Interdigitated aluminium and titanium sensors for assessing epithelial barrier functionality by electric cell-substrate impedance spectroscopy (ECIS)

Electric cell-substrate impedance spectroscopy (ECIS) enables non-invasive and continuous read-out of electrical parameters of living tissue. The aim of the current study was to investigate the performance of interdigitated sensors with 50 μm electrode width and 50 μm inter-electrode distance made of gold, aluminium, and titanium for monitoring the barrier properties of epithelial cells in tissue culture. At first, the measurement performance of the photolithographic fabricated sensors was characterized by defined reference electrolytes. The sensors were used to monitor the electrical properties of two adherent epithelial barrier tissue models: renal proximal tubular LLC-PK1 cells, representing a normal functional transporting epithelium, and human cervical cancer-derived HeLa cells, forming non-transporting cancerous epithelial tissue. Then, the impedance spectra obtained were analysed by numerically fitting the parameters of the two different models to the measured impedance spectrum. Aluminium sensors proved to be as sensitive and consistent in repeated online-recordings for continuous cell growth and differentiation monitoring assensors made of gold, the standard electrode material. Titanium electrodes exhibited an elevated intrinsic ohmic resistance incomparison to gold reflecting its lower electric conductivity. Analysis of impedance spectra through applying models and numerical data fitting enabled the detailed investigation of the development and properties of a functional transporting epithelial tissue using either gold or aluminium sensors. The result of the data obtained, supports the consideration of aluminium and titanium sensor materials as potential alternatives to gold sensors for advanced application of ECIS spectroscopy

Impedimetric analysis of biological cell monolayers before and after exposure to nanosecond pulsed electric fields

Models and methods for the interpretation of impedance spectra for normal and cancer cells before and after electrical stimulation, focusing on nanosecond pulsed electric fields (nsPEFs), were investigated to describe salient features and their development that were observed in dedicated in situ experimental studies. For the first time a non-invasive, real-time and label-free method was established to explore temporal changes and their underlying physical processes of adherent cells for characteristics of cell-cell connections and the extracellular matrix.Modelle und Methoden zur Interpretation von Impedanzspektren für normale und Krebszellen vor und nach elektrischer Stimulation, mit dem Fokus auf Nanosekunden-gepulste elektrische Feldern, wurden untersucht, um herausragende Merkmale und deren Entwicklung zu beschreiben, die in speziellen In-situ-Experimenten beobachtet wurden. Zum ersten Mal wurde eine nicht-invasive, zeitnahe und markierungsfreie Methode entwickelt, um zeitliche Veränderungen und diesen zugrunde liegenden physikalischen Prozessen hinsichtlich der Eigenschaften von Zell-Zell-Verbindungen und der extrazellulären Matrix zu untersuchen

Wideband bioimpedance meter with the adaptive selection of frequency grid

Широкосмуговий вимірювач біоімпедансу з адаптивним вибором сітки частот. Для діагностики функціонального стану і структури біооб'єктів з слабко вираженими неоднорідностями важливо швидко та точно визначати амплітудно- і фазочастотну характеристики. Для забезпечення цього в приладі використовується синтезатор частоти, амплітудно-фазовий детектор в інтегральному виконанні, а також активні електроди, за допомогою яких джерело струму і вхідні буферні підсилювачі з малою вхідною ємністю розташовуються в безпосередній близькості до досліджуваного біооб’єкту. Це дозволяє проводити вимірювання на частотах до 5 МГц з похибкою вимірювання не більше 5 % по модулю імпедансу і не більше 2-х градусів за фазою.Introduction. For the diagnosis of functional state and structure of biological objects with weakly expressed irregularities it is important quickly and accurately to determine the amplitude- and phase-frequency characteristics. Therefore, the purpose of the article is a representation of the results of the development of biological objects high-speed impedance meter with the ability to select adaptive grid measuring frequencies in the extended band. Structure of the impedance meter. Developed instrument is designed to measure the impedance of the object on fourelectrode method. The device uses a frequency synthesizer amplitude-phase detector integrally fabricated and active electrodes, by which the voltage controlled current source and the input buffer amplifiers with low input capacitance, are located in close proximity to the studied bioobject. This allowed to make measurements at frequencies up to 5 MHz. Instruments characteristics. To test the device characteristics the frequency characteris-tics of the test object (RC-chain) impedance were measured. It is composed of 5 precision resistors and capacitors. Parameters of the elements were measured preliminarily by labora-tory inductance, capacitance and resistance meter E7-12. The dependence of the measure-ment errors of the developed device in the frequency range from 1 kHz to 5 MHz is not more than 5% of the modulus of the impedance and not more than 2° of the phase.Для диагностики функционального состояния и структуры биообъектов с слабо выраженными неоднородностями важно быстро и точно определять амплитудно- и фазочастотную характеристики. Для обеспечения этого в приборе используется синтезатор частоты, амплитудно-фазовый детектор в интегральном исполнении, а также активные электроды, с помощью которых источник тока управляемый напряжением и входные буферные усилители с малой входной ѐмкостью располагаются в непосредственной близости к исследуемому биообъекту. Такие решения позволили производить измерения на частотах до 5 МГц с погрешностью не более 5% по модулю импеданса и не более 2-х градусов – по фазе

Real-time bioimpedance measurements of stem cellbased disease models-on-a-chip

In vitro disease models are powerful platforms for the development of drugs and novel therapies. Stem-cell based approaches have emerged as cutting-edge tools in disease modelling, allowing for deeper insights into previously unknown disease mechanisms. Hence the significant role of these disease-in-a-dish methods in therapeutics and translational medicine. Impedance sensing is a non-invasive, quantitative technique that can monitor changes in cellular behaviour and morphology in real-time. Bioimpedance measurements can be used to characterize and evaluate the establishment of a valid disease model, without the need for invasive end-point biochemical assays. In this work, two stem cell-based disease models-on-a-chip are proposed for acute liver failure (ALF) and age-related macular degeneration (AMD). The ALF disease model-on-a-chip integrates impedance sensing with the highly-differentiated HepaRG cell line to monitor in real-time quantitative and dynamic response to various hepatotoxins. Bioimpedance analysis and modelling has revealed an unknown mechanism of paracetamol hepatotoxicity; a temporal, dose-dependent disruption of tight junctions (TJs) and cell-substrate adhesion. This disruption has been validated using ultrastructural imaging and immunostaining of the TJ-associated protein ZO-1. Age-related macular degeneration (AMD) is the leading cause of blindness in the developed world with a need for disease models for its currently incurable forms. Human induced pluripotent stem cells (hiPSCs) technology offers a novel approach for disease modelling, with the potential to impact translational retinal research and therapy. Recent developments enable the generation of Retinal Pigment Epithelial cells from patients (hiPSC-RPE), thus allowing for human retinal disease in vitro studies with great clinical and physiological relevance. In the current study, the development of a tissue-on- a-chip AMD disease model has been established using RPE generated from a patient with an inherited macular degeneration (case cell line) and from a healthy sibling (control cell line). A reproducible Electric Cell-substrate Impedance Sensing (ECIS) electrical wounding assay was conducted to mimic RPE damage in AMD. First, a robust and reproducible real-time quantitative monitoring over a 25-day period demonstrated the establishment and maturation of RPE layers on microelectrodes. A spatially-controlled RPE layer damage that mimicked cell loss in AMD was then initiated. Post recovery, significant differences in migration rates were found between case and control cell lines. Data analysis and modelling suggested this was due to the lower cell-substrate adhesion of the control cell line. These findings were confirmed using cell adhesion biochemical assays. Moreover, different-sized, individually-addressed square microelectrode arrays with high spatial resolution were designed and fabricated in-house. ECIS wounding assays were performed on these chips to study immortalized RPE migration. Migration rates comparable to those obtained with ECIS circular microelectrodes were determined. The two proposed disease-models-on-a-chip were then used to explore the therapeutic potential of the antioxidant N-Acetyl-Cysteine (NAC) on hiPSC-RPE and HepaRG cell recovery. Addition of 10 mM NAC at the end of a 24h paracetamol challenge caused a slight increase in the measured impedance, suggesting partial cell recovery. On the other hand, no effect on case hiPSC-RPE migration has been observed. More experiments are needed to examine the effect of different NAC concentrations and incubation periods. The therapeutic potential of electrical stimulation has also been explored. A preliminary study to evaluate the effect of electrical stimulation on RPE migration has been conducted. An externally applied direct current electric field (DC EF) of 300 mV/mm was found to direct the migration of the immortalized RPE cell line (hTERT-RPE1) perpendicular to the EF. The cells were also observed to elongate and to realign their long axes perpendicular to the applied EF. The proposed tissue-on-a-chip disease models are powerful platforms for translational studies. The potential of such platforms has been demonstrated through revealing unknown effects of acetaminophen on the liver as well as providing deeper insights into the underlying mechanisms of macular degeneration. Combining stem cell technology with impedance sensing provides a high throughput platform for studying patient-specific diseases and evaluating potential therapies

Impact of substrate topology, chemical stimuli and Janus nanoparticles on cellular properties

Cellular behavior is influenced by many biochemical but also physical factors in the direct cellular environment. Thereby, cells not only react to external cues, the interaction between cells and their environment is also dependent on the properties of the cell itself. The endocytosis of nanoparticles for example depends on the intermolecular forces between plasma membrane and particle as well as on the mechanical properties of the membrane. In the first part of this thesis I focus on the interaction between inorganic Janus nanoparticles, a new type of nanomaterials, which possess amphiphilic properties, and model membranes. In coarse grain simulations it has been demostrated that incubation of membranes with these particles lead either to pore formation in the lipid bilayer or to tubulation and vesiculation by long-range attractive interaction between particles bound to the membrane. Conducting surface plasmon resonance spectroscopy experiments I show that the binding energy of the used inorganic Janus particles to a solid supported monolayer could be sufficient to induce tubulation of tension-free membranes but is to small to provide the energy necessary to form a vesicle. This result is confirmed by fluorescence microscopic examination of giant unilamellar vesicles serving as a model system for the plasmamembrane, which were treated with Janus particles. Vesicles incubated with Janus particles show inwards directed membrane tubes, while incubation of vesicles with isotropic control particles had no effect on the membrane or could be attributed to an osmotic gradient. However, uptake experiments into living cells and cytotoxicity assays show no obvious difference between spherical particles and Janus particles, which hints for a negligible contribution of nanoparticle-induced tubulation or vesiculation to cellular uptake of nanoparticles and cytotoxicity. On the one hand mechanical properties of the cell influence the interaction between the cell and its environment. On the other hand, mechanical properties of cells change in response to environmental cues. Therefore, in the next part, atomic force microscopy-based microrheology is used to measure frequency-dependent mechanical properties of cells in different conditions. Fixation of cells with different chemical fixatives and transformation of epithelial cells to mesenchymal cells lead to more solid-like mechanical properties, while interaction with the actin cytoskeleton lead to more fluid-like properties. A comparison between malignant cells and non-malignant cells shows that malignant cells are more fluid-like compared to their non-malignant counterparts. Furthermore, the influence of substrate topology on cellular mechanics and cytoskeletal arrangement is examined. Changing physical properties of the substrate such as stiffness or topography has been shown to affect plenty of cellular processes like migration, proliferation, morphology or differentiation. Here, I investigate the impact of porous substrates on cellular morphology, cytoskeletal organization and elasticity in the context of confluent epithelial monolayers. I found that cells eventually self-organize to match the geometry of the pore pattern and remodel their actin cytoskeleton to reinforce their adhesion zone. Cells fluidize with increasing pore size up to 2 µm but eventually become stiffer if grown on very large pores up to 5 µm. The adhesion of cells to substrates is further researched by application of metal-induced energy transfer fluorescence lifetime imaging, which is used for the first time for this purpose. The fluorescence lifetime of a fluorophore in proximity to a metal layer is a function of the distance between fluorophore and metal layer. Applying a quantitative model of this interaction facilitates locating the fluorophore with nanometer precision in the axial direction up to 200 nm above the metal layer. By staining of the plasmamembrane I was able to image to basal membrane of three different cell lines and follow spreading of cells with high axial resolution. The introduced method is not restricted to measurement of cell/substrate distance and can be used for applications, which necessitate axial nanometer resolution in a range up to 200 nm

Development and Evaluation of Biocompatible Engineered Nanoparticles for Use in Ophthalmology

The synthesis and design of biocompatible nanoparticles for targeted drug delivery and bioimaging requires knowledge of both their potential toxicity and their transport. For both practical and ethical reasons, evaluating exposure via cell studies is a logical precursor to in vivo tests. As a step towards clinical trials, this work extensively investigated the toxicity of gold nanoparticles (Au NPs) and carbon dot (CD) nanoparticles as a prelude to their in vivo application, focusing specifically on ocular cells. As a further step, it also evaluated their whole-body transport in mice. The research pursued two approaches in assessing the toxicity of engineered nanoparticles and the suitability of their use in targeted delivery and bioimaging applications: (1) In vitro (using retinal pigment epithelial, corneal, and lens epithelial cells (2) In vivo (mouse whole body studies). Part. 1. In the in vitro assessments of Part 1, the biocompatibilities of spherical, rod, and cubic shaped Au NPs were compared for different exposure concentrations. Spherical Au NPs were evaluated in particular detail, and a possible toxicity mechanism was proposed, based on the findings of a colorimetric assay, electrical impedance measurements, and confocal imaging analysis. The assay measured the activity of succinate hydrogenase, a mitochondrial enzyme, while electrical impedance spectroscopy quantified the strength of cell-cell and cell-substrate attachment, a proxy of viability. Finally, confocal imaging analysis verified that the NPs were internalized and confirmed the degree of their toxicity. Collectively, the data indicated that surface area concentration was the critical toxicity parameter. Subsequently, to create biocompatible Au NPs, a unique end-thiolation of hyaluronic acid was adapted to create homogenously coated Au NPs. The end-thiolated hyaluronate (HS-HA) coating not only improved the biocompatibility of the Au NPs but also enhanced the internalization rate of the larger Au NPs, which could not enter the cells otherwise. The first part of this research also studied the synthesis of biocompatible deep red-emissive CDs for bioimaging applications. For this purpose, a central-composite design response surface methodology (CCD-RSM) was utilized. A scalable isolation-free microwave pyrolysis method for synthesizing deep red-emissive nitrogen-doped carbon dots (nCDs) from citric acid and ethylenediamine was successfully developed and optimized. The formation of C‒N and the presence of pyrrolic N content proved to be keys to creating red-emissive nCDs. Confocal images demonstrated that the nanoparticles could enter healthy corneal, retinal, and lens epithelial ocular cells, as well as cancerous Chinese Hamster Ovary cells. Part 2. Building on the results of in-vitro testing of the engineered Au NPs and nCDs, in Part 2 we developed protocols for injecting both types of NPs in-vivo. Prior to any intravenous or intravitreal injections, a preliminary study tested the ability of Au NPs to cross the tight junctions between retinal pigment epithelial cells. Transwell® permeable supports were used to simulate the blood-retinal barrier (BRB). The results showed that 20 nm Au NPs successfully crossed the permeable supports covered with confluent retinal pigment epithelial cells. Based on this finding, both intravitreal and intravenous injections of nascent and HS-HA coated Au NPs were tested. The intravitreal injections caused retinal detachment, very probably due to the mechanical intrusion of the injection needle and the volume, albeit small, of the injected NPs. Far more significant and encouraging, intravenously injected coated and uncoated NPs successfully crossed the BRB. As a result of the intravenous injections, it was observed that both coated and uncoated Au NPs were able to cross the blood-retinal barrier. As expected, the numbers of HS-HA-coated Au NPs were significantly higher in specific parts of the retina that contain more CD44 expressing cells, which have cell surface receptors for internalizing HA. Finally, based on the confocal imaging analysis, the NP concentration in each retinal layer was quantified as a function of time, post-injection. The NPs reached the retina in less than 5 minutes and reached a maximum concentration within approximately 20 minutes. Due to the enhanced retention and permeability effect of NPs, 8.5% of the uncoated and 12.1% of the HA-coated NPs that reach the retina remained after 24 hours. Next, nCDs with and without the HA coating were injected subcutaneously into post-mortem mouse and porcine eye globes. Ex-vivo porcine eye images showed that intravitreally injected nCDs had effectively diffused through the vitreous to the cornea, and post-mortem whole-body mouse images also demonstrated that the nCDs are suitable for bioimaging, excitable in the NIR region with the sensitivity of 15%. Cumulatively, our observations indicate that HA coated NPs could potentially deliver other payloads such as DNAs, mRNAs, proteins, siRNAs, and drugs into the cells which overexpress CD44 receptors, for example, cancerous and inflammatory cells, thus providing a platform for targeted treatment and imaging of many severe vision-threatening diseases and degenerative conditions

Impedance-Based Analysis of the Cellular Response to Microparticles: Theory, Assay Development and Model Study

This thesis provides a model study on the information content of multimodal impedance-based assays to assess the impact of microscale particles on cell physiology of mammalian cells. In three main chapters, different approaches to this topic are presented and discussed: The first chapter focused on the simulation of several scenarios within cell-based assays. These simulations are all based on the so-called ECIS model, originally introduced by Giaever and Keese (1991), describing the impedance contribution of cell-covered gold-film electrodes. This theoretical part of the thesis should help to support the interpretation of impedance data. First, opening and closing of cell junctions (Rb) for different types of barrierforming cell layers were simulated and the accompanying changes in the complex impedance were extracted at various frequencies. The simulation data for some model epithelial and endothelial cell types showed that the relationship between resistance and barrier tightness may undergo inversion for frequencies above the cell-type specific threshold. Moreover, the influence of incomplete electrode coverage or inhomogeneity within the cell layer was studied systematically. For all experiments a good correlation between the simulated data and the experimental support was found. The aim of the second project was to establish a new opto-electrical assay to investigate the dye transfer via gap junctions into neighboring cells. The principle of this new assay was based on loading a selected cell population with Lucifer Yellow by in situ electroporation. The cell-type specific adjustment of the ac pulse parameters for a temporary permeabilization of the plasma membrane improved the incorporation of Lucifer Yellow into the cytoplasm without affecting NRK cell viability. The assay also required the optimization of the gold-film electrode layout which enabled the application of the ac pulses, the non-invasive impedance recordings before and after pulse application and the microscopic analysis of dye transfer from cells on the electrode into adjacent cells. The final electrode layout (8W4E-GJ) contained four “semi-elliptical” electrodes which were separated by a photopolymer-free gap to facilitate microscopic analysis without any interference from the red autofluorescence of the photopolymer. The development of an appropriate experimental protocol yielded on electroporation in Ca2+-free buffer and the application of two sequential ac pulses, as it was found to enhance the uptake efficiency into primary-loaded NRK cells. The opto-electrical assay was successfully applied to analyze the effect of the well-known gap junctional intercellular communication inhibitor 2-APB. The analysis of dye transfer via gap junctions was based on confocal fluorescence micrographs documenting dye transfer from the electrode into the photopolymer-free gap. The analysis was further improved by the application of the red-fluorescent TRITC dextran as co-electroporated reference dye, which was trapped in the cytoplasm of primary-loaded cells due to its molecular size. The image analysis of the position-dependent intensities of both dyes (TRITC dextran and Lucifer Yellow) allowed a quantification of gap junctional intercellular communication. The third chapter contains all sub-projects dealing with a multimodal and label-free analysis of the impact of micrometer-sized silica particles (Ø = 2 μm) on vitality, migration, proliferation and gap junctional intercellular communication of adherent NRK cells in vitro. A sequence of different impedimetric assays, all based on the well-established ECIS technique, was applied for the analysis of particle impact on cell physiology. Microscopic studies addressing the particle uptake revealed the presence of membrane-coated particles in the cytoplasm of NRK cells. Further evidence for particle uptake was gathered from ToFSIMS analysis that showed a densely-packed particle distribution around the cell nucleus in cells with intact plasma membranes. Time-resolved ECIS measurements revealed no acute cytotoxicity of silica particles as well as no influence on cell migration. Furthermore, the influence of silica particles on NRK proliferation was studied impedimetrically. No differences in the time-course of proliferation were found for particle-loaded or control cells. To study the influence of internalized particles on gap junctional intercellular communication the new optoelectrical assay was applied. Dye transfer to NRK cells in the periphery of the electrode was insignificantly different in absence and presence of silica particles. The results were supported by classical techniques, like FRAP analysis, scrape loading or parachute assay. Superior to other assays, the developed opto-electrical assay allowed for analysis of cell adhesion and cellular response to the presence of particle during an exposure time of 24 h prior to the dye transfer study. This enables the investigation of the impact of internalized particles on different cell-related parameters like viability, motility and gap junctional intercellular communication within one cell population

Impedance analysis of endothelial cells undergoing orbital shear stress.

Understanding the endothelium at the cellular level could further knowledge of the cardiovascular system as a whole and could therefore lead to advances in the prevention and treatment of cardiovascular disease. Electric cell-substrate impedance sensing is an in vitro technique that can be used for observing the behavior of endothelial cells in real-time using a fluid flow environment to simulate the circulatory system. This study examined the effect of fluid shear stress on human umbilical vein endothelial cells using electrochemical impedance spectroscopy (impedance sensing). Circuit models were fit to empirical data to measure cell layer resistance and capacitance changes, and to determine if data trends follow those of previously published findings. Information derived from transendothelial electrical resistance measurements, about changes in cell layer permeability when subjected to varying shear stress conditions within an orbiting circular well, was used to draw conclusions aided by microscopic images of the cells

Development of real-time cellular impedance analysis system

The cell impedance analysis technique is a label-free, non-invasive method, which simplifies sample preparation and allows applications requiring unmodified cell retrieval. However, traditional impedance measurement methods suffer from various problems (speed, bandwidth, accuracy) for extracting the cellular impedance information. This thesis proposes an improved system for extracting precise cellular impedance in real-time, with a wide bandwidth and satisfactory accuracy. The system hardware consists of five main parts: a microelectrode array (MEA), a stimulation circuit, a sensing circuit, a multi-function card and a computer. The development of system hardware is explored. Accordingly, a novel bioimpedance measurement method coined digital auto balancing bridge method, which is improved from the traditional analogue auto balancing bridge circuitry, is realized for real-time cellular impedance measurement. Two different digital bridge balancing algorithms are proposed and realized, which are based on least mean squares (LMS) algorithm and fast block LMS (FBLMS) algorithm for single- and multi-frequency measurements respectively. Details on their implementation in FPGA are discussed. The test results prove that the LMS-based algorithm is suitable for accelerating the measurement speed in single-frequency situation, whilst the FBLMS-based algorithm has advantages in stable convergence in multi-frequency applications. A novel algorithm, called the All Phase Fast Fourier Transform (APFFT), is applied for post-processing of bioimpedance measurement results. Compared with the classical FFT algorithm, the APFFT significantly reduces spectral leakage caused by truncation error. Compared to the traditional FFT and Digital Quadrature Demodulation (DQD) methods, the APFFT shows excellent performance for extracting accurate phase and amplitude in the frequency spectrum. Additionally, testing and evaluation of the realized system has been performed. The results show that our system achieved a satisfactory accuracy within a wide bandwidth, a fast measurement speed and a good repeatability. Furthermore, our system is compared with a commercial impedance analyzer (Agilent 4294A) in biological experiments. The results reveal that our system achieved a comparable accuracy to the commercial instrument in the biological experiments. Finally, conclusions are given and the future work is proposed

Microfluidic platform for impedance characterization of endothelial cells under fluid shear stress.

Endothelium dysfunction has been associated with many pathophysiological processes leading to cardiovascular diseases. Studying endothelium behavior is vital to understand the onset, prevention, and treatment of such diseases. Electrical impedance spectroscopy has been shown to provide a real-time in vitro evaluation of cell behavior including cell monolayer permeability. However, the majority of published work has been primarily with static cell culture models or macro-scale models that do not properly represent the physiological sizes, structures, and environmental conditions of human blood vessels. Within this dissertation, the design, fabrication, characterization, and application of a microfluidic impedance platform is presented for the in vitro characterization of HUVECs undergoing different hydrodynamic shear stress conditions (static, 2.5, 17.6 and 58.1 dyne/cm2). Electrodes diameters of 50, 100, and 200 µm were incorporated to monitor different subpopulations sizes of HUVECs. Initial characterization experiments with relevant biological solutions indicated that electrodes smaller than 50 µm in diameter suffered from significant interfacial impedance and were unsuitable for the sensing application. Impedance spectra (102-106 Hz) were collected for HUVECs at the different shear conditions for 14 hours. Equivalent circuit fits were implemented to derive the different electrical cell monolayer parameters including the trans-endothelial resistance, cell membrane capacitance, constant phase element, and the resistance of cell culture medium. Results confirmed that while the trans-endothelial resistance and cell membrane capacitance were suitable measurements for cell permeability and confluency respectively, the constant phase element did not identify any discernible cell behavior. Resistance of cell culture medium was strongly influenced by cell attachment and values should be extracted from control cell-free measurements. Initial trans-endothelial resistance measurements showed a shear magnitude dependent increase at the sudden onset of flow. This increase was greatest for the largest shear condition (58.1 dyne/cm2). After 14 hours of shear, trans-endothelial resistance measurements were largest for HUVECs sheared at 58.1 dyne/cm2 and lowest for the 17.6 dyne/cm2 shear condition and the difference showed to be statically significant (p \u3c0.05). Monitored HUVECs were stained for nuclei, F-actin and VE-cadherin. Quantification of immunofluorescence of VE-cadherin showed a similar trend to the extracted trans-endothelial resistance values. Immunofluorescence images of F-actin showed significant cytoskeleton remodeling of sheared HUVECs. While cells sheared at 17.6 dyne/cm2 aligned parallel to the direction of flow, HUVECs sheared at 58.1 dyne/cm2 were angled in the direction of flow and sometimes even perpendicular to flow direction