Electric Cell-substrate Impedance Sensing System Research Articles

A novel “microbiota-host interaction model” to study the real-time effects of fermentation of non-digestible carbohydrate (NDCs) on gut barrier function

In this study, an in vitro co-culture model using an electric cell-substrate impedance sensing system (ECIS) for testing the impact of real-time fermentation of non-digestible carbohydrates (NDCs) by the intestinal microbiota on gut barrier function was established. We applied Lactobacillus plantarum WCFS1 as a model intestinal bacterium and alginate-pectin as immobilization polymers as well as a source of NDCs to determine the impact of pectin fermentation on the barrier function of T84 gut epithelial cells. In the first design, L. plantarum WCFS1 was encapsulated in an alginate capsule followed by embedding in an agar layer to mimic a firm mucus layer that might be present in the colon. In this experimental design, the presence of the agar layer interfered with the transepithelial electrical resistance (TEER) measurement of T84 cells. Subsequently, we removed the agar layer and used encapsulated bacteria in an alginate gel and found that the TEER measurement was adequate. The encapsulation of the L. plantarum WCFS1 does avoid direct contact with cells. Also, the encapsulation system allows higher amounts of packing densities of L. plantarum WCFS1 in a limited space which can limit the oxygen concentration within the capsule and therefore create anaerobic conditions. To test this design, T84 cells were co-incubated with L. plantarum alginate-capsules supplemented with graded loads of fermentable pectin (0, 4, and 8 mg/ml per capsule) to investigate the effect of pectin fermentation on gut barrier function. We observed that as the pectin content in the L. plantarum capsules increased, pectin showed a gradually stronger protective effect on the TEER of the gut epithelium. This could partly be explained by enhanced SCFA production as both lactate and acetate were enhanced in L. plantarum containing alginate capsules with 8 mg/ml pectin. Overall, this newly designed in vitro co-culture model allows for studying the impact of bacteria-derived fermentation products but also for studying the direct effects of NDCs on gut barrier function in a relatively high-throughput way.

Circulating Levels of Ang/Tie2 and VEGF-a Pathway Mediators Are Associated with Clinical Severity, Endothelial Barrier Disruption and Coagulation Activation in COVID-19

Background: the pathogenesis of severe COVID-19 involves the deregulated activation of different compartments of immunothrombosis, which are otherwise important for pathogen eradication and tissue repair. Coagulation activation, angiogenesis and alterations of endothelial barrier (EB) are elements of immunothrombosis that have been shown to be involved in the pathogenesis of COVID-19. Angiopoietins (Ang) 1 and 2 and their receptor Tie2 and VEGF-A are well-known pro-angiogenic mediators that, during inflammation also mediate EB disruption. Recently, it has also been demonstrated that the Ang/Tie2 pathway is involved in coagulation activation. Here we explored whether increased levels of angiogenesis/EB regulators (which have been previously associated with disease severity in COVID-19) are also associated with both EB disruption and coagulation activation in this condition. Methods: the study population consisted of 30 patients with COVID-19 confirmed by RT-PCR and presenting typical CT findings admitted due to hypoxemia. Thirty sex- and age-matched healthy individuals were recruited at the same time, from the same geographic region. Patients were part of a clinical trial (REBEC: U1111-1250-1843) but samples were obtained before any study intervention, within 24 hours from diagnosis confirmation. Circulating levels of angiogenesis/EB regulation mediators and coagulation biomarkers were measured by commercial assays (immunological or functional). Monolayers of endothelial cells from umbilical veins (HUVECs) or lung (HULECs) were used for measurement of EB integrity using an impedance sensor system (ECIS, Electric Cell-substrate Impedance Sensing System). Cells were stimulated with serum from patients or healthy individuals and EB integrity was continuously monitored for 36 hours. Clinical outcomes were obtained from the digital medical records. Results: mean length of hospital stay (LOS) was 12.9 ± 9.8 days. Twelve patients (40%) required intensive care (ICU) and 28/30 patients survived. Mean D-dimer was 3,609 ± 14,440 ng/mL. Circulating levels of Ang1, Ang2, sTie2 and VEGF-A were all significantly increased in patients compared to healthy individuals (Ang1: 463.2 ± 194.6 vs 237.4 ± 104.9 pg/mL, p<0.0001; Ang2: 1,926 (1,275 - 3,134) vs 1,215 (9 - 1,440 pg/mL), p<0.0001; Tie2: 10,753 ± 2,377 vs 8,603 ± 1,851 pg/mL, p<0.0001 and VEGF-A: 94.7 (73.4 - 116.0) vs 45.9 (39.7 - 57.0 pg/mL), p<0.0001.). In contrast, soluble VE-cadherin levels were decreased in patients compared to healthy individuals (1,234 ± 318 vs 1,539 ± 363 ng/mL, P=0.001). Serum from COVID-19 patients induced decreases of EB integrity in monolayers of both HUVECs and HULECs as early as 15 minutes, lasting up to 5 hours after stimulation (figure 1). The magnitude of EB disruption was correlated with clinically relevant outcomes such as time of ICU stay and LOS (figure 1). Interestingly, levels of Ang1, Ang2 and soluble VE-cadherin levels were also significantly correlated with the magnitude of EB disruption, as well as with biomarkers of coagulation activation such as fibrinogen, Von Willebrand Factor antigen levels, PAI-1, P-selectin and urokinase receptor (uPAR). Conclusions: Ang-1/Ang-2 mediated Tie2 signaling has been shown to be important for the fine regulation of barrier integrity and coagulation activation at the endothelial level, which are two critical elements of immunothrombosis. Our results provide evidence supporting that the interplay between these processes can play a role in the mechanisms driving COVID-19 severity, and suggest that targeting the Ang/Tie2 and VEGF-A pathways could be attractive strategies to modulate not only changes of the alveolar-capillary barrier, but also of coagulation activation in COVID-19.Figure 1. In (a), endothelial barrier (EB) integrity of HUVEC monolayers upon stimulation by serum from COVID-19 patients and healthy individuals (n=27-30 per group). The lower the normalized resistance, the higher the magnitude of EB disruption. Significant differences (* to ****) are evident from 15 min to 5 hours (Anova corrected for multiple comparisons). In the lower panels, the correlation of EB disruption with clinically relevant outcomes such as length of hospital stay (b) and days of intensive care (c) are shown. Negative correlations (Spearman test) indicate that the magnitude of EB disruption is associated with worse outcomes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cigarette or E‐cigarette content alters autophagy and permeability of lung endothelium

Cigarette smoking (CS) is the major cause of Chronic Obstructive Pulmonary Disease (COPD). E-cigarettes are considered by some as a safer alternative; however, inhalation of nicotine-containing e-cigarettes can also cause pathophysiologic changes, and “vaping” of some substances has led to severe lung damage. Our group has previously identified cortactin (CTTN), a central regulator of the actin cytoskeleton, as an important modulator of lung endothelial cell (EC) function. Our present study seeks to characterize the role of CTTN in lung endothelial dysfunction upon CS and e-cigarette challenge. Human lung EC were exposed to CS (40 µg/ml, 24 h) and cell lysates were subjected to Western blotting for autophagy markers p62 and LC3B-II. CS exposure increased both p62 and LC3B-II expression, suggesting autophagic flux inhibition. Further CTTN down-regulation by siRNA altered CS-induced p62 and LC3B-II expression. Excess mitochondrial ROS (mitoROS) production is associated with mitochondrial dysfunction and altered autophagy. Here, the mitoROS production in lung ECs was assessed using the MitoSOX assay. CS (40 µg/ml, 2 h) challenge significantly increased mitoROS production, which was potentiated by cortactin siRNA. Blocking mitoROS with Mito-TEMPO (10 µM, 3 h), a cell-permeable antioxidant, decreased mitochondrial superoxide production. We next assessed the role of Mito-TEMPO on E-cigarette-induced endothelial permeability. Lung EC exposure to E-cigarette extract (50 µg/ml) resulted in disruption of endothelial permeability, assessed by Electric Cell-substrate Impedance Sensing system (ECIS). Pretreatment with Mito-TEMPO provided barrier protection upon e-cigarette challenge. These results suggest a key role of mitoROS in e-cigarette-induced endothelial permeability. In conclusion, this study identified new mechanisms by which CS/e-cigarette exposure induces EC dysfunction and provides evidence for a previously undescribed role for CTTN in these responses.

A portable standalone wireless electric cell-substrate impedance sensing (ECIS) system for assessing dynamic behavior of mammalian cells

In this study, we have developed a wireless, portable, standalone, and simple electric cell-substrate impedance sensing (ECIS) system to analyze in-depth functional aspects of cellular functions on the surface of a co-planar metal electrode coated on conventional glass substrate using a low-cost circuitry and correlated it with an equivalent electrical circuit (EEC) model. Low-cost circuitry was used for studying the dynamic behavior of the mouse myoblast cells (C2C12) in a culture chamber. Further, the developed ECIS system was connected with 8-bit Arduino UNO microcontroller board for establishing a compact sized measuring unit, which can be placed inside a CO2 incubator to provide proper environmental condition for the biological cells during the entire measuring time. Integrating ZigBee RF module with the 8-bit Arduino UNO microcontroller board provides a wireless communication network. Theoretical calculation of the lumped-elemental electrical parameters associated with cell-electrolyte interface and metal-electrolyte interface was calculated. The calculation was performed by fitting the experimental impedance data to EEC model using least mean square method to determine the dynamic and vital functions of the mammalian cells such as proliferation (in real-time) with a change in intrinsic electrical parameters associated at any particular time point. Impedance measurements and the lumped-elemental electrical parameter were correlated with the respective microscopic images. The developed ECIS system was found to enable measuring of the extent of cellular proliferation over time. The compactness of the developed ECIS system integrated with the ZigBee RF module and the 8-bit Arduino UNO microcontroller board facilitates its utilization even when placed in the CO2 incubator for a prolonged time.

Using Electric Cell-Substrate Impedance Sensing (ECIS) as a novel method to evaluate bacterial urinary tract infections

Abstract Urinary tractinfections (UTIs) are commonly acquired bacterial infections that cause significant morbidity, worsened by the rise of antimicrobial resistance among uropathogenic bacteria. Understanding the dynamics of bacterial UTIs is essential to the proper use of antimicrobial therapies in patient treatment. In this study, Electric Cell-Substrate Impedance Sensing (ECIS) technology was used to monitor bacterial-mediated cytotoxicity in-vitro enabling evaluation of antibiotics. Using the ECIS system, real-time changes of barrier function were recorded for human bladder epithelial cells infected with either antibiotic sensitive or resistant bacteria. This study employed four common uropathogens: Pseudomonas aeruginosa, Escherichia coli, Enterococcus species, and Klebsiella pneumoniae. The effect of different antimicrobials, antibiotic titers, and organism concentrations were evaluated. The addition of an antibiotic helped confirm and differentiate sensitive and resistant bacterial strains. Antibiotic concentrations at sensitive and resistant breakpoints were not as effective at high organism concentrations but were completely effective at lower more clinically relevant bacterial loads. The data showed bacterial strains resistant to co-cultured antibiotic took longer on average to degrade the cell monolayer than sensitive strains. Overall, these results indicate that antibiotic resistance is often associated with a fitness cost. Our findings support the use of ECIS to augment routine susceptibility data allowing the direct phenotypic measurement of bacterial virulence and the effects of antibiotic treatment on these strains.

An evaluation of the impact of clinical bacterial isolates on epithelial cell monolayer integrity by the electric Cell-Substrate Impedance Sensing (ECIS) method

Virulence is the relative capacity of a pathogenic microorganism to cause damage in susceptible host cells such as those found in airway passages and the gut. In this study, the effect of clinical bacterial isolates on the monolayer integrity of cultured human alveolar basal epithelial cells (A549) was evaluated using the Electric Cell-Substrate Impedance Sensing (ECIS) system. ECIS is a morphological biosensor which records electrical properties of cell-covered microelectrodes in an AC circuit including impedance (ohm), resistance (ohm), and capacitance (μFarad). In the current study, fluctuations in the electrical properties of cell-covered microelectrodes reflect dynamic changes in cell morphology resulting from disrupted cell monolayers following exposure to bacteria. Using the ECIS system, real-time changes of cell morphology and disruption of monolayer integrity of cell-cultures in vitro were revealed for A549 cells infected with either Pseudomonas aeruginosa, ESBL Escherichia coli, Staphylococcus aureus (MRSA), or Enterococcus (VRE). We determined empirically that the optimal signal response was obtained for resistance (ohm) measurements at 4000 hertz. Following infection of A549 cells, the data revealed that Pseudomonas aeruginosa resulted in little change in microelectrode resistance (ohm @4 kHz) as compared to pathogen-free controls within the first 12 h. In contrast, E. coli, MRSA, and VRE caused significant changes in electrode resistance (ohm @4 kHz) values in the infected cells compared to controls over the first 5 h. Resistance (ohm @4 kHz) changes were also observed in cell monolayers infected with different bacterial concentrations for all isolates over 24 h. The highest concentration of bacteria caused the measured resistance (ohm @4 kHz) to drop faster than its' immediate lower concentration, suggesting a dose-dependent effect. Compared to live bacteria, cells exposed to heat-killed bacteria did not show significant changes in resistance (ohm @4 kHz) over 48 h post-exposure.Functionally, cytokine responses were different between cells treated with live and heat-killed bacteria. Of note, live bacteria induced IFNγ, IL-13, and IL-1β production in A549 cells, whereas heat-killed bacteria induced IL-8 production suggesting a differential interaction with cells that could reveal the underlying causes of resistance (ohm @4 kHz) changes. Our findings indicate that ECIS provides a means to quantify, automate, and measure bacterial virulence, which may have broader implications governing the course of treatment compared to traditional methods alone.

Fucosyltransferase 4 and 7 mediates adhesion of non-small cell lung cancer cells to brain-derived endothelial cells and results in modification of the blood\u2013brain-barrier: in vitro investigation of CD15 and CD15s in lung-to-brain metastasis

PurposeMetastatic non-small cell lung (NSCLC) cancer represents one of the most common types of brain metastasis. The mechanisms involved in how circulating cancer cells transmigrate into brain parenchyma are not fully understood. The aim of this work was to investigate the role of fucosylated carbohydrate epitopes CD15 and sialyated CD15s in cancer adhesion to brain-derived endothelial cells and determine their influence in blood–brain barrier (BBB) disruptionMethodsThree distinct, independent methods were used to measure brain endothelial integrity and include voltohmmeter (EVOM™), impedance spectroscopy (CellZscope®) and electric cell-substrate impedance sensing system (ECIS™). Two fucosyltransferases (FUT4 and 7) responsible for CD15 and CD15s synthesis were modulated in four human cancer cell lines (three lung cancer and one glioma).ResultsOverexpression of CD15 or CD15s epitopes led to increase in adhesion of cancer cells to cerebral endothelial cells compared with wild-type and cells with silenced CD15 or CD15s (p < 0.01). This overexpression led to the disruption of cerebral endothelial cell monolayers (p < 0.01). Knockdown of FUT4 and FUT7 in metastatic cancer cells prevented disruption of an in vitro BBB model. Surprisingly, although the cells characterised as ‘non-metastatic’, they became ‘metastatic’ -like when cells were forced to over-express either FUT4 or FUT7.ConclusionsResults from these studies suggest that overexpression of CD15 and CD15s could potentiate the transmigration of circulating NSCLC cells into the brain. The clinical significance of these studies includes the possible use of these epitopes as biomarkers for metastasis.

Web-based User Interface for Electrical Cell-substrate Impedance Sensing System

Web-based User Interface for Electrical Cell-substrate Impedance Sensing System

DAQ based Impedance Measurement System for Low Cost and Portable Electrical Cell-Substrate Impedance Sensing

Electric cell-substrate impedance sensing (ECIS), a technique for label-free and real-time detection of cells, is emerging as an alternative or assistive method to traditional biochemical assays for diagnostic and pharmaceutical applications. In this work, we developed a DAQ based impedance measurement system for low cost and portable ECIS applications. The alternating input signal for ECIS was generated from the digital-to-analog converter (DAC) channel of the DAQ board and applied through the current limited resistor to the cells on the electrode. The responding signal was recorded by the analog-to-digital converter (ADC) channel of the DAQ board, and its amplitude and phase were analyzed by software lock-in amplifier. The feasibility of the developed ECIS system was evaluated by impedance measurement and DAPI analysis of C2C12 cell growth on the indium tin oxide microelectrode, and by impedimetric and MTT cell viability assay of HEK293 cells exposed to H2O2.

Fragmental Frequency Analysis Method to Estimate Electrical Cell Parameters From Bioimpedance Study

This paper introduces an alternate approach utilizing the fragmental frequency analysis method for analyzing bioimpedance data to estimate electrical cell parameters. Impedance of cervical cancer cells (HeLa) in phosphate buffer saline media is measured using an electric cell-substrate impedance sensing (ECIS) device and impedance analyzer. The measured impedance data were visualized by modeling an equivalent electrical circuit of the system considering the dominancy of individual parameters of the ECIS system and analyzed in different frequency zones. The present approach eliminates the convergence inaccuracy in fitting the experimental impedance data with fitting software arising due to invalid initial conditions, and a large number of data points. This method provides high-frequency characterization, modeling of ECIS system, knowledge of effect of ECIS model parameters with frequency, and an alternate way to calculate model parameters.

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

Mizoribine inhibits the proliferation of renal stem/progenitor cells by G1/S arrest during renal regeneration.

Immunosuppressive agents are generally administered to treat kidney diseases. However, it is unclear whether renal stem/progenitor cells are directly affected by the immunosuppressive agents. We used normal rat kidney cells, ureteric bud cells and rat kidney stem/progenitor cells in this study. Mizoribine (MZR), cyclophosphamide (CPA) and cyclosporine (CyA) were added to the culture media of these cells. We evaluated the effects of these immunosuppressive agents on cell proliferation using an electrical cell-substrate impedance sensing system (ECIS) and their effects on the process of renal regeneration using the ischemia-reperfusion (I/R) injury rat model. The ECIS data showed that proliferation of each of the 3 types of cells was significantly suppressed by MZR. MZR treatment enhanced renal tubular injury in ischemia-reperfusion (I/R) injured rats, and significantly decreased levels of M-phase cells and Nestin-positive cells. These results suggested that MZR inhibits the cell cycle of renal stem/progenitor cells;thus, physicians should take note that MZR might affect not only inflammation but also renal regeneration.

Assessment of cadmium-induced hepatotoxicity and protective effects of zinc against it using an improved cell-based biosensor

Cadmium (Cd) is a toxic environmental pollutant which creates risks to human health. Liver is one of the major sites of Cd accumulation in the organism and target organs under chronic and acute exposure of Cd. It was estimated that Zn2+ supplementation can prevent apoptosis induced by a variety of agents such as Cd2+. In this paper, an improved electric cell-substrate impedance sensing (ECIS) assay was developed to investigate the Cd2+-induced hepatotoxicity and the effects of Zn2+ at different concentrations on it in vitro. Normal liver L-02 cells were cultured onto interdigitated electrodes. With ECIS technology the dynamic responses of L-02 cells were monitored in a non-invasive and label-free manner. The results showed a clear dose- and time-depending toxic effect of Cd2+ on L-02 cells. The half-inhibition concentration of Cd2+ was 20μM after 24h of exposure. The results also revealed that enhanced Zn2+ consumption (10–20μM) may be beneficial for preventing Cd2+-induced hepatotoxicity. However, its excessive intake (40μM) may intensify the hepatotoxicity. Furthermore, for improving the detection sensitivity of the system, a cell immobilization approach using self-assembly techniques was applied to bind cells on electrodes for improving the detection sensitivity of the system. This study demonstrated that the ECIS system holds promise as a utility platform for real-time studying cell status and evaluating chemical effect with high detection sensitivity.

Impedance-Based Cell Culture Platform To Assess Light-Induced Stress Changes with Antagonist Drugs Using Retinal Cells

This Article describes an unprecedented, simple, and real-time in vitro analytical tool to measure the luminous effect on the time responses function of retinal ganglion cells (RGC-5) by electric cell substrate impedance sensing (ECIS) system. The ECIS system was used for the continuous measurement of different color light-induced effects on the response of cells that exposed to protective drugs. The measurement suggests that the association of photo-oxidative stress was mediated by reactive oxygen species (ROS), which plays a critical role that leads to cell stress, damages, and retinopathy, resulting in eye degenerative diseases. Continuous light radiation caused time-dependent decline of RGC-5 response and resulted in photodamage within 10 h due to adenosine 5'-triphosphate depletion and increased ROS level, which is similar to in vivo photodamage. The ECIS results were correlated with standard cell viability assay. ECIS is very helpful to determine the protective effects of analyzed drugs such as β-carotene, quercetin, agmatine, and glutathione in RGC-5 cells, and the maximum drug activity of nontoxic safer drug concentrations was found to be 0.25, 0.25, 0.25, and 1.0 mM, respectively. All drugs show protection against light radiation toxicity in a dose-dependent manner; the most effective drug was found to be glutathione. The proposed system identifies the phototoxic effects in RGC-5 and provides high throughput drug screening for photo-oxidative stress during early stages of drug discovery. This study is convenient and potential enough for the direct measurements of the photoprotective effect in vitro and would be of broad interest in the field of therapeutics.

High-mobility group box-1 protein activates inflammatory signaling pathway components and disrupts retinal vascular-barrier in the diabetic retina

Extracellular high-mobility group box-1 (HMGB-1) functions as a pro-inflammatory cytokine and exhibits angiogenic effects. The purpose of this study was to investigate the expression of HMGB-1 signaling pathway components in the retinas of diabetic rats and to examine the effect of intravitreal administration of HMGB-1 on the retinas of rats. The retinas of diabetic and intravitreally injected HMGB-1 rats were studied using immunohistochemistry, Western blotting, co-immunoprecipitation and enzyme-linked immunosorbent assay. The effect of HMGB-1 on retinal endothelial cell barrier function was evaluated using electrical cell-substrate impedance sensing system (ECIS). Diabetes induced significant upregulation of the expression of HMGB-1, receptor for advanced glycation end products (RAGE), ERK1/2 and nuclear transcription factor Kappa B (NF-κB), whereas the expression of toll-like receptor 2 (TLR2) and occludin was significantly downregulated. Co-immunoprecipitation studies revealed significant increase in interaction between HMGB-1 and RAGE. HMGB-1 reduced transendothelial electrical resistance of bovine retinal endothelial cells. Intravitreal administration of HMGB-1 to normal rats induced significant upregulation of intercellular adhesion molecule-1 (ICAM-1), soluble intercellular adhesion molecule-1 (sICAM-1), HMGB-1, RAGE, ERK1/2, and NF-κB, and significantly increased retinal vascular permeability, whereas the expression of TLR2 and occludin was downregulated. Oral administration of glycyrrhizin, a specific inhibitor of HMGB-1, attenuated diabetes-induced upregulation of HMGB-1 expression, NF-κB activation and downregulation of occludin expression. Our findings provide evidence that in the diabetic retina, HMGB-1 possibly interacts with RAGE and activates ERK1/2 and NF-κB to generate an inflammatory response and disrupt retinal vascular barrier.

A computational modeling and analysis in cell biological dynamics using electric cell-substrate impedance sensing (ECIS)

In this paper, a study of computational modeling and multi-scale analysis in cell dynamics is presented. Our study aims at: (1) deriving and validating a mathematical model for cell growth, and (2) quantitatively detecting and analyzing the biological interdependencies across multiple observational scales with a variety of time and frequency resolutions. This research was conducted using the time series data practically measured from a novel on-line cell monitoring technique, referred to as electric cell-substrate impedance sensing (ECIS), which allows continuously tracking the cellular behavior such as adhesion, proliferation, spreading and micromotion. First, comparing our ECIS-based cellular growth modeling analysis results with those determined by hematocytometer measurement using different time intervals, we found that the results obtained from both experimental methods consistently agreed. However, our study demonstrated that it is much easier and more convenient to operate with the ECIS system for on-line cellular growth monitoring. Secondly, for multi-scale analysis our results showed that the proposed wavelet-based methodology can effectively quantify the fluctuations associated with cell micromotions and quantitatively capture the biological interdependencies across multiple observational scales. Note that although the wavelet method is well known, its application into the ECIS time series analysis is novel and unprecedented in computational cell biology. Our analyses indicated that the proposed study on ECIS time series could provide a hopeful start and great potentials in both modeling and elucidating the complex mechanisms of cell biological systems.

Use of cellular electrical impedance sensing to assess in vitro cytotoxicity of anticancer drugs in a human kidney cell nephrotoxicity model

Nephrotoxicity is one of the major concerns for anticancer drug safety because most drugs are metabolized and excreted by the kidneys. Convenient tools able to perform rapid in vitro cytotoxicity analysis and identify drug side effects in kidney cells during early phases of drug discovery could be beneficial to drug development programs. Here we developed an electrical cell-substrate impedance sensing system (ECIS) capable of continuously measuring the dosage and time response of human proximal tubular epithelial (HK2) cells exposed to four drugs throughout the experimental period. These drugs induced HK2 cell apoptosis/death in a dose-dependent manner, although with very different dose-response effects. DDP (50 μM) was the most cytotoxic and induced obvious HK2 cell apoptosis rapidly after exposure. The other three drugs had much lower cytotoxicity, even at concentrations approaching 1 mM. The results obtained from our ECIS system correlated well with conventional in vitro assays such as flow cytometry and cell viability assays. Notably, the continuous and automatic measurements provided by ECIS system allow for better resolution for drugs with different temporal toxicity profiles. Furthermore, we investigated the effect of DDP's antidotes, glutathione and sodium subsulfite, on DDP-induced cytotoxicity, both of which decreased nephrotoxicity of DDP in a dose-dependent manner. Overall this study illustrates the convenience of ECIS for direct, continuous assessment of the cytotoxicity of anticancer drugs in vitro. ECIS has the potential to become a useful, non-invasive analytical method for early evaluation of drugs and antidotes of toxins.

Real-time electrical measurement of L929 cellular spontaneous and synchronous oscillation.

Nonexcitable cell types, fibroblasts of heart muscle or astrocytes, are well known for their spontaneous Ca2+ oscillations. On the other hand, murine fibroblast (L929) cells are known to be deficient in cell–cell adhesive proteins and therefore lack gap junctions for cellular communication. However, these cells exhibit a unique property of collectively synchronized and spontaneous oscillation, as revealed by real-time monitoring of cells cultured on a 250-μm diameter microelectrode for more than 3 days using an electrical cell-substrate impedance-sensing system (ECIS). Live-cell imaging is a widely used technique for oscillation detection, but it has limitations relating to cellular physiological environment maintenance for microscopic analysis and for prolonged periods of study. The present research emphasizes an electrical-sensing technique (ECIS) capable of overcoming the most important issues inherent in live-cell imaging systems for the detection of L929 cellular spontaneous and synchronized oscillation in real-time for longer periods. Possible mechanisms involved in L929 oscillation were elucidated to be periodic extension/contraction of lamellipodia continued as blebbing, which is produced by signals from the actomyosin complex initiated by connexin hemichannel opening and adenosine triphosphate (ATP) release. By applying the connexin hemichannel inhibitor, flufenamic acid, the hindrance of ATP release and calcium transients were analyzed to elucidate this hypothesis.

Effects of osmolarity on human epithelial conjunctival cells using an electrical technique

The purpose of this study is to report the effect of different media osmolarity on a cell line monolayer of normal human conjunctival epithelia (IOBA-NHC) using Electric Cell-substrate Impedance Sensing (ECIS). We built our own ECIS system. We fabricated biocompatible microelectrodes. We used a monolayer of IOBA-NHC cells with media at different osmolarities (315, 360, 446, and 617mOsm/l). When there is an increase in hyperosmolarity, there is a slight decrease in the measured resistance of the naked microelectrode (without cells), whereas its capacitance remained practically unchanged. The evaluation of resistance and capacitance of a microelectrode covered by a monolayer of IOBA-NHC in relation to a naked microelectrode showed no difference in the standard media (315mOsm/l), a small difference with 360mOsm/l, and significant differences with hyperosmolarities of 446mOsm/l and 610mOsm/l. The resistance with a confluent cell monolayer is up to three times greater compared to the value of the resistance of the naked electrode with standard media. Both resistance and capacitance measurements for the cell monolayer were sensitive to changes in osmolarity.