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 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.
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.
1. Cell preparation
2. Setup of RTCA experiment
3. Viral infection in HUVECs
4. Data analysis and exporting data
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 infection control (brown line; Figure 4). As for HUVECs infected with a higher dose of ZIKV P6-740 at an MOI of 1 (blue line), the CI value started to drop as early as at 11 HPI. At 24 HPI, there was a 5% and 7% drop in the normalized CI value (0.949, 0.929) when ECs were infected with ZIKV at an MOI of 0.1 and 1, respectively. A 50% reduction in the normalized CI value was recorded at 96 HPI and at 66.75 HPI upon ZIKV infection at an MOI of 0.1 and 1, respectively. The normalized CI value reached its lowest point at 107 HPI, where there was a reduction of 51% and 60% (0.4915, 0.3984) upon ZIKV infection at an MOI of 0.1 and 1, respectively. The normalized CI value was found to have a 4% and 13% increase from 107 HPI onward until the end of the experiment at 168 HPI (0.5128, 0.4571). The black line shows the effects of thrombin used as a positive control, where the CI values remained low throughout the experiment, mimicking the disruption of tight junctions, and induced vascular leakage35.
Figure 1: Human umbilical vein endothelial cells (HUVECs). The figure is in 40x magnification to view the rate of confluency. Please click here to view a larger version of this figure.
Figure 2: Screenshots of the Cell and Treatment tab under the Layout tab on the RTCA software. (A) In the Layout tab, click the Cell tab. (B) To enter target cell information and determine the well type for each well, highlight the well and type the target cell name, number, and color at the bottom of the software. Click Apply. (C) Click the Layout tab under the Treatment tab. (D) To enter treatment information, highlight the well and type in the treatment name, concentration of treatment, unit, and color. Click Apply. Please click here to view a larger version of this figure.
Figure 3: Screenshots of the Data Analysis tab in the RTCA software for cell index normalization. (A) In the Y Axis drop-down box, select Normalized Cell Index. (B) In the X Axis section, select the normalization time. (C) Screenshot of the Schedule tab in the RTCA software. The normalization time is indicated at the Step_2 total time. Please click here to view a larger version of this figure.
Figure 4: Normalized cell index versus time plot of Zika virus (ZIKV) infection in human umbilical vein endothelial cells (HUVECs). The specialized gold-microelectrode plate was coated with 20 µg/mL collagen type 1. The HUVECs were seeded at a density of 1.0 x 104 cells/well. ZIKV P6-740 (MOI: 0.1, 1) was used for infection. Light blue: ZIKV P6-740 (MOI: 0.1); blue: ZIKV P6-740 (MOI: 1); black: thrombin as the positive control (20 U/mL); brown: negative infection control. Each data point signifies the average and was analyzed in triplicate. Please click here to view a larger version of this figure.
Supplementary Figure 1: Schematic drawing on the technique used in the electrical biosensor impedance based RTCA. A side view of the well plate; at the bottom of the wells there are gold-plated negative and positive terminals. As the instrument measures CI in the microwell that contains culture media that allows to conduct electricity, the electron flows from the negative to the positive terminal. When it is a blank control, electron flow is smooth, hence no impedance is recorded. When the cells are seeded, and as more and more cells attach to bottom of well, impedance in electron flow is present and recorded by the computer. Using this impedance value, the instrument converts it to a CI value, indicating the number of cells attached to bottom of the well (adhesion). Created with BioRender. Please click here to download this File.
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 breakdown of the endothelial barrier38,39. CPEs in HUVECs can be described as monolayer cell detachment and floating from the tissue culture vessel. The infected cell morphological changes, such as detachment and floating of adherent cells, can be continuously detected using the RTCA method. This technique is based on electrical impedance, which allows the monitoring of cells in real time up to every few seconds. It allows the quantification of cell proliferation, morphological changes, and in the case of HUVECs, it can be used to monitor changes in endothelial integrity and cell barrier function40 upon stimulation.
In this study, the critical step is the setup of experiment, where the 96-well resistor plate is utilized. This is to ensure that all connections of the analyzer are well connected to the plate. In RTCA, the measured impedance value is converted as a CI value to differentiate the differences in cell number, adhesion degree, cellular morphology, and cell viability41. The drop in the recorded impedance by the RTCA analyzer, which translates into a drop in the CI value, suggests a reduced cell number, weaker adhesion degree, and substantial changes in cellular morphology42. Therefore, it is also important to test the suitability of cell lines and optimum cell numbers to be used in the RTCA experiment. It is critical to determine if the desired CI index can be achieved before the execution of testing on the cells using the RTCA system. Additionally, this also helps to determine if the RTCA system is suitable to monitor cell growth and cell death profiles of selected cell types. In our study, a steep drop in CI values was observed when HUVECs were infected with ZIKV P6-740 at MOI of 0.1 and 1. The steep drop in CI value, hence suggesting that HUVEC cellular morphological changes occurred following infection, and this led to the disruption of tight junctions and the EC vascular integrity.
Conventionally, CPEs caused by the viral infection of cells in vitro are estimated based on microscopic observation of the infected cells. Additionally, some other assays, such as the plaque assay, allow the visualization of CPEs (such as cell detachment) and are used to measure the extent of the CPEs42. However, these methods have limitations in that they only provide information on discrete time points of the cellular morphological changes (endpoint assays). Other assays, such as immunofluorescence staining, rely on combinations of specific antibodies tagged with a fluorophore to visualize the morphological changes43. Although the immunofluorescence assay has a broad capability that permits the visualization of many components in any given tissue or cell type, this technique does not allow real-time monitoring and detection during viral infection. In contrast, the RTCA system records the real-time cell morphological changes during the viral infection, allowing the capture of transient effects, such as transient vascular leakage, that could be missed by endpoint assays. For example, endpoint assays would fail to detect the data and morphological changes as early as 60 HPI, 80 HPI, and 100 HPI. In addition to RTCA, trans-epithelial electrical resistance (TEER) measurement is another commonly used method to monitor cell-to-cell interactions, where the electric resistance across the cell monolayer is measured. However, the TEER requires several repeated measurements over time in order to monitor live cell activities and is often performed on a smaller number of samples or wells at a time. As a result, TEER typically has lower throughput compared to RTCA, which can run up to 96 wells simultaneously44.
Despite its benefits, the RTCA system has several limitations. The main limitation of the RTCA system is the costly equipment and consumables. The specialized gold-microelectrode plates used by the RTCA system are relatively costly, single-use, and disposable45. Additionally, the cell morphological changes cannot be imaged and captured on a recorder (camera), nor can the assay capture the accumulation of the virus antigen or changes in the cell surface protein, such as VE-cadherin in HUVECs46. As a result, the RTCA cannot visualize the cell morphological changes that would provide additional useful information to elucidate the disease pathogenesis. During the assay or model establishment, additional staining verification, such as VE-cadherin staining for HUVECs, would be useful to validate the measurements obtained. Furthermore, the RTCA system is limited to adherent cell lines, as it requires the attachment of cells at the bottom of the wells for the impedance to be recorded. Therefore, the reported assay cannot be used for non-adherent cell lines46.
In conclusion, we have described a real-time cell analyzing protocol using the RTCA instrument to monitor changes in endothelial cells upon ZIKV infection in 96-well format. This method offers several advantages over conventional endpoint assays, in that it allows for a noninvasive and non-label intensive approach for continuously monitoring cellular morphological changes in real time. This assay can also be used to analyze vascular integrity changes of other cell lines in different experimental setups.
The authors have nothing to disclose.
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).
1.5 mL microcentrifuge tube | Nest | 615601 | |
37 °C incubator with 5% CO2 | Sanyo | MCO-18AIC | |
75 cm2 tissue culture flask | Corning | 430725U | |
Antibiotic solution penicillin-streptomycin (P/S) | Sciencell | 0503 | |
Biological safety cabinet, Class II | Holten | HB2448 | |
Collagen Type 1 | SIgma-Aldrich | C3867 | |
Endothelial cell growth supplement (ECGS) | Sciencell | 1052 | |
Endothelial cell medium (ECM) | Sciencell | 1001 | |
E-plate 96 | Agilent Technologies, Inc. | 05232368001 | |
Fetal bovine serum (FBS) | Sciencell | 0025 | |
Hanks' Balanced Salt Solution (HBSS) | Sigma-Aldrich | H9394 | |
Hemocytometer | Laboroptik LTD | Neubauer improved | |
Human Umbilical Vein Endothelial Cells (HUVECs) | Sciencell | 8000 | |
Inverted microscope | ZEISS | TELAVAL 31 | |
Latitude 3520 Laptop | Dell | – | |
Multichannel micropipette (10 – 100 µL) | Eppendorf | 3125000036 | |
Multichannel micropipette (30 – 300 µL) | Eppendorf | 3125000052 | |
Reagent reservior | Tarsons | T38-524090 | |
RTCA resistor plate 96 | Agilent Technologies, Inc. | 05232350001 | |
RTCA Software Pro (Version 2.6.1) | Agilent Technologies, Inc. | 5454433001 | |
Single channel pipettes (10 – 100 µL) | Eppendorf | 3123000047 | |
Single channel pipettes (100 – 1000 µL) | Eppendorf | 3123000063 | |
Single channel pipettes (20 – 200 µL) | Eppendorf | 3123000055 | |
xCELLigence Real-time cell analyzer SP (Model: W380) | Agilent Technologies, Inc. | 00380601030 | https://www.agilent.com/en/product/cell-analysis/real-time-cell-analysis/rtca-analyzers/xcelligence-rtca-sp-single-plate-741232 |