This research received support from the Ministry of Higher Education Malaysia under the Higher Institution Centre of Excellence (HICoE) program (MO002-2019) and funding under the Long-Term Research Grant Scheme (LRGS MRUN Phase 1: LRGS MRUN/F1/01/2018).

1. Cell preparation
<ol>
	<li>Preparation of HUVECs
	<ol>
		<li>Grow 7.5 x 105 HUVEC cells/mL in a 75 cm2 (coated with 20 &#181;g/mL collagen type 1) cell culture flask containing 12 mL of endothelial cell medium (ECM) supplemented with 10% fetal bovine serum (FBS), 0.01% endothelial cell growth supplement (ECGS) that contains growth factors, hormones, and proteins necessary for the culture of HUVECs, and 0.01% penicillin-streptomycin (P/S). Incubate the cells in a cell culture incubator at 37 &#176;C with 5% CO2 (Figure 1).</li>
	</ol>
	</li>
</ol>
2. Setup of RTCA experiment
<ol>
	<li>Resistor verification run on RTCA
	<ol>
		<li>Double-click the RTCA software icon in the desktop to launch the application. A login window will appear, type in the username and password to login.</li>
		<li>Place a&#160;96-well RTCA resistor plate with the A1 well facing inward into the RTCA station located in the cell culture incubator.</li>
		<li>In the Exp Notes tab of the RTCA software, type the experiment name, device SN, and device type (QC plate).</li>
		<li>In the Layout tab under the Cell tab of the RTCA software, highlight all the wells and right-click on the highlighted wells to make sure all the wells are turned on (grayed out).</li>
		<li>In the Schedule tab of the RTCA software, click Step_1. Enter Sweeps: 10, and Interval: 30 s. Click Apply. Click Start/Continue in the top left corner of the software to start the verification run. 
		NOTE: Make sure the connection of the wells to the RTCA station is okay by observing &#34;Plate Scanned. Connections OK&#34; at the bottom of the page.</li>
	</ol>
	</li>
	<li>Resistor verification run for data analysis
	<ol>
		<li>In the Cell Index tab, check the data upon completion of the run. All the CI values should be less than 0.063.</li>
		<li>In the lower part of the Cell Index tab, check the raw scan data. The raw scan data should exhibit repeated value patterns of 40.0 &#177; 2.0 (rows A and H), 67.5 &#177; 2.5 (rows B and G), 93.6 &#177; 2.9 (rows C and F), and 117.6 &#177; 3.2 (rows D and E).</li>
		<li>To stop the verification run, click Plate &#62; Release Plate. The 96-well RTCA resistor plate is now ready to be removed from the RTCA station.</li>
	</ol>
	</li>
	<li>Obtaining background reading on RTCA
	<ol>
		<li>Wash the collagen type 1-coated specialized gold-microelectrode plate (coated with 20 &#181;g/mL collagen type 1) with 60 &#181;L/well of Hank&#39;s balanced salt solution (HBSS; with sodium bicarbonate, without calcium chloride and magnesium sulfate) 2x.</li>
		<li>Discard the remaining HBSS and add 50 &#181;L/well of ECM. Place the specialized gold-microelectrode plate containing ECM with the A1 well facing inward into the RTCA station located in the cell culture incubator.</li>
		<li>In the Exp Notes tab of the RTCA software, type the experiment name, device SN, and device type (96-well format).</li>
		<li>In the Layout tab under the Cell tab of the RTCA software, highlight the wells that will be used and right-click on the highlighted wells to make sure all the wells are turned on (grayed out).</li>
		<li>In the Schedule tab of the RTCA software, click Step_1, and click Start/Continue in the top left corner of the software to obtain a background reading. 
		&#8203;NOTE: Make sure the connection of the wells is correct by observing &#34;Plate Scanned. Connections OK&#34; at the bottom of the page.</li>
		<li>Once the background reading is obtained, the specialized gold-microelectrode plate is now ready for cell seeding and ready to be removed from the RTCA station.</li>
	</ol>
	</li>
	<li>HUVEC preparation in the specialized gold-microelectrode plate
	<ol>
		<li>Seed the HUVECs in the specialized gold-microelectrode plate with 50 &#181;L/well of conditioned medium, 1 x 104 cells/well (conditioned medium: ECM supplemented with 10% FBS, 0.01% ECGS, and 0.01% P/S). Place the plate back into the RTCA station with the A1 well facing inward for overnight incubation in a cell culture incubator at 37 &#176;C with 5% CO2. 
		NOTE: A cell count was performed on the cell suspension with a dilution factor of two using a hemocytometer.</li>
		<li>In the Schedule tab, click Add a Step in the top left corner. Click on Step_2 and change the Sweeps: 601, and Interval: 2 min. Click Apply. Click on Step_2 and click Start/Continue in the top left corner of the software.</li>
		<li>After 20 h of incubation and when the CI values in the Well Graph tab show a plateau, the plate is ready for infection. In the Schedule tab of the RTCA software, click Abort Step.</li>
		<li>The specialized gold-microelectrode plate is now ready to be removed from the RTCA station for subsequent steps.</li>
	</ol>
	</li>
</ol>
3. Viral infection in HUVECs
<ol>
	<li>Virus dilution
	<ol>
		<li>The ZIKV stock culture is kept at -80 &#176;C. For this study, remove the virus stock kept in the cryogenic vials and dilute the ZIKV stock with serum-free ECM in 1.5 mL centrifuge tubes to achieve multiplicity of infections (MOIs) of 0.1 and 1. 
		NOTE: The ZIKV stock was previously prepared by performing propagation in Vero cells and harvesting the ZIKV supernatant34.</li>
	</ol>
	</li>
	<li>Infection of the cell monolayer
	<ol>
		<li>Remove and discard the conditioned medium (ECM supplemented with 10% FBS, 0.01% ECGS, and 0.01% P/S) from each well. Rinse each well with 60 &#181;L of HBSS 2x. Insert the HBSS with the aid of the side of the well; do not shoot the cell monolayer.</li>
		<li>Working from the highest to lowest dilution, add 40 &#181;L of each diluted virus sample in serum-free ECM into the well. Triplicate the well for each dilution and maintain six wells without infection (negative control and positive control thrombin). Do not shoot the cell monolayer; instead, add the diluted virus with the aid of the side of the well. Use a fresh pipet tip for each insertion. 
		&#8203;NOTE: Thrombin was chosen as the positive control as it can increase the endothelial permeability of HUVECs rapidly.</li>
		<li>Incubate the plate in a cell culture incubator at 37 &#176;C with 5% CO2 to allow virus adsorption. Swish the plate 5x every 15 min to allow virus adsorption throughout the cell monolayer.</li>
	</ol>
	</li>
	<li>Addition of the overlay cell culture medium
	<ol>
		<li>After 1 h of incubation, remove and discard the virus suspension from the lowest concentration to the highest.</li>
		<li>Wash the infected cells with 60 &#181;L of HBSS 2x. Overlay the well with ECM supplemented with 2% FBS. For the positive control wells, dilute the thrombin to 20 U/mL with ECM supplemented with 2% FBS, and overlay the wells with thrombin-containing ECM medium. Do not shoot the cell monolayer; instead, add overlay media with the aid of the side of the well.</li>
	</ol>
	</li>
	<li>Placement of the infected specialized gold-microelectrode plate back in RTCA station
	<ol>
		<li>In the Schedule tab, click Add a Step in the top left corner. Click on Step_3 and change the Sweeps:&#160;3,601 and Interval:&#160;2 min. Click Apply.</li>
		<li>Click OK in the pop-up window that appears. Place the infected specialized gold-microelectrode plate with the A1 well facing inward into the RTCA station located in the cell culture incubator.</li>
		<li>Click on Step_3 and click Start/Continue in the top left corner of the software.</li>
	</ol>
	</li>
</ol>
4. Data analysis and exporting data
<ol>
	<li>Addition of experimental information of each well
	<ol>
		<li>In the Layout tab, click the Cell tab (Figure 2A). To enter the target cell information and determine the well type for each well, highlight the well and type in the target cell name, number, and color at the bottom of the software. Click Apply (Figure 2B).</li>
		<li>To select and determine the well type for each well in the Cell tab, highlight the well, select well type (sample, positive control, or negative control), and click Set.</li>
		<li>In the Layout tab, click on the Treatment tab (Figure 2C). To enter treatment information, highlight the well and type in the treatment name, concentration of treatment, unit, and color. Click Apply (Figure 2D).</li>
		<li>To select and determine the well type for each well on the Treatment tab, highlight the well, select well type (sample, positive control, or negative control), and click Set. 
		NOTE: For each selection, multiple wells can be highlighted at the same time.</li>
	</ol>
	</li>
	<li>Normalization of CI value
	<ol>
		<li>Right-click on the right of the Data analysis tab and select Normalized Cell Index from the Y Axis drop-down box (Figure 3A). In the X Axis section at the bottom, select the normalized time as the last measured time point where the specialized gold-microelectrode plate was removed for infection (Figure 3B). 
		NOTE: The normalization process carried out by the software removes most of the variability due to the differences in well seeding and attachment, as long as the chosen normalization point is before addition of the virus and/or treatment. Hence, the optimal normalization time point should be the last time point before virus and/or treatment addition. The time point to normalize can be checked in the Schedule tab under Step_2. The total time indicated is the time point to normalize (Figure 3C).</li>
	</ol>
	</li>
	<li>Plotting obtained data using RTCA software
	<ol>
		<li>In the Data analysis tab, select the wells to be added into the graph by highlighting the wells and clicking &#60;&#60; Add. The normalized cell index against the time graph is now ready to be copied and exported. Make sure to tick the Average tick box.</li>
	</ol>
	</li>
	<li>Exporting data using RTCA software
	<ol>
		<li>In the top left corner of the RTCA software, click Plate &#62; Export Experiment Info&#8230;. Check the box to select the data to be exported. Click OK. 
		NOTE: Raw data (raw CI values) can be exported when Cell index &#62; All is ticked.</li>
		<li>Select the folder to save the export file. Click SAVE. A pop-up window will appear indicating the exporting of experimental information. Once exporting is done, another window will appear indicating the exported experiment information is ready to be viewed.</li>
	</ol>
	</li>
</ol>

The authors declare that they have no competing interests.

Cytocidal viral infection in permissive cells is usually associated with substantial changes in cell morphology and biosynthesis36. The virus-induced cellular morphological changes, such as cell shrinking, cell enlargement, and/or syncytia formation, are also known as cytopathic effects (CPEs), characteristic to certain viral infections37. In ECs, the CPE was described as the destruction of cells and breakdown of the tight junctions in between each EC, hence causing the bre...

Endothelial cells (ECs) line the inner surface of all blood and lymphatic vessels. They are connected by adherens, tight gap junctions, creating a semi-permeable barrier between blood or lymph and the surrounding tissues1,2. The intact endothelial cell-cell junctions are critical for regulating the transport of macromolecules, solutes, and fluids, crucial for the physiological activities of the corresponding tissues and organs3. The endothelial barrier is dynamic and modulated by specific stimuli4. Disruption or loss of endothelial barrier function is a hallmark of disease pathophysiology in acute and chronic inflammation in the pathogenesis of many diseases5,6,7, including viral infection8,9,10,11. For flaviviruses, it was found that the non-structural protein NS1 can alter endothelial permeability, for example, via disruption of the endothelial glycocalyx-like layer as a result of increased expression and activation of cathepsin L, sialidases, and endoglycosidase heparinase9. In the disease state, the integrity of the endothelial monolayer is affected, and cell-cell junctions are disrupted, which may lead to endothelial injury and dysfunction12 and eventually widespread pathological manifestations across multiple organ systems13,14. Viral infection of ECs results in changes in the cell signaling pathways and cellular gene expression profile, which may affect the fluid barrier functions, cause disruption of the ECs, and induce vascular leakage10,15. Zika virus (ZIKV) infection of ECs has been found to disrupt the junctional integrity that causes changes in vascular permeability and lead to endothelial barrier dysfunction, resulting in vascular leakage10,15. Studies have shown that ZIKV is linked to severe birth defects; however, not much is known about the exact mechanism by which this occurs16,17. Understanding endothelial barrier changes during an infection is therefore critical to understand the disease mechanism.
ECs are currently used as an important experimental in vitro vascular model system for various physiological and pathological processes18,19,20,21. Human umbilical vein endothelial cells (HUVECs) have been extensively used as a primary, non-immortalized cell system to study the vascular endothelium and vascular biology in vitro22,23,24,25. HUVECs were first isolated for in vitro culture in the early 1970s from the umbilical vein of the human umbilical cord26. HUVECs have a cobblestone-like morphology that is easily made to proliferate in the laboratory. Due to shear stress after being maintained for long periods in the confluent state, elongation of the cell shape is observed. HUVECs can be characterized by the presence of Weibel and Palade bodies (WPB), pinocytic vesicles, small amounts of fibrils located near the nucleus, mitochondria with tubular shapes which rarely show ramifications, an ellipsoid nucleus with a fine granular pattern of condensed chromatin, and one to three nucleoli present in each nucleus27. Although HUVECs show numerous morphological characteristics, the identification of HUVECs cannot be achieved by optic examination alone. Assays, such as immunofluorescence staining of vascular endothelial (VE)-cadherin, is commonly used for the monitoring of vascular integrity in HUVECs28,29,30,31.
This study describes the real-time cell analysis (RTCA) protocol of the infection of HUVECs. The RTCA system uses specialized gold-coated plates containing microelectrodes that provide a conductive surface for the cell attachment. When the cells attach to the microelectrode surface, a barrier that impedes electron flow through the microelectrodes is formed. Measurement of the impedance generated by microelectrodes can be used to gain information on some cellular processes, including cell attachments, proliferation, and migration32. The reported assay is an impedance-based cellular assay that, coupled with a real-time cell analyzer, provides continuous measurement of live cell proliferation, morphology changes (such as cell shrinking) and cell attachment quality in a noninvasive and label-free manner. In this study, the real-time cell analyzer is used to monitor the endothelial integrity and morphological changes of HUVECs during ZIKV infection. Briefly, the instrument measures electron flow transmitted in specialized gold-microelectrode-coated microtiter plates in the presence of a culture medium (electrically conductive solution). Cell adherence or changes in cell number and morphology disturb the electron flow and cause impedance variations that can be captured by the instrument (Supplementary Figure 1). The difference between HUVEC growth, proliferation, and adherence before and after ZIKV infection is recorded in real time by an electrical impedance signal and then translated to cell index (CI) value. The CI value from the pre-infection is used as the baseline for CI value normalization. The changes in CI values reflect cellular morphological changes or cell adherence loss upon the infection33. The study results show that the RTCA protocol allows the detection of transient effects or cell morphological changes during viral infection compared to an endpoint assay, such as immunofluorescence staining of VE-cadherin. The RTCA method could be useful for analyzing the HUVEC vascular integrity changes upon infection by other viruses.

<table><tbody><tr><td>1.5 mL microcentrifuge tube</td><td>Nest</td><td>615601</td><td></td></tr><tr><td>37 &amp;deg;C incubator with 5% CO2</td><td>Sanyo</td><td>MCO-18AIC</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flask&amp;nbsp;</td><td>Corning</td><td>430725U</td><td></td></tr><tr><td>Antibiotic solution penicillin-streptomycin (P/S)</td><td>Sciencell</td><td>0503</td><td></td></tr><tr><td>Biological safety cabinet, Class II</td><td>Holten</td><td>HB2448</td><td></td></tr><tr><td>Collagen Type 1</td><td>SIgma-Aldrich</td><td>C3867</td><td></td></tr><tr><td>Endothelial cell growth supplement (ECGS)</td><td>Sciencell</td><td>1052</td><td></td></tr><tr><td>Endothelial cell medium (ECM)</td><td>Sciencell</td><td>1001</td><td></td></tr><tr><td>E-plate 96</td><td>Agilent Technologies, Inc.</td><td>05232368001</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sciencell</td><td>0025</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS)</td><td>Sigma-Aldrich</td><td>H9394</td><td></td></tr><tr><td>Hemocytometer</td><td>Laboroptik LTD</td><td>Neubauer improved</td><td></td></tr><tr><td>Human Umbilical Vein Endothelial Cells (HUVECs)</td><td>Sciencell</td><td>8000</td><td></td></tr><tr><td>Inverted microscope</td><td>ZEISS</td><td>TELAVAL 31</td><td></td></tr><tr><td>Latitude 3520 Laptop</td><td>Dell&amp;nbsp;</td><td>-</td><td></td></tr><tr><td>Multichannel micropipette (10 - 100 &amp;micro;L)</td><td>Eppendorf</td><td>3125000036</td><td></td></tr><tr><td>Multichannel micropipette (30 - 300 &amp;micro;L)</td><td>Eppendorf</td><td>3125000052</td><td></td></tr><tr><td>Reagent reservior</td><td>Tarsons</td><td>T38-524090</td><td></td></tr><tr><td>RTCA resistor plate 96</td><td>Agilent Technologies, Inc.</td><td>05232350001</td><td></td></tr><tr><td>RTCA Software Pro (Version 2.6.1)</td><td>Agilent Technologies, Inc.</td><td>5454433001</td><td></td></tr><tr><td>Single channel pipettes (10 - 100 &amp;micro;L)</td><td>Eppendorf</td><td>3123000047</td><td></td></tr><tr><td>Single channel pipettes (100 - 1000 &amp;micro;L)</td><td>Eppendorf</td><td>3123000063</td><td></td></tr><tr><td>Single channel pipettes (20 - 200 &amp;micro;L)</td><td>Eppendorf</td><td>3123000055</td><td></td></tr><tr><td>xCELLigence Real-time cell analyzer SP (Model: W380)</td><td>Agilent Technologies, Inc.</td><td>00380601030</td><td>https://www.agilent.com/en/product/cell-analysis/real-time-cell-analysis/rtca-analyzers/xcelligence-rtca-sp-single-plate-741232</td></tr></tbody></table>

monitoring changes in human umbilical vein endothelial cells upon viral infection using impedance-based real-time cell analysis

Endothelial cells line the inner surface of all blood and lymphatic vessels, creating a semi-permeable barrier regulating fluid and solute exchange between blood or lymph and their surrounding tissues. The ability of a virus to cross the endothelial barrier is an important mechanism that facilitates virus dissemination in the human body. Many viruses are reported to alter endothelial permeability and/or cause endothelial cell barrier disruption during infection, which is able to cause vascular leakage. The current study describes a real-time cell analysis (RTCA) protocol, using a commercial real-time cell analyzer to monitor endothelial integrity and permeability changes during Zika virus (ZIKV) infection of the human umbilical vein endothelial cells (HUVECs). The impedance signals recorded before and after ZIKV infection were translated to cell index (CI) values and analyzed. The RTCA protocol allows the detection of transient effects in the form of cell morphological changes during a viral infection. This assay could also be useful for studying changes in the vascular integrity of HUVECs in other experimental setups.

Before ZIKV infection, the CI recorded for the HUVEC monolayer cultured on a specialized gold-microelectrode coated microtiter plate was above 8, suggesting strong adherence properties. Plates or wells with a CI index less than 8 should be discarded. The HUVECs were then infected with ZIKV at an MOI of 0.1 and 1, and the cell impedance was recorded for 7 days. The CI value of HUVECs infected with ZIKV P6-740 at an MOI of 0.1 (light blue line) started to drop at 15 h post-infection (HPI), compared to the negative infectio...

Watch this Scientific Journal Video about Monitoring Changes in Human Umbilical Vein Endothelial Cells upon Viral Infection Using Impedance-Based Real-Time Cell Analysis at JoVE.com

Monitoring Changes in Human Umbilical Vein Endothelial Cells upon Viral Infection Using Impedance-Based Real-Time Cell Analysis

The current study describes a protocol to monitor the changes in human umbilical vein endothelial cells (HUVECs) during viral infection using a real-time cell analysis (RTCA) system.

Endothelial cells line the inner surface of all blood and lymphatic vessels, creating a semi-permeable barrier regulating fluid and solute exchange ...

monitoring-changes-human-umbilical-vein-endothelial-cells-upon-viral

Research

JoVE Journal

Biology

339 Views.  Universiti Malaya. Dit protocol maakte de continue detectie van voorbijgaand effect mogelijk in de vorm van enkele morfologische veranderingen tijdens een virale infectie, zoals loslating en zweven van aanhangende cellen. Dit protocol maakte een niet-invasieve en niet-arbeidsintensieve aanpak mogelijk voor het continu monitoren van cellulaire morfologische veranderingen en het vastleggen van voorbijgaande effecten zoals vasculaire lekkage die konden worden gemist door een eindpunttest. Dit protocol kan worden g...

Video: Monitoring Changes in Human Umbilical Vein Endothelial Cells upon Viral Infection Using Impedance-Based Real-Time Cell Analysis

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

Medicine

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Immunology and Infection

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are either time-consuming or unable to detect small variations. Presented here is a protocol for the precise quantification of viral titer by analyzing electrical impedance variations of infected cells in real-time. Cellular impedance is measured through gold microelectrode biosensors located under the cells in microplates, in which magnitude depends on the number of cells as well as their size and shape. This protocol allows real-time analysis of cell proliferation, viability, morphology and migration with enhanced sensitivity. Also provided is an example of a practical application by quantifying the decay of influenza A virus (IAV) submitted to various physicochemical parameters affecting viral infectivity over time (i.e., temperature, salinity, and pH). For such applications, the protocol reduces the workload needed while also generating precise quantification data of infectious virus particles. It allows the comparison of inactivation slopes among different IAV, which reflects their capacity to persist in given environment. This protocol is easy to perform, is highly reproducible, and can be applied to any virus producing cytopathic effects in cell culture.

Reported here is a protocol for the quantification of infectious viral particles using real-time monitoring of electrical impedance of infected cells. A practical application of this method is presented by quantifying influenza A virus decay under different physicochemical parameters mimicking environmental conditions.

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are ...

Cell-Based Electrical Impedance Platform to Evaluate Zika Virus Inhibitors in Real Time

Cell-based electrical impedance (CEI)&#160;technology measures changes in impedance caused by a growing or manipulated adherent cell monolayer on culture plate wells embedded with electrodes. The technology can be used to monitor the consequences of Zika virus (ZIKV) infection and adherent cell replication in real time, as this virus is highly cytopathogenic. It is a straightforward assay that does not require the use of labels or invasive methods and has the benefit of providing real-time data. The kinetics of ZIKV infection are highly dependent on the employed cell line, virus strain, and multiplicity of infection (MOI), which cannot be easily studied with conventional endpoint assays. Furthermore, the CEI assay can also be used for the evaluation and characterization of antiviral compounds, which can also have dynamic inhibitory properties over the course of infection. This methods article gives a detailed explanation of the practical execution of the CEI assay and its potential applications in ZIKV research and antiviral research in general.

Here, we demonstrate the use of cell-based electrical impedance (CEI) as a very easy and straightforward method to study Zika virus infection and replication in human cells in real time. Furthermore, the CEI assay is useful for the evaluation of antiviral compounds.

Cell-based electrical impedance (CEI)&#160;technology measures changes in impedance caused by a growing or manipulated adherent cell monolayer on culture ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Cellular Impedance-Based Analysis for Monitoring of Cellular Processes in Real-Time

Source: Labadie, T., et al., <a href="https://app.jove.com/v/61133">Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis.</a> J. Vis. Exp. (2021)
In this video, we demonstrate the process of real-time monitoring of cellular events, including the proliferation of adherent cells, via a cellular impedance assay. Here, the adherent cells act as insulators, restricting the current flow between the electrodes and increasing the impedance.

Source: Labadie, T., et al., Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis. J. Vis. Exp. (2021)
In this video, we ...

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Cell-based assays

Impedance-Based Cellular Assay to Measure Multiplicity of Infection of Influenza Virus

Source: Labadie, T., et al., <a href="https://app.jove.com/v/61133">Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis.</a> J. Vis. Exp. (2021)
In this video, we discuss the correlation between the CIT50 and the multiplicity of viral infections. The virus-infected cells display cytopathic effects, causing their death and detachment from the gold microelectrode, which in turn, reduces the impedance.

Monitoring Changes in Human Umbilical Vein Endothelial Cells upon Viral Infection Using Impedance-Based Real-Time Cell Analysis

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