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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

1. Cell preparation

  1. Preparation of HUVECs
    1. Grow 7.5 x 105 HUVEC cells/mL in a 75 cm2 (coated with 20 µg/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.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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).

....

Materials

NameCompanyCatalog NumberComments
1.5 mL microcentrifuge tubeNest615601
37 °C incubator with 5% CO2SanyoMCO-18AIC
75 cm2 tissue culture flask Corning430725U
Antibiotic solution penicillin-streptomycin (P/S)Sciencell0503
Biological safety cabinet, Class IIHoltenHB2448
Collagen Type 1SIgma-AldrichC3867
Endothelial cell growth supplement (ECGS)Sciencell1052
Endothelial cell medium (ECM)Sciencell1001
E-plate 96Agilent Technologies, Inc.05232368001
Fetal bovine serum (FBS)Sciencell0025
Hanks' Balanced Salt Solution (HBSS)Sigma-AldrichH9394
HemocytometerLaboroptik LTDNeubauer improved
Human Umbilical Vein Endothelial Cells (HUVECs)Sciencell8000
Inverted microscopeZEISSTELAVAL 31
Latitude 3520 LaptopDell -
Multichannel micropipette (10 - 100 µL)Eppendorf3125000036
Multichannel micropipette (30 - 300 µL)Eppendorf3125000052
Reagent reserviorTarsonsT38-524090
RTCA resistor plate 96Agilent Technologies, Inc.05232350001
RTCA Software Pro (Version 2.6.1)Agilent Technologies, Inc.5454433001
Single channel pipettes (10 - 100 µL)Eppendorf3123000047
Single channel pipettes (100 - 1000 µL)Eppendorf3123000063
Single channel pipettes (20 - 200 µL)Eppendorf3123000055
xCELLigence Real-time cell analyzer SP (Model: W380)Agilent Technologies, Inc.00380601030https://www.agilent.com/en/product/cell-analysis/real-time-cell-analysis/rtca-analyzers/xcelligence-rtca-sp-single-plate-741232

References

  1. Alberts, B., et al. Cell junctions. Molecular Biology of the Cell. 4th Edition. , (2002).
  2. Pugsley, M. K., Tabrizchi, R. The vascular system: An overview of structure and function. Journal of Pharmacological and Toxicological Methods. 44 (2....

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