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

In Vitro Wounding Models Using the Electric Cell-Substrate Impedance Sensing (ECIS)-Zθ Technology

Biosensors ◽

10.3390/bios8040090 ◽

2018 ◽

Vol 8 (4) ◽

pp. 90 ◽

Cited By ~ 1

Author(s):

Andrea Gu ◽

Dan Kho ◽

Rebecca Johnson ◽

E. Graham ◽

Simon O’Carroll

Keyword(s):

Barrier Function ◽

Cell Monolayer ◽

Immunohistochemical Analysis ◽

Cell Loss ◽

Impedance Sensing ◽

Viable Cells ◽

Cell Substrate ◽

Underlying Causes ◽

A Cell

Electric Cell-Substrate Impedance Sensing (ECIS) can produce reproducible wounding models by mechanically disrupting a cell monolayer. This study compared in vitro wound-healing using human cerebral microvascular endothelial cells (hCMVEC) with both single electrode (8W1E) and multiple electrodes (8W10E+) arrays. Measurements of hCMVEC migration and barrier functions were conducted, revealing variable levels of barrier disruption could be achieved by altering the duration and magnitude of the applied current. In all scenarios, the barrier (Rb) did not recover the strength observed prior to injury. Localization of junctional proteins following wounding were analyzed by immunocytochemistry. Following wounding, cell migration was generally faster on the 8W10E+ than the 8W1E array. Immunohistochemical analysis revealed non-viable cells remained on the 8W1E electrodes but not the 8W10E+ electrodes. However, viable cells partially remained on the 8W10E+ electrodes following wounding. In addition, the 8W10E+ electrodes demonstrated variation in cell loss across electrodes within the same well. This suggests the type of wounding is different on the two array types. However, our data show both arrays can be used to model incomplete barrier recovery and therefore both have potential for testing of drugs to improve endothelial barrier function. This is the first time that the possibility of using the 8W10E+ array as a wounding model is addressed. We highlight the differences in wounding produced between the two arrays, and can be used to study the underlying causes for impaired barrier function following CNS injuries.

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Abstract 17144: miR-146a-5p Induces Pulmonary Barrier Disruption via Macrophage-Derived TNFa

Circulation ◽

10.1161/circ.142.suppl_3.17144 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Huang Huang ◽

Jiang Hu ◽

Jing Zhu ◽

Wei Chao ◽

Lin Zou

Keyword(s):

Barrier Function ◽

Barrier Dysfunction ◽

Array Analysis ◽

Impedance Sensing ◽

Barrier Disruption ◽

Sensing System ◽

Cell Substrate ◽

Pulmonary Arterial ◽

Arterial Endothelial Cells ◽

Substantial Morbidity

Introduction: Sepsis-induced acute lung injury (ALI) has substantial morbidity and mortality. Diffused alveolar damage as a result of increased endothelial cell (EC) permeability is a hallmark of ALI. We have previously demonstrated that certain uridine-rich extracellular (ex) miRNAs such as miR-146a activate innate immune response and possess a potent proinflammatory property, but their effect on EC function is largely unknown. Hypothesis: Pulmonary EC barrier function is impaired by miR-146a-5p. Methods: Conditioned media (CM) were collected from macrophages (Mφ) cultures treated with lipofectamine (lipo) or miR-146a-5p (50 nM) for 24 h. EC barrier function was analyzed in human pulmonary arterial endothelial cells (HPAECs) by transendothelial electric resistance (TER) using an electric cell-substrate impedance sensing system. Results: Direct miR-146a-5p treatment of HPAECs did not cause any change in TER even though it induced robust IL-6 and CXCL2 production in Mφ. To test whether miR-146a-5p affects EC function indirectly, we incubated HPAECs with miR146a-CM and observed a more than 50% reduction in TER ( Fig. A ). Lipo-CM had no such effect. This barrier effect was accompanied by VE-cadherin internalization and disrupted VE-cadherin lining following 8 hours of incubation ( Fig. B) . To determine the potential mediators in the CM responsible for the EC barrier disruption, we performed a cytokine array analysis of 111 cytokines in the CM and identified 15 differentially expressed cytokines. Among them, we found that TNFα neutralizing Ab (1μg/ml), but not control IgG (1μg/ml), partially blocked the miR-146a-CM-induced EC barrier disruption with improved VE-Cadherin integrity ( Fig. B ). Conclusion: Our data demonstrated miR-146a-5p induces HPAEC barrier dysfunction via an indirect mechanism involving Mφ production of TNFα.

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Electric Cell-Substrate Impedance Sensing To Monitor Viral Growth and Study Cellular Responses to Infection with Alphaherpesviruses in Real Time

mSphere ◽

10.1128/msphere.00039-17 ◽

2017 ◽

Vol 2 (2) ◽

Cited By ~ 13

Author(s):

Matthew R. Pennington ◽

Gerlinde R. Van de Walle

Keyword(s):

Real Time ◽

Cellular Response ◽

Cellular Responses ◽

Cellular Damage ◽

Impedance Sensing ◽

Novel Technologies ◽

Antiviral Therapies ◽

Mucosal Surfaces ◽

Cell Substrate ◽

Hsv 1

ABSTRACT Alphaherpesviruses, including those that commonly infect humans, such as HSV-1 and HSV-2, typically infect and cause cellular damage to epithelial cells at mucosal surfaces, leading to disease. The development of novel technologies to study the cellular responses to infection may allow a more complete understanding of virus replication and the creation of novel antiviral therapies. This study demonstrates the use of ECIS to study various aspects of herpesvirus biology, with a specific focus on changes in cellular morphology as a result of infection. We conclude that ECIS represents a valuable new tool with which to study alphaherpesvirus infections in real time and in an objective and reproducible manner. Electric cell-substrate impedance sensing (ECIS) measures changes in an electrical circuit formed in a culture dish. As cells grow over a gold electrode, they block the flow of electricity and this is read as an increase in electrical impedance in the circuit. ECIS has previously been used in a variety of applications to study cell growth, migration, and behavior in response to stimuli in real time and without the need for cellular labels. Here, we demonstrate that ECIS is also a valuable tool with which to study infection by alphaherpesviruses. To this end, we used ECIS to study the kinetics of cells infected with felid herpesvirus type 1 (FHV-1), a close relative of the human alphaherpesviruses herpes simplex virus 1 (HSV-1) and HSV-2, and compared the results to those obtained with conventional infectivity assays. First, we demonstrated that ECIS can easily distinguish between wells of cells infected with different amounts of FHV-1 and provides information about the cellular response to infection. Second, we found ECIS useful in identifying differences between the replication kinetics of recombinant DsRed Express2-labeled FHV-1, created via CRISPR/Cas9 genome engineering, and wild-type FHV-1. Finally, we demonstrated that ECIS can accurately determine the half-maximal effective concentration of antivirals. Collectively, our data show that ECIS, in conjunction with current methodologies, is a powerful tool that can be used to monitor viral growth and study the cellular response to alphaherpesvirus infection. IMPORTANCE Alphaherpesviruses, including those that commonly infect humans, such as HSV-1 and HSV-2, typically infect and cause cellular damage to epithelial cells at mucosal surfaces, leading to disease. The development of novel technologies to study the cellular responses to infection may allow a more complete understanding of virus replication and the creation of novel antiviral therapies. This study demonstrates the use of ECIS to study various aspects of herpesvirus biology, with a specific focus on changes in cellular morphology as a result of infection. We conclude that ECIS represents a valuable new tool with which to study alphaherpesvirus infections in real time and in an objective and reproducible manner.

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Electrical cell-substrate impedance sensing as a non-invasive tool for cancer cell study

The Analyst ◽

10.1039/c0an00560f ◽

2011 ◽

Vol 136 (2) ◽

pp. 237-245 ◽

Cited By ~ 109

Author(s):

Jongin Hong ◽

Karthikeyan Kandasamy ◽

Mohana Marimuthu ◽

Cheol Soo Choi ◽

Sanghyo Kim

Keyword(s):

Cancer Cell ◽

Impedance Sensing ◽

Non Invasive ◽

Cell Substrate ◽

Cell Study

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Comparison of Leading Biosensor Technologies to Detect Changes in Human Endothelial Barrier Properties in Response to Pro-Inflammatory TNFα and IL1β in Real-Time

Biosensors ◽

10.3390/bios11050159 ◽

2021 ◽

Vol 11 (5) ◽

pp. 159

Author(s):

James J. W. Hucklesby ◽

Akshata Anchan ◽

Simon J. O'Carroll ◽

Charles P. Unsworth ◽

E. Scott Graham ◽

...

Keyword(s):

Human Brain ◽

Real Time ◽

Barrier Properties ◽

Endothelial Barrier ◽

Endothelial Monolayer ◽

Impedance Sensing ◽

Endothelial Monolayers ◽

Impedance Data ◽

Cell Substrate ◽

Limited Frequency

Electric Cell-Substrate Impedance Sensing (ECIS), xCELLigence and cellZscope are commercially available instruments that measure the impedance of cellular monolayers. Despite widespread use of these systems individually, direct comparisons between these platforms have not been published. To compare these instruments, the responses of human brain endothelial monolayers to TNFα and IL1β were measured on all three platforms simultaneously. All instruments detected transient changes in impedance in response to the cytokines, although the response magnitude varied, with ECIS being the most sensitive. ECIS and cellZscope were also able to attribute responses to particular endothelial barrier components by modelling the multifrequency impedance data acquired by these instruments; in contrast the limited frequency xCELLigence data cannot be modelled. Consistent with its superior impedance sensing, ECIS exhibited a greater capacity than cellZscope to distinguish between subtle changes in modelled endothelial monolayer properties. The reduced resolving ability of the cellZscope platform may be due to its electrode configuration, which is necessary to allow access to the basolateral compartment, an important advantage of this instrument. Collectively, this work demonstrates that instruments must be carefully selected to ensure they are appropriate for the experimental questions being asked when assessing endothelial barrier properties.

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