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.     

Investigation of the Effective Action Distance Between Hematopoietic Stem/Progenitor Cells and Human Adipose-Derived Stem Cells During Their In Vitro Co-culture

The in vitro suitable action distance between umbilical cord blood-derived hematopoietic stem/progenitor cells and its feeder cell, human adipose-derived stem cells, during their co-culture, was investigated through a novel transwell co-culture protocol, in which the distance between the two culture chambers where each cell type is growing can be adjusted from 10 to 450 μm. The total cell number was determined with a hemacytometer, and the cell morphology was observed under an inverted microscope each day. After 7 days of co-culture, the fold-expansion, surface antigen expression of CD34(+) and CFU-GM assay of the hematopoietic mononuclear cells (MNCs) were analyzed. The results showed that there was an optimal communication distance at around 350 μm between both types of stem cells during their in vitro co-culture. By using this distance, the UCB-MNCs and CD34(+) cells were expanded by 15.1?±?0.2 and 5.0?±?0.1-fold, respectively. It can therefore be concluded that the optimal action distance between stem cells and their supportive cells, when cultured together for 7 days, is of around 350 μm.     

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.     

CELL DENSITY DEPENDENCE OF ULTRAVIOLET LIGHT ENHANCED REACTIVATION OF Herpes simplex TYPE I AND THE LARGE PLAQUE EFFECT IN C3H/10T1/2 MOUSE FIBROBLASTS

C3H/10T1/2 mouse fibroblasts were grown to different cell densities either by plating at low density and allowing different growth periods, or by plating at a series of increasing densities and allowing the same growth period. These plates were UV irradiated at 7.5 J/m2 or mock irradiated and 24 h later infected with UV-irradiated Herpes simplex type I virus which had been UV irradiated at 50 or 125 J/m2 or mock irradiated. The numbers and sizes of plaques were measured and these data used to calculate the extent of UV-enhanced host cell reactivation, the capacity enhancement, the large plaque effect (LPE) and the small plaque effect (SME). The influence of cell density on these phenomena was similar for both series of density experiments. Ultraviolet-enhanced host cell reactivation could be demonstrated only for cultures of lower density. The capacity of the cells for Herpes simplex type I virus decreased with cell density, but UV irradiated cells showed an increase in capacity with cell density. Plaque sizes decreased in all cases with cell density but the LPE and SPE were not significantly altered. The greatest variation in the above parameters occurred just as the cells were approaching confluence, where most host cell reactivation experiments are carried out. We conclude that the reproducibility of such experiments depends critically on cell density, a dependence which may be relevant to mechanistic interpretations of the UV-dependent phenomena.     

Cell-Cell Communication Between Fibroblast and 3T3-L1 Cells Under Co-culturing in Oxidative Stress Condition Induced by H<Subscript>2</Subscript>O<Subscript>2</Subscript>

The present study was carried out to understand the interaction between fibroblast and 3T3-L1 preadipocyte cells under H₂O₂-induced oxidative stress condition. H₂O₂ (40 μM) was added in co-culture and monoculture of fibroblast and 3T3-L1 cell. The cells in the lower well were harvested for analysis and the process was carried out for both cells. The cell growth, oxidative stress markers, and antioxidant enzymes were analyzed. Additionally, the mRNA expressions of caspase-3 and caspase-7 were selected for analysis of apoptotic pathways and TNF-α and NF-κB were analyzed for inflammatory pathways. The adipogenic marker such as adiponectin and PPAR-γ and collagen synthesis markers such as LOX and BMP-1 were analyzed in the co-culture of fibroblast and 3T3-L1 cells. Cell viability and antioxidant enzymes were significantly increased in the co-culture compared to the monoculture under stress condition. The apoptotic, inflammatory, adipogenic, and collagen-synthesized markers were significantly altered in H₂O₂-induced co-culture of fibroblast and 3T3-L1 cells when compared with the monoculture of H₂O₂-induced fibroblast and 3T3-L1 cells. In addition, the confocal microscopical investigation indicated that the co-culture of H₂O₂-induced 3T3-L1 and fibroblast cells increases collagen type I and type III expression. From our results, we suggested that co-culture of fat cell (3T3-L1) and fibroblast cells may influence/regulate each other and made the cells able to withstand against oxidative stress and aging. It is conceivable that the same mechanism might have been occurring from cell to cell while animals are stressed by various environmental conditions.     

Effects of vascular endothelial growth factor (VEGF) and chondroitin sulfate A on human monocytic THP-1 cell migration

Angiogenesis serves as a crucial factor in disease development and progression, such as cancer metastasis, and monocyte migration is one of the key steps for angiogenesis. Therapeutic modulation of angiogenesis is a promising new therapeutic avenue under investigation. In this study, effects of vascular endothelial growth factor (VEGF) and chondroitin sulfate A on monocyte migration were investigated. Human monocytic THP-1 cells were from Riken Cell Bank (Tsukuba, Japan) and vascular endothelial cells (VECs) were obtained from swine thoracic aorta. The migration experimental system was adapted from Falcon™ Cell Culture Inserts with pore sizes of 3 and 8 μm cultured endothelial cells or not on the insert polyethylene terephthalate (PET) membranes. Four VEGF concentrations (0, 10, 50 and 100 ng/ml) and three concentrations of chondroitin sulfate A (0, 1.25 and 5.0 mg/ml) were used to investigate their effects on THP-1 cell migration ability through PET membranes and VECs monolayer. The THP-1 cell migration was evaluated by counting the number of migrated cells related to the total number of cells under a microscope. We counted the migration cells every 1 h on a Tatai-type hemocytometer using an inverted microscope for total 7 h. For inserts with pore sizes of 3 and 8 μm, the THP-1 cell migration increased with VEGF concentrations; however, cell migration decreased with the chondroitin sulfate A concentration. Our results demonstrated that VEGF accelerated monocyte migration through endothelial monolayer and chondroitin sulfate A is an effective inhibitor of monocyte migration for angiogenesis.     

Study of bioadhesion on a flat plate with a yeast/glass model system

The attachment of microorganisms to a surface is a critical first step of biofilm fouling in membrane processes. The shear-induced detachment of baker's yeast in adhesive contact with a plane glass surface was thus experimentally studied, using a specially designed shear stress flow chamber. The yeast was marketed either as rod-shaped pellets (type I yeast) or as spherical pellets (type II yeast). A complete series of experiments for measuring the shear stress necessary to detach a given proportion of individual yeast cells of type I or II was performed under different environmental conditions (ionic strength, contact time). In parallel, the surface physicochemical properties of the cells (surface charge, hydrophobicity, and electron donor and electron acceptor components) were determined. For the first type of yeast cells, which were rather hydrophilic, adhesion to the glass plate was weak. This was due to both electrostatic effects and hydrophilic repulsion. Furthermore, adhesion was not sensitive to any variation of the ionic strength. For yeast of the second type, adhesion was drastically increased. This could be explained by their physicochemical surface properties and especially their hydrophobic and electron acceptor components, which caused strong attractive van der Waals and Lewis acid-base interactions, counterbalancing the electrostatic repulsion. For increasing ionic strengths, adhesion was greater, due to lower electrostatic repulsion. The results were quantified through the definition of a critical wall shear stress ( tau w 50% ) required to detach 50% of the yeast cells initially deposited on the glass surface. The influence of the contact time was also evaluated and it was shown that, whatever the type of yeast, macromolecules such as proteins were released into the extracellular medium due to cell lysis and could contribute to the formation of a conditioning film. As a result, the cells were more strongly stuck to the glass plate.     

2,4,6‐trichlorophenyl acrylate emulsion‐templated porous polymers (PolyHIPEs). Morphology and reactivity studies

A series of monolithic crosslinked polymers with 2,4,6‐trichlorophenyl acrylate as a reactive component was prepared by free radical polymerization of the internal phases of high internal phase emulsions (HIPEs). The volume ratio of water to oil phase (void volume; 60–90%), crosslinker type (divinylbenzene or ethylenglycol dimethacrylate) and quantity (30–50 mol %) and type of porogenic solvent (chlorobenzene, toluene, chloroform, dichloroethane) were altered to study these effects on the structure and reactivity of the monolithic polymers. The polymer supports were characterized by scanning electron microscopy (SEM), FTIR spectroscopy, elemental analysis and mercury intrusion porosimetry. SEM images revealed an open cellular structure with voids between 1 and 12 μm and window sizes between 0.3 and 3 μm. The porogen had an influence on the surface area, being larger with added porogen and the influence being highest with toluene. Adding toluene also influenced the void size, increasing the average diameter from ～2 μm (no porogen) to ～12 μm (added toluene). Monolithic supports were functionalized by reaction of the ester moieties with tris(2‐aminoethyl)amine derivative and by hydrolysis of the ester groups to carboxylic acids. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 4043–4053, 2007     

Breast tumor cell detection at single cell resolution using an electrochemical impedance technique

Gold micro-electrodes with various diameters (25, 50, 75, 100 and 250 μm) were manufactured using standard micro-fabrication techniques and optimized for counting of MCF-7 cells (breast tumor cells) with single cell resolution. For specific cell capture, anti-EpCAM was immobilized on 11-mercaptoundecanoic acid (11-MUA)-3-mercaptopropionic acid (3-MPA) mixed self-assembled monolayer (SAM) modified gold surface of micro-electrodes. Electrodes were characterized using optical, cyclic voltammetry and electrochemical impedance spectroscopic (EIS) techniques. Cell capture response recorded using EIS suggested that optimum electrode dimensions should be analogous to desired cell size. For MCF-7 cells with an average diameter of 18 ± 2 μm, an electrode with 25 μm diameter was established as the optimum electrode size for precise single cell recognition and enumeration. In EIS investigation, the 25 μm electrode exhibited an impedance change of ~2.2 × 10(7) Ω in response to a single tumor cell captured on its surface. On the other hand other electrodes (250, 100, 75 and 50 μm) showed much less response for a single tumor cell. In future, the use of high density arrays of such electrodes with surface modifications will result in miniaturized lab on a chip devices for precise counting of MCF-7 cells with single cell resolution.     

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.     

剪切作用下PA1010/PP共混物的形态与性能研究

通过动态保压注射成型方法制备了聚酰胺1010/聚丙烯(PA1010/PP)共混物,并研究了形态与性能的关系.力学性能测试结果表明在熔体冷却过程中施加剪切可以大大提高共混物的拉伸强度、拉伸模量和缺口冲击强度,当PP的质量分数为20%时,共混物的缺口冲击强度达到21.3kJ/m2,是静态样的3倍多,拉伸强度达到50.9MPa,是静态样的1.5倍.扫描电镜(SEM)结果表明在动态保压样的横断面可以观察到剪切诱导的形态,中间是芯层,围绕着芯层的是剪切层,最外面是皮层,相区尺寸显著减小、分散相分散更趋均匀,特别是PP的质量分数为20%时,相区尺寸从原来的约3.9μm降低到约1.4μm.动态保压样机械性能的提高归因于剪切作用下独特相形态的形成,分子链沿流动方向的取向是拉伸强度提高的主要原因,而剪切使分散相颗粒变小和剪切层中分子链的取向是冲击强度提高的主要原因.     

Two-dimensional flow magnetophoresis of microparticles

A new two-dimensional micro-flow magnetophoresis device was constructed in a superconducting magnet (10 T) using triangular shaped pole pieces, which could apply a magnetic strength, B(dB/dx), in the range of ca. 0-14,000 T(2) m(-1) across a capillary cell. Polystyrene particles with diameters of 1, 3, and 6 μm were used as test samples in a paramagnetic medium of 1 M MnCl(2) to evaluate the performance of this method. Microparticles migrated across the capillary along the edge of the pole pieces, and then flowed through the gap in the pole piece at a position defined as the migration distance, depending on the magnetic susceptibility and the size of particles as well as the flow rate. The most effective flow rate to exhibit the largest resolution among the particles was theoretically predicted and experimentally confirmed. By this method, the magnetic susceptibilities of individual deoxygenated and non-deoxygenated red blood cells were measured from the relative migration distance.     

Effect of Cortisol on Calpains in the C2C12 and 3T3-L1 Cells

The present study was carried out to understand the effect of cortisol on calpain system in the C2C12 and 3T3-L1 adipocyte cells under co-culture system. Cells were co-cultured by using transwell inserts with a 0.4 μm porous membrane to separate C2C12 and 3T3-L1 preadipocyte cells. Each cell type was grown independently on the transwell plates. Following cell differentiation, inserts containing 3T3-L1 cells were transferred to C2C12 plates. Ten microgram per milliliter of cortisol was added to the medium. Following treatment for 3 days, the cells in the lower well were harvested for analysis. Calpains such as μ-calpain, m-calpain, and calpastatin were selected for the analysis. RT-PCR results indicated the significant increase in the mRNA expression of μ-calpain, m-calpain, and calpastatin. In addition, the confocal microscopical investigation indicated the cortisol treatment increases calpain expression in the C2C12 and 3T3-L1 cells. Taking all these together, cortisol treatment with co-culture system shows most reliable status of calpains expression in the cells, which is quite distinct from one-dimensional monocultured cells.     

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.     

Removal of PCR inhibitors using dielectrophoresis as a selective filter in a microsystem

Diagnostic PCR has been used to analyse a wide range of biological materials. Conventional PCR consists of several steps such as sample preparation, template purification, and PCR amplification. PCR is often inhibited by contamination of DNA templates. To increase the sensitivity of the PCR, the removal of PCR inhibitors in sample preparation steps is essential and several methods have been published. The methods are either chemical or based on filtering. Conventional ways of filtering include mechanical filters or washing e.g. by centrifugation. Another way of filtering is the use of electric fields. It has been shown that a cell will experience a force when an inhomogeneous electric field is applied. The effect is called dielectrophoresis (DEP). The resulting force depends on the difference between the internal properties of the cell and the surrounding fluid. DEP has been applied to manipulate cells in many microstructures. In this study, we used DEP as a selective filter for holding cells in a microsystem while the PCR inhibitors were flushed out of the system. Haemoglobin and heparin - natural components of blood - were selected as PCR inhibitors, since the inhibitory effects of these components to PCR have been well documented. The usefulness of DEP in a microsystem to withhold baker's yeast (Saccharomyces cerevisiae) cells while the PCR inhibitors haemoglobin and heparin are removed will be presented and factors that influence the effect of DEP in the microsystem will be discussed. This is the first time dielectrophoresis has been used as a selective filter for removing PCR inhibitors in a microsystem.     

ELECTRORHEOLOGICAL PROPERTIES OF DIATOMITE/SILICONE OIL SUSPENSIONS UNDER DC FIELDS

In this study, electrorheological （ER） behavior of suspensions prepared from 3.0 and 9.0 μm diatomite particulate, dispersed in insulating silicone oil （SO） medium was investigated. Sedimentation stabilities of suspensions （c = 5 wt%） prepared using these diatomite powders were determined to be 32 days （d = 3 μm） and 24 days （d = 9 μm）, respectively. ER activity of all the suspensions was observed to increase with increasing electric field strength, concentration and decreasing shear rate. Shear stress of diatomite suspensions increased linearly with increasing concentrations of the particles and with the applied electric field strength. Electric field viscosity of all the suspensions decreased sharply with increasing shear rate and particle size, showing a typical shear thinning non-Newtonian visco-elastic behavior. Effects of high temperature and polar promoter onto ER activity ofdiatomite/SO system were also investigated.     

Classification of cell types using a microfluidic device for mechanical and electrical measurement on single cells

This paper presents a microfluidic system for cell type classification using mechanical and electrical measurements on single cells. Cells are aspirated continuously through a constriction channel with cell elongations and impedance profiles measured simultaneously. The cell transit time through the constriction channel and the impedance amplitude ratio are quantified as cell's mechanical and electrical property indicators. The microfluidic device and measurement system were used to characterize osteoblasts (n=206) and osteocytes (n=217), revealing that osteoblasts, compared with osteocytes, have a larger cell elongation length (64.51 ± 14.98 μm vs. 39.78 ± 7.16 μm), a longer transit time (1.84 ± 1.48 s vs. 0.94 ± 1.07 s), and a higher impedance amplitude ratio (1.198 ± 0.071 vs. 1.099 ± 0.038). Pattern recognition using the neural network was applied to cell type classification, resulting in classification success rates of 69.8% (transit time alone), 85.3% (impedance amplitude ratio alone), and 93.7% (both transit time and impedance amplitude ratio as input to neural network) for osteoblasts and osteocytes. The system was also applied to test EMT6 (n=747) and EMT6/AR1.0 cells (n=770, EMT6 treated by doxorubicin) that have a comparable size distribution (cell elongation length: 51.47 ± 11.33 μm vs. 50.09 ± 9.70 μm). The effects of cell size on transit time and impedance amplitude ratio were investigated. Cell classification success rates were 51.3% (cell elongation alone), 57.5% (transit time alone), 59.6% (impedance amplitude ratio alone), and 70.2% (both transit time and impedance amplitude ratio). These preliminary results suggest that biomechanical and bioelectrical parameters, when used in combination, could provide a higher cell classification success rate than using electrical or mechanical parameter alone.     

Characterization of Enzymatically Induced Aggregation of Casein Micelles in Natural Concentration by in Situ Static Light Scattering and Ultra Low Shear Viscosimetry

The aggregation of casein micelles in undiluted skim milk after the addition of chymosin was studied by static light scattering and ultra low shear viscometry. The static light scattering measurements were made with two different sample thicknesses, 72 and 16 μm. The scattering data were analyzed by indirect Fourier transformation and by the polydispersity inversion technique which led to pair distance distribution functions and size distribution function, respectively. The minimum scattering angle was 1 degrees, which allows for the determination of particle sizes up to a maximum diameter of 12 μm. The fractal dimension determined from double logarithmic plots of intensity versus scattering vector resulted in values between 1.9 and 2.0. The influence of multiple scattering was determined by comparison of the measurements with the different sample thicknesses. The measurements show no significant influence of multiple scattering when the transmission is above 0.85. Due to the very complex and porous structure of the casein aggregates the Rayleigh-Debye-Gans scattering theory has been used in the data analysis. Measurements with a new instrument using ultra low shear showed good agreement with theory. Copyright 1999 Academic Press.