Name: Monitoring Changes in Human Umbilical Vein Endothelial Cells upon Viral Infection Using Impedance-Based Real-Time Cell Analysis (Video) | JoVE Journal
Uploaded: 2023-05-05T14:00:00.000+00:00
Duration: 7 min 56 s
Description: Learn real-time cell analysis for monitoring HUVEC morphological changes during viral infection using impedance-based RTCA. This non-invasive method captures transient effects like cell detachment and vascular leakage missed by endpoint assays.

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5 minutes

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

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...

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

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Learn real-time cell analysis for monitoring HUVEC morphological changes during viral infection using impedance-based RTCA. This non-invasive method captures transient effects like cell detachment and vascular leakage missed by endpoint assays.

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

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