The effects of shear stress on isolated receptor-ligand interactions of Staphylococcus epidermidis and human plasma fibrinogen using molecularly patterned microfluidics

Staphylococcus epidermidis is an opportunistic pathogen that has been implicated in hospital-acquired infections, specifically related to implanted intravascular devices. S. epidermidis adhesion is a mechanism of colonization, leading to pathogenesis. Here we demonstrate an easily fabricated and robust parallel microfluidic platform to investigate the physiologically-relevant effects of fluid shear on S. epidermidis adhesion to human fibrinogen (hFg) with increased experimental throughput. In situ molecular patterning using fluid flow boundaries allows for isolation of the molecular interactions in highly defined shear stress environments, while keeping the device operation simple and reproducible. We characterize two modes of attachment of S. epidermidis to hFg coated surfaces. Single colonies adhere in high fractions at low shear stresses (~1 dyne cm(-2)) and adhesion decays with increasing shear. However, clusters of bacteria adhere the highest at median wall shear stress (up to 10 dyne cm(-2)), and adhesion subsequently decays above this critical shear stress. This initial characterization suggests a previously unobserved phenomenon of shear activated cell-cell adhesion in S. epidermidis, which acts to increase the overall attachment strength to hFg. Both of these modes of attachment are dependant upon the presence of intact hFg, indicating that adhesion is resultant from specific molecular recognition between the bacteria and human fibrinogen. This platform provides new insight into complex host-pathogen interactions, and will allow for further investigation of colonization and pathogenesis in more physiologically relevant conditions.     

Using a co-culture microsystem for cell migration under fluid shear stress

We have successfully developed a microsystem to co-cultivate two types of cells with a minimum defined gap of 50 μm, and to quantitatively study the impact of fluid shear stress on the mutual influence of cell migration velocity and distance. We used the hydrostatic pressure to seed two different cells, endothelial cells (ECs) and smooth muscle cells (SMCs), on opposite sides of various gap sizes (500 μm, 200 μm, 100 μm, and 50 μm). After cultivating the cells for 12 h and peeling the co-culture microchip from the culture dish, we studied the impacts of gap size on the migration of either cell type in the absence or presence of fluid shear stress (7 dyne cm(-2) and 12 dyne cm(-2)) influence. We found that both gap size and shear stress have profound influence on cell migration. Smaller gap sizes (100 μm and 50 μm) significantly enhanced cell migration, suggesting a requirement of an effective concentration of released factor(s) by either cell type in the gap region. Flow-induced shear stress delayed the migration onset of either cell type in a dose-dependent manner regardless of the gap size. Moreover, shear stress-induced decrease of cell migration becomes evident when the gap size was 500 μm. We have developed a co-culture microsystem for two kinds of cells and overcome the conventional difficulties in observation and mixed culture, and it would have more application for bio-manipulation and tissue repair engineering.     

A multi-shear perfusion bioreactor for investigating shear stress effects in endothelial cell constructs

Tissue engineering research is increasingly relying on the use of advanced cultivation technologies that provide rigorously-controlled cell microenvironments. Herein, we describe the features of a micro-fabricated Multi-Shear Perfusion Bioreactor (MSPB) designed to deliver up to six different levels of physiologically-relevant shear stresses (1-13 dyne cm(-2)) to six cell constructs simultaneously, during a single run. To attain a homogeneous fluid flow within each construct, flow-distributing nets photo-etched with a set of openings for fluid flow were placed up- and down-stream from each construct. Human umbilical vein endothelial cells (HUVECs) seeded in alginate scaffolds within the MSPB and subjected to three different levels of shear stress for 24 h, responded accordingly by expressing three different levels of the membranal marker Intercellular Adhesion Molecule 1 (ICAM-1) and the phosphorylated endothelial nitric oxide synthetase (eNOS). A longer period of cultivation, 17 d, under two different levels of shear stress resulted in different lengths of cell sprouts within the constructs. Collectively, the HUVEC behaviour within the different constructs confirms the feasibility of using the MSPB system for simultaneously imposing different shear stress levels, and for validating the flow regime in the bioreactor vessel as assessed by the computational fluid dynamic (CFD) model.     

A multichannel dampened flow system for studies on shear stress-mediated mechanotransduction

Shear stresses are powerful regulators of cellular function and potent mediators of the development of vascular disease. We have designed and optimized a system allowing the application of flow to cultured cells in a multichannel format. By using a multichannel peristaltic pump, flow can be driven continuously in the system for long-term studies in multiple isolated flow loops. A key component of the system is a dual-chamber pulse dampener that removes the pulsatility of the flow without the need for having an open system or elevated reservoir. We optimized the design parameters of the pulse dampening chambers for the maximum reduction in flow pulsation while minimizing the fluid needed for each isolated flow channel. Human umbilical vein endothelial cells (HUVECs) were exposed to steady and pulsatile shear stress using the system. We found that cells under steady flow had a marked increased production of eNOS and formation of actin stress fibers in comparison to those under pulsatile flow conditions. Overall, the results confirm the utility of the device as a practical means to apply shear stress to cultured cells in the multichannel format and provide steady, long term flow to microfluidic devices.     

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.     

Study of cell transmigration using a co-culture microsystem under shear stress

This study reports a novel cell co-culture technique using micro-molding in capillaries (MIMIC) technology that was utilized to observe the transmigration conditions of two types of cells with and without fluidic shear stress. Besides, the gap size of co-culture device could achieve shortest and not mixture. Endothelial cells (ECs) and smooth muscle cells (SMCs) were used in our experiment. In addition, concentrations of two cell are 8000 cells/μL (ECs) and 9000 cells/μL (SMCs), respectively, the shear stress is 7 dyne/cm₂, and the isolation distance between two types of cell are 50 and 200 μm. It is found that in the smaller culture space (50 μm) condition, ECs and SMCs would induce mutually, which would further make cell migration; in larger culture space (200 μm) condition, no inducing reaction took place between ECs and SMCs. It will have more advantages in bio-manipulation and tissue repair engineering.     

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.     

Crystal growth in syndiotactic poly(vinyl alcohol) hydrogels

Aqueous solutions of syndiotacticity-rich poly(vinyl alcohol) (s-PVA) form gels easily. The optimum condition of growth of the calcium tartrate crystal formed by diffusing calcium chloride into hydrogels containing tartaric acid was studied with use ofs- PVA of a syndiotacticity of 56 % and a degree of polymerization of 1460. The crystal grew in the gel of the concentrations of 2 % s-PVA and of 0.5 N tartaric acid at pH=4. The relation between the formation of Liesegang rings and shear modulus of a gel was studied by diffusing silver nitrate into gels containing potassium chromate. The distance between rings decreased with increasing shear modulus of a gel in the range from 670 to 7500 dyne/cm². The Liesegang rings were not formed for the shear modulus gel for 280 and 16200 dyne/cm₂.     

Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow

Development of an in vitro living cell-based model of the intestine that mimics the mechanical, structural, absorptive, transport and pathophysiological properties of the human gut along with its crucial microbial symbionts could accelerate pharmaceutical development, and potentially replace animal testing. Here, we describe a biomimetic 'human gut-on-a-chip' microdevice composed of two microfluidic channels separated by a porous flexible membrane coated with extracellular matrix (ECM) and lined by human intestinal epithelial (Caco-2) cells that mimics the complex structure and physiology of living intestine. The gut microenvironment is recreated by flowing fluid at a low rate (30 μL h(-1)) producing low shear stress (0.02 dyne cm(-2)) over the microchannels, and by exerting cyclic strain (10%; 0.15 Hz) that mimics physiological peristaltic motions. Under these conditions, a columnar epithelium develops that polarizes rapidly, spontaneously grows into folds that recapitulate the structure of intestinal villi, and forms a high integrity barrier to small molecules that better mimics whole intestine than cells in cultured in static Transwell models. In addition, a normal intestinal microbe (Lactobacillus rhamnosus GG) can be successfully co-cultured for extended periods (>1 week) on the luminal surface of the cultured epithelium without compromising epithelial cell viability, and this actually improves barrier function as previously observed in humans. Thus, this gut-on-a-chip recapitulates multiple dynamic physical and functional features of human intestine that are critical for its function within a controlled microfluidic environment that is amenable for transport, absorption, and toxicity studies, and hence it should have great value for drug testing as well as development of novel intestinal disease models.     

Patterning cells and shear flow conditions: convenient observation of endothelial cell remoulding, enhanced production of angiogenesis factors and drug response

We present a method that allows patterning cells and shear flow conditions for endothelial cell based assays. This method is novel in combining (1) cell culture on the surface of a substrate both topographically and chemically patterned; (2) multi-shear flow assays after covering the cell substrate with a microfluidic cover plate containing microchannels of different channel widths, and (3) conventional immunostaining assays after removal of the cover plate. This method has the advantage of performing cell cultures and immunoassays in standard cell biology environments with open access, facilitating the formation of confluent cell layers and the observation of cell responses to shear-flow and drug stimulations. To obtain multi-shear stress conditions, a single channel with stepwise increasing channel widths was patterned on the surfaces of both the substrate and the microfluidic cover plate. As results, we observed excellent viability of endothelial cells in the whole range of applied shear stresses (0-25 dyn cm(-2)) and shear stress dependent cytoskeleton remoulding, activation of von Willebrand factor (vWF), and re-organisation of angiogenesis factors such as tetra peptide acetyl-Ser-Asp-Lys-Pro (AcSDKP) of endothelial cells. To validate this approach for drug analysis, we also studied drug effects under shear stress conditions. Our results indicate that the drug effect of combretastatin A-4, an anti-tumour vascular targeting drug, could be significantly enhanced under shear flow conditions.     

A glucose biosensor based on chitosan-Prussian blue-multiwall carbon nanotubes-hollow PtCo nanochains formed by one-step electrodeposition

In this paper, a simple one-step electrodeposition method is described to fabricate chitosan-Prussian blue-multiwall carbon nanotubes-hollow PtCo nanochains (CS-PB-MWNTs-H-PtCo) film onto the gold electrode surface, then glucose oxidase (GOD) and Nafion were modified onto the film subsequently to fabricate a glucose biosensor. The morphologies and electrochemistry of the composite were investigated by using Fourier transform infrared (FTIR) spectrometry, scanning electron microscopy (SEM) and electrochemical techniques including cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), respectively. The performances of the biosensor have been investigated by chronoamperometry method under the optimized conditions. This biosensor showed a linear response to glucose range from 1.5 μM to 1.12 mM with a detection limit of 0.47 μM (S/N=3), a high sensitivity of 23.4 μA mM(-1) cm(-2), and a fast response time. The apparent Michaelis-Menten constant (K(M)(app)) was 1.89 mM. In addition, the biosensor also exhibited strong anti-interference ability, excellent stability and good reproducibility.     

An oxygen cathode operating in a physiological solution

We report the electroreduction of O(2) to water under physiological conditions (pH 7.4, 0.15 M NaCl, 37.5 degrees C) at a current density of 5 mA cm(-2) and at a potential only 0.18 V reducing versus that of the reversible O(2)/H(2)O electrode at pH 7.4. The immobilized electrocatalyst enabling the reduction is the electrostatic adduct of bilirubin oxidase from Myrothecium verrucaria, a polyanion at pH >4.1, and the polycationic redox copolymer of polyacrylamide and poly (N-vinylimidazole) complexed with [Os (4,4'-dichloro-2,2'-bipyridine)(2)Cl](+/2+), cross-linked on carbon cloth. The current density of the rotating electrodes was O(2) transport limited up to 8.8 mA cm(-2); their kinetic limit was reached at 9.1 mA cm(-2). The operational life of the electrodes depended on their angular velocity, which defined not only the current density but also the mechanical shear stress stripping the electrocatalyst. When the electrodes were rotated at 300 rpm and were poised at -256 mV versus the potential of the reversible O(2)/H(2)O electrode, their 2.4 mA cm(-2) initial current density decreased to 1.3 mA cm(-2) after 6 days of continuous operation at 37.5 degrees C.     

Size dependence of SiO2 particles enhanced glucose biosensor

A wide size range of SiO₂ particles were synthesized and were used as enzyme immobilization carriers to fabricate glucose biosensors. The size of the particles was in the range of 17-520 nm. These biosensors could be operated under physiological conditions (0.1 M phosphate buffer, pH 7.2). Particle size could affect the performance of SiO₂ modified glucose biosensors drastically. The smaller particles had higher performance. The smallest SiO₂ modified biosensor could work well in the glucose concentration range of 0.02-10 mM with a correlation coefficient of 0.9993. Its sensitivity was 2.08 μA/mM and the detection limit was 1.5 μM glucose.     

A glucose/oxygen enzymatic fuel cell based on redox polymer and enzyme immobilisation at highly-ordered macroporous gold electrodes

Highly ordered macroporous electrodes are prepared by electro-deposition of gold through a polystyrene sphere template. Drop-coating redox polymer and either glucose oxidase, for the anode, or Melanocarpus albomyces laccase, for the cathode on the macroporous gold provides film-coated electrodes for assembly of membrane-less glucose/oxygen enzymatic fuel cells (EFC) in pH 7.4 buffer containing 10 mM glucose and 0.15 M NaCl. Under these conditions the maximum power density of 17 μW cm(-2) for EFCs using films adsorbed to planar gold electrodes increased to 38 μW cm(-2) for films adsorbed to 2? sphere gold macroporous electrodes.     

Evaluation of UVA-Mediated Oxidative Damage to Proteins and Lipids in Extracorporeal Photoimmunotherapy

The combination of UVA and 8-methoxypsoralen (8-MOP) is known for the ability to produce reactive oxygen species (ROS) that react subsequently with DNA, lipids and proteins. In most studies concerned with UVA effects mediated by free radicals, UVA doses higher than those exhibiting beneficial clinical results in extracorporeal photoimmunotherapy (ECPI) were used. The present study was undertaken to determine markers of oxidative stress in plasma and cells from the buffy coat using conditions relevant for ECPI (cumulative UVA dose at the sample level < or = 2 J/cm2). Plasma exposed to UVA of 20 J/cm2 resulted in protein oxidation as well in crosslinking and fragmentation revealed by electrophoresis. Exposure of the buffy coat and plasma to considerably lower doses of UVA (up to 2 J/cm2) combined with various 8-MOP concentrations resulted neither in an increase of malondialdehyde as a marker of lipid peroxidation nor in a changed electrophoretic protein pattern. In these same experiments the total antioxidative capacity decreased to 65% of the initial value, suggesting that the antioxidative defense of plasma is able to cope with oxidative stress under ECPI conditions. These results were confirmed by data from 10 patients with scleroderma or cutaneous T-cell lymphoma during ECPI treatment. The present results suggest that, although ROS are formed during ECPI, gross oxidative damage does not occur. It is, however, possible, that specific effects mediated by oxygen radicals may co-trigger the photoimmunomodulatory effects of ECPI.     

A novel non-enzymatic glucose sensor modified with Fe2O3 nanowire arrays

Fe(2)O(3) was generally considered to be biologically and electrochemically inert, and its electrocatalytic functionality has been rarely realized directly in the past. In this work, Fe(2)O(3) nanowire arrays were synthesized and electrochemically characterized. The as prepared Fe(2)O(3) nanomaterial was proved to be an ideal electrode material due to the intrinsic peroxidase-like catalytic activity. The Fe(2)O(3) nanowire array modified glucose sensor exhibited excellent biocatalytic performance towards the oxidation of glucose with a response time of <6 s, a linear range between 0.015-8 mM, and sensitivity of 726.9 μA mM(-1)cm(-1). Additionally, a high sensing selectivity towards glucose oxidation in the presence of ascorbic acid (AA) and dopamine (DA) has also been obtained at their maximum physiological concentrations, which makes the Fe(2)O(3) nanomaterial promising for the development of effective electrochemical sensors for practical applications.     

The protective role of melatonin under heavy metal-induced stress in Melissa Officinalis L.

Heavy metals, due to their inability to degrade, pose a serious environmental and nutritional problem. The accumulation of essential and non-essential heavy metals in living organisms reduces normal growth and development, resulting in acute poisoning, disease and even death of organisms. Melatonin is a very important multifunctional molecule in protecting plants from oxidative stress due to its ability to directly neutralize reactive oxygen species (ROS). Also, melatonin has a chelating property, which may contribute in reducing metal-induced toxicity. In this paper, the protective role of melatonin in counteracting metal-induced free radical generation was highlighted. Using the HPLC-FLD technique melatonin was identified and quantified in the roots and leaves of lemon balm ( Melissa officinalis L.), grown under photoperiod conditions. Furthermore, the response of plants pre-treated with exogenous 0.1 mM melatonin to the increased zinc (Zn) and cadmium (Cd) concentrations was observed, with changes in mineral (Ca, Mg), physiological and antioxidant status of the plant during heavy metals stress. The obtained melatonin concentrations were the highest published for dry plants so far. Elevated Cd and Zn levels in soil caused alternation in biochemical and physiological parameters of lemon balm leaves and roots. However, melatonin pre-treatment increased plant tolerance to heavy metals stress. Increased Cd and Zn uptake and their translocation into the leaves were also improved, indicating the possible use of melatonin in phytoremediation.     

Interfacial tension of blends containing thermotropic liquid crystalline polymers

A method is proposed to determine the interfacial tension of immiscible blends containing a liquid crystalline polymer (LCP) and a flexible-molecule polymer, under flow conditions. The method is based on Taylor's theorem for immiscible fluids, i.e., that a suspended drop of liquid A in liquid matrix B is deformed in shear or elongational flow in proportion to the ratio of interfacial to viscous stresses. Taylor's theorem, as originally derived, applies to low concentrations, Newtonian fluids and small deformations. Thus, the theorem was modified to account for “Power Law” fluids in elongational flow and large deformations, more applicable to the system under investigation. The elongational viscosities of the LCP and the flexible polymer (polycarbonate) as a function of elongational rate were determined using converging type flow. The two polymers exhibited a Power-Law behavior in elongational flow and, hence, the experimental constitutive equations of state were used to quantify the viscous stresses. The interfacial stresses were modified for large deformations by taking into consideration the deformed shape and hence increased surface area of the elongated LCP particle. Using the modified expression, the interfacial tension of LCP and PC was determined to be in the range of 5–6.6 dyne/cm.     

Enhanced electrochemiluminescence from luminol at multi-walled carbon nanotubes decorated with palladium nanoparticles: a novel route for the fabrication of an oxygen sensor and a glucose biosensor

Incorporation of palladium nanoparticles on the surface of multi-walled carbon nanotubes and modification of glassy carbon electrode with the prepared nano-hybrid material led to the fabrication of a novel electrode. The modified electrode showed attractive electrocatalytic activity and sensitizing effect on luminol-O(2) and luminol-H(2)O(2) electrochemiluminescence (ECL) reactions at neutral media. The sensitized luminol-O(2) and luminol-H(2)O(2) reactions were successfully applied for the ECL determination of dissolved O(2) and glucose, respectively. Under the optimal conditions for luminol-O(2) system, the ECL signal intensity of luminol was linear with the concentration of dissolved oxygen in the range between 0.08 and 0.94 mM (r=0.9996) and for luminol-H(2)O(2) system, the ECL signal intensity of luminol was linear with the concentration of glucose in the range between 0.1 and 1000 μM (r=0.9998). The limits of detection (S/N=3) for dissolved oxygen and glucose were 0.02 mM and 54 nM, respectively. The relative standard deviations (RSD) for repetitive measurements of 0.50 mM oxygen (n=10) and 10 μM glucose (n=30) were 3.5% and 0.3%, respectively. Also, under the optimal conditions for luminol-H(2)O(2) system, the ECL signal intensity of luminol was linear with the concentration of H(2)O(2) in the range between 1 nM and 0.45 mM (r=0.9997). The limit of detection (S/N=3) for H(2)O(2) detection was 0.5 nM and the relative standard deviation for repetitive measurements of 10 μM H(2)O(2) (n=10) was 0.8%.     

Bioactive Compounds Extracted from Ecklonia cava by Using Enzymatic Hydrolysis Protects High Glucose-Induced Damage in INS-1 Pancreatic β-Cells

Pancreatic ??-cells are very sensitive to oxidative stress and this might play an important role in ??-cell death in diabetes. In the present study, we investigated whether the brown alga Ecklonia cava has protective effects against high glucose-induced damage in INS-1 pancreatic ??-cells. For that purpose, we prepared an enzymatic hydrolysate from E. cava (EHE) by using the carbohydrase, Celluclast. High-glucose (30?mM) treatment induced glucotoxicity, whereas EHE prevented cells from high glucose-induced damage then restoring cell viability was significantly increased. Furthermore, lipid peroxidation, intracellular reactive oxygen species (ROS) and nitric oxide (NO) were overproduced as the result of the treatment by high glucose; however, these lipid peroxidation, ROS and NO generations were effectively inhibited by addition of EHE in a dose-dependent manner. Moreover, EHE treatment increased activities of antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) in high glucose pretreated INS-1 pancreatic ??-cells. EHE slightly reduced the expression of pro-apoptotic protein Bax induced by high glucose but increased the expression of Bcl-2, an anti-apoptotic protein. These findings indicate that EHE might be used as potential nutraceutical agent which will protect the glucotoxicity caused by hyperglycemia-induced oxidative stress associated with diabetes.