Name: monitoring changes in human umbilical vein endothelial cells upon viral infection using impedance-based real-time cell analysis
Uploaded: 2023-05-05T14:00:00
Description: 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

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 in...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Nest</td>
			<td>615601</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C incubator with 5% CO2</td>
			<td>Sanyo</td>
			<td>MCO-18AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 tissue culture flask&#160;</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&#39; 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&#160;</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel micropipette (10 - 100 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3125000036</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel micropipette (30 - 300 &#181;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 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3123000047</td>
			<td></td>
		</tr>
		<tr>
			<td>Single channel pipettes (100 - 1000 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3123000063</td>
			<td></td>
		</tr>
		<tr>
			<td>Single channel pipettes (20 - 200 &#181;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 ...

This protocol allowed the continuous detection of transient effect in the form of some morphological changes during a viral infection, such as detachment and floating of adherent cells. This protocol allowed for a non-invasive and non-labor intensive approach for continuous monitoring cellular morphological changes and capturing transient effect like vascular leakage that could be missed by endpoint assay. This protocol can be used to analyze vascular integrity changes of other cell lines in different experimental setups if the cells are adherent cells.After preparing the HUVECs, launch the RTCA software application by double clicking the icon on the desktop. Type in the username and password into the login window. Place a 96 well RTCA resistor plate with the A1 well facing inward into the RTCA station located in the cell culture incubator.Then go to the experiment notes tab of the RTCA software and type the experiment name, device serial number, and device type. In the layout tab under the cell tab of the RTCA software, highlight all the wells, and right click on the highlighted wells to ensure all the wells are turned on. Next, open the schedule tab of the RTCA software.Click Step_1, and set sweeps to 10 and interval to 30 seconds. Then click start continue in the top left corner of the software to begin the verification run. After the run, open the cell index tab to check the data.All the cell index values should be less than 0.063. Then check the raw scan data in the lower part of the cell index tab for repeated value patterns as described in the manuscript. To finish the verification run, click plate, and select release plate.Remove the 96 well RTCA resistor plate from the RTCA station. To obtain background reading on RTCA, wash the collagen type one coated specialized gold microelectrode plate twice with 60 microliters per well of HBSS. Discard the remaining HBSS, and add 50 milliliters per well of extracellular matrix.Then place the plate into the RTCA station located in the cell culture incubator with the A1 well facing inward. In the exp notes tab of the RTCA software, type in the experiment name, device serial number, and device type. Navigate to the cell tab of the RTCA software and select the layout tab.Highlight the wells to be used, and right click on the highlighted wells to make sure all the wells are turned on. Now go to the schedule tab of the RTCA software. Click Step_1, and click start or continue in the top left corner to obtain a background reading.The plate is now ready for cell seeding and removal from the RTCA station. Afterward, seed 10 to the fourth cells per well of HUVECs in the gold microelectrode plate with 50 microliters per well of conditioned medium. Place the plate back into the RTCA station located in the cell culture incubator, with the A1 well facing inward.Then in the schedule tab of the software, click add a step in the top left corner. Click on Step_2. Change the sweeps to 601, the interval to two minutes, and click apply.Click on Step_2, and click start or continue in the top left corner of the software. After 20 hours of incubation when the cell index 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.The plate is now ready to be removed from the RTCA station. To infect the HUVECs, retrieve the Zika virus stock from the minus 80 degrees Celsius freezer. Dilute the Zika virus stock with serum-free extracellular matrix in 1.5 milliliter centrifuge tubes to achieve multiplicities of infection of 0.1 and one.Next, discard the conditioned medium from each well, and rinse each well with 60 microliters of HBSS while using the side of the well. Add 40 microliters of diluted virus sample into the well, starting from the highest to lowest dilution, using the side of the well. Incubate the plate in a cell culture incubator at 37 degrees Celsius with 5%carbon dioxide to allow virus absorption.Swirl the plate five times every 15 minutes to allow virus absorption throughout the cell monolayer. After one hour of incubation, remove and discard the virus suspension from the lowest concentration to the highest. Wash the infected cells twice with 60 milliliters of HBSS.Overlay the wells with extracellular matrix supplemented with 2%FBS. For the positive control wells, dilute the thrombin in 20 units per milliliter with extracellular matrix supplemented with 2%FBS, and overlay the wells with thrombin containing extracellular matrix medium, using the side of the well. Now click on the schedule tab, and add a step in the top left corner.Navigate to Step_3, and modify the sweeps to 3, 601, and the interval to two minutes. Click apply, then click OK in the popped up window. Place the infected specialized gold microelectrode plate with the A1 well facing inward into the RTCA station located in the culture incubator.Finally, click on Step_3, and click start continue in the top left corner of the software. The cell index value of HUVECs infected with Zika virus P6-740 at a multiplicity of infection of 0.1 started to drop at 15 hours post infection, compared to the negative infection control. The HUVECs infected with a higher dose of Zika virus P6-740 at a multiplicity of infection of one showed a drop in the cell index at 11 hours post infection.At 24 hours post infection, there was a 5%and 7%drop in the normalized cell index value when the endothelial cells were infected at a multiplicity of infection of 0.1 and one respectively. A 50%reduction in the normalized cell index value was recorded at 96 hours post infection and 66.75 hours post infection upon infection at a multiplicity of infection of 0.1 and one respectively. The normalized cell index value reached its lowest point at 107 hours post infection, with a reduction of 51%and 60%upon infection at a multiplicity of infection of 0.1 and one respectively.The normalized cell index value was found to have a 4%and 13%increase from 107 hours post infection onward until the end of the experiment at 168 hours post infection. Before each RTCA experiment, a resistor verification run using the 96 well RTCA plate is essential to ensure each biosensor works well to produce reliable cell index values.

Research

JoVE Journal

Biology

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