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

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 tissues^1,2. 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 incubator at 37 °C with 5% CO₂ (Figure 1).

2. Setup....

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.......

Materials

Name	Company	Catalog Number	Comments
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