems in quantitative cellular assays using live cells. Commonly applied flow conditions used in microfluidics were evaluated using the quantitative intracellular Ca2+ analysis of Chinese hamster ovary (CHO) cells as a model system. Above certain thresholds of shear stress, hydrodynamically induced intracellular Ca2+ fluxes were observed which mimic the responses induced by chemical stimuli, such as the agonist uridine 5'-triphosphate tris salt (UTP). This effect is of significance given the increasing application of microfluidic devices in high-throughput cellular analysis for biophysical applications and pharmacological screening.     

A novel strategy to graft RGD peptide on biomaterials surfaces for endothelization of small-diamater vascular grafts and tissue engineering blood vessel

To improve the performance of small-diamater vascular grafts, endothelization of biomaterials surfaces and tissue engineering are more promising strategies to fabricate small-diamater vascular grafts. In this study, a Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) peptide was grafted on the surfaces of poly(carbonate urethane)s (PCUs), with photoactive 4-benzoylbenzoic acid (BBA) by UV irradiation. The photoactive peptides (BBM-GRGDSP) were synthesized with classical active ester of peptide synthesis. The modified surfaces of PCU with the photoactive RGD peptides were characterized by water contact angle measurement and X-ray Photoelectron Spectroscopy (XPS), which results suggested that the peptides were successfully grafted on the PCU surfaces. The effect of these modified surfaces on endothelial cells (ECs) adhesion and proliferation was examined over 72 h. PCU surfaces coupled with the synthetic photoactive RGD peptides, as characterized with phase contrast microscope and the metabolic activity (MTT) assay enhanced ECs proliferation and spreading with increasing concentration of RGD peptides grafted on their surfaces. Increased retention of ECs was also observed on the polymers surfaces under flow shear stress conditions. The results demonstrated that GRGDSP peptides grafted on the surfaces of polymers with photoactive 4-benzoylbenzoic acids could be an efficient method of fabrication for artificial small-diamater blood vessels. The modified polymer is expected to be used for small-diamater vascular grafts and functional tissue engineered blood vessels to improve ECs adhesion and retention on the polymer surfaces under flow shear stress conditions.     

SHEAR-FLOW AND MATERIAL INSTABILITIES IN PARTICULATE SUSPENSIONS AND GRANULAR MEDIA

This article is a review of recent theoretical work on shear flow instabilities of particulate suspensions and dry granular medium. Attention is devoted largely to steady homogeneous unbounded simple shearing flows, as a generalization of the classical Kelvin problem for Newtonian fluids, with a view towards identifying material or constitutive instabilities arising from the coupling of stress to particulate concentration and temperature fields. After reviewing the most common constitutive models, a unified linear-stability treatment is given for suspensions and granular media, based on an assumed 'short-memory' response of stress and various fluxes to perturbations on materially steady and uniform base states. A two-dimensional stability analysis of inertialess suspension flow indicates the possibility of particle-depleted shear bands. A comprehensive three-dimensional analysis of rapid granular flow reveals transverse 'layering and spanwise 'corrugations' as possible modes of instability. The latter appear to result from a kind of material instability, although not the simple short-wavelength instability found for suspensions. Based on the current theoretical treatments, several new studies are recommended, including the effects of granular dilatancy and yield stress, three-dimensional disturbances in suspensions and the effects of gravity in granular now.     

TIRF-Rheometer for Measuring Protein Adsorption under High Shear Rates: Constructional and Fluid Dynamic Aspects

In total internal reflection fluorescence (TIRF) measurements an exponentially decaying evanescent wave of light (285–290 nm) excites the Trp residues in a protein to fluoresce at 350 nm when it is adsorbed to a transparent surface. A major problem in the measurement of protein adsorption kinetics in such systems is that the protein has to diffuse through a boundary layer to reach the surface. The thickness of such a boundary layer can be reduced by shearing the fluid phase. In the classical TIRF adsorption chamber only a unidirectional flow of buffer through the chamber is possible. In such a chamber the shear rates and shear stresses vary across the cross section and only low shear rates are obtainable. Therefore based on a rheological system for studying fluid shear stress on cultured cells a TIRF-chamber was constructed which allowed the installment of a rotating cone (max. rate: 1200 rpm) and plate viscometer-type variable shear device. In this case a flow field can be set up in which the shear rates and shear stresses are approximately constant. Cone angles (α) between 1.0–2.5 ° allowed shear rates (γ) between 0 and 7200 s⁻¹. The TIRF-rheometer can be employed in two different modes in the form of: (a) a closed system (no fluid flow through the rheometer chamber), (b) an open system with continuous buffer flow through the chamber. The flow conditions were checked by observing the dissolution of a small spot of dried Coomassie blue as a function of the shear rate and time. A significant secondary flow was found with all cone angles and was dependent on the square root of the shear rate. Ink injection studies demonstrate that mixing times in the chamber below two seconds are obtainable. The TIRF Rheometer thus provides a means for studying the shear dependence of the adsorption of blood proteins and the generation of thin to ultra thin boundary layers for the measurement of protein adsorption kinetics relevant to biomaterials.     

On-chip evaluation of shear stress effect on cytotoxicity of mesoporous silica nanoparticles

In this work, nanotoxicity in the bloodstream was modeled, and the cytotoxicity of sub-50 nm mesoporous silica nanoparticles to human endothelial cells was investigated under microfluidic flow conditions. Compared to traditional in vitro cytotoxicity assays performed under static conditions, unmodified mesoporous silica nanoparticles show higher and shear stress-dependent toxicity to endothelial cells under flow conditions. Interestingly, even under flow conditions, highly organo-modified mesoporous silica nanoparticles show no significant toxicity to endothelial cells. This paper clearly demonstrates that shear stress is an important factor to be considered in in vitro nanotoxicology assessments and provides a simple device for pursuing this consideration.     

Study of shear behavior of granular materials by 3D DEM simulation of the triaxial test in the membrane boundary condition

This paper studies the shear behavior of granular materials by using a three-dimensional (3D) discrete element method (DEM) simulation of the triaxial test. The experimental triaxial tests were conducted on glass beads samples for verification. DEM simulations of the triaxial test were carried out in the membrane boundary condition consisting of 37,989 membrane particles. A new method that divides the irregular sample shape into two parts of cones and parts of three-dimensional simplexes is used to follow the volume change of irregular deformation of samples. The free rotatable upper platen is considered during the shearing process, which influences the shear behavior of samples especially in the residual stage and formations of a single shear band or X-shape shear band. The confining pressures have been demonstrated to influence the rotation angle and angular velocity of the upper platen. Moreover, the timing of replacing a rigid wall boundary condition with the membrane boundary condition is investigated, which affects the porosity of samples before shearing and the mechanical strength. The DEM model in the membrane boundary condition reflects well the evolution of irregular sample deformation and shear band in the shearing process. From the perspective of micro structures, the normal force decreases and the tangential stress increases during the shearing stage. This study greatly improves the accuracy of DEM simulations of the triaxial test in the membrane boundary condition.     

SHEAR-FLOW AND MATERIAL INSTABILITIES IN PARTICULATE SUSPENSIONS AND GRANULAR MEDIA

ABSTRACT

This article is a review of recent theoretical work on shear flow instabilities of particulate suspensions and dry granular medium. Attention is devoted largely to steady homogeneous unbounded simple shearing flows, as a generalization of the classical Kelvin problem for Newtonian fluids, with a view towards identifying material or constitutive instabilities arising from the coupling of stress to particulate concentration and temperature fields. After reviewing the most common constitutive models, a unified linear-stability treatment is given for suspensions and granular media, based on an assumed ‘short-memory’ response of stress and various fluxes to perturbations on materially steady and uniform base states. A two-dimensional stability analysis of inertialess suspension flow indicates the possibility of particle-depleted shear bands. A comprehensive three-dimensional analysis of rapid granular flow reveals transverse ‘layering and spanwise ’corrugations’ as possible modes of instability. The latter appear to result from a kind of material instability, although not the simple short-wavelength instability found for suspensions. Based on the current theoretical treatments, several new studies are recommended, including the effects of granular dilatancy and yield stress, three-dimensional disturbances in suspensions and the effects of gravity in granular now.     

Flow Rate Affects Nanoparticle Uptake into Endothelial Cells

Nanoparticles are commonly administered through systemic injection, which exposes them to the dynamic environment of the bloodstream. Injected nanoparticles travel within the blood and experience a wide range of flow velocities that induce varying shear rates to the blood vessels. Endothelial cells line these vessels, and have been shown to uptake nanoparticles during circulation, but it is difficult to characterize the flow-dependence of this interaction in vivo. Here, a microfluidic system is developed to control the flow rates of nanoparticles as they interact with endothelial cells. Gold nanoparticle uptake into endothelial cells is quantified at varying flow rates, and it is found that increased flow rates lead to decreased nanoparticle uptake. Endothelial cells respond to increased flow shear with decreased ability to uptake the nanoparticles. If cells are sheared the same way, nanoparticle uptake decreases as their flow velocity increases. Modifying nanoparticle surfaces with endothelial-cell-binding ligands partially restores uptake to nonflow levels, suggesting that functionalizing nanoparticles to bind to endothelial cells enables nanoparticles to resist flow effects. In the future, this microfluidic system can be used to test other nanoparticle–endothelial cell interactions under flow. The results of these studies can guide the engineering of nanoparticles for in vivo medical applications.     

非圆形磁性颗粒对磁流变脂剪切应力的影响及其计算模型研究

磁流变脂是继磁流变液和磁流变弹性体之后,又一个具有巨大发展潜力的磁流变智能材料。参照作者提出的圆形颗粒的建模过程,建立了非圆形磁性颗粒磁流脂的剪切应力模型,并以六边形磁性颗粒磁流变脂为例,推导出六边形磁性颗粒磁流变脂剪切应力公式。该模型对半径和边长相同时剪切屈服应力与磁场强度的关系,体积相同时剪切屈服应力与磁场强度的关系,零场下的剪切力应力进行了模拟计算。结果表明同体积下的非圆形磁性颗粒磁流变脂的剪切应力比圆形磁性颗粒磁流变脂大,且随接触边长的减少剪切应力亦减少,但都比圆形磁性颗粒磁流变脂的剪切应力大。即使零场条件下,非圆形磁性颗粒磁流变脂的剪切应力比圆形磁性颗粒磁流变脂大,说明球形磁流变液不是最佳选择。所以,非圆形磁性颗粒的磁流变脂机理研究对高性能磁流变液制备及其应用具有极其重要的指导意义。     

Biomechanical regulation of vascular smooth muscle cell functions: from in vitro to in vivo understanding

Vascular smooth muscle cells (VSMCs) have critical functions in vascular diseases. Haemodynamic factors are important regulators of VSMC functions in vascular pathophysiology. VSMCs are physiologically active in the three-dimensional matrix and interact with the shear stress sensor of endothelial cells (ECs). The purpose of this review is to illustrate how haemodynamic factors regulate VSMC functions under two-dimensional conditions in vitro or three-dimensional co-culture conditions in vivo. Recent advances show that high shear stress induces VSMC apoptosis through endothelial-released nitric oxide and low shear stress upregulates VSMC proliferation and migration through platelet-derived growth factor released by ECs. This differential regulation emphasizes the need to construct more actual environments for future research on vascular diseases (such as atherosclerosis and hypertension) and cardiovascular tissue engineering.     

部分凝固合金的等温流变特性

文章研究了用同轴双筒流变仪测定在 Couette 剪切流动中Sn-15％Pb 合金部分凝固时的流交性质。结果表明:当固相分数接近极限固相分数时,半固态浆液具有宾汉流体的流变学特征:宾汉屈服现象和剪切应力与切变速率曲线线性化。这是由于固相颗粒形成了不连续网。而当浆液中固相分数较低时,它的流变性更象假塑性流体。研究发现:在一定的剪切条件下,固相颗粒不是彼此分离的,而是存在聚集。在等温剪切流动中固相颗粒群的形成与崩溃,对部分凝固合金的流变性质有重要影响。     

Ultra-Low-Powered Aqueous Shear Stress Sensors Based on Bulk EG-CNTs Integrated in Microfluidic Systems

Novel aqueous shear stress sensors based on bulk carbon nanotubes (CNTs) were developed by utilizing microelectricalmechanical system (MEMS) compatible fabrication technology. The sensors were fabricated on glass substrates by batch assembling electronics-grade CNTs (EG-CNTs) as sensing elements between microelectrode pairs using dielectrophoretic technique. Then, the CNT sensors were permanently integrated in glass–polydimethylsiloxane (PDMS) microfluidic channels by using standard glass–PDMS bonding process. Upon exposure to deionized (DI) water flow in the microchannel, the characteristics of the CNT sensors were investigated at room temperature under constant current (CC) mode. The specific electrical responses of the CNT sensors at different currents have been measured. It was found that the electrical resistance of the CNT sensors increased noticeably in response to the introduction of fluid shear stress when low activation current (≪1 mA) was used, and unexpectedly decreased when the current exceeded 5 mA. We have shown that the sensor could be activated using input currents as low as 100 $mu$A to measure the flow shear stress. The experimental results showed that the output resistance change could be plotted as a linear function of the shear stress to the one-third power. This result proved that the EG-CNT sensors can be operated as conventional thermal flow sensors but only require ultra-low activation power ($sim 1$ $mu$W), which is $sim 1000$ times lower than the conventional MEMS thermal flow sensors.