Antiviral Drug Studies

Anti-viral Drugs - virus in orange
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A major focus of virology is the development of antiviral drugs, a class of medication used to treat viral infections. Antiviral drug development is challenging as compounds must selectively inhibit viral replication without damaging the host. Viruses evolve rapidly, continuously presenting new targets for antiviral drugs and making previous drugs less effective. With the recent outbreaks of SARS-CoV-2 (COVID-19), Ebola, Zika, influenza, and other viral infections, efforts to develop broad-spectrum antivirals, which are effective against a wide range of viral infections, have increased to treat emerging and re-emerging viral infections.

Axion BioSystems' Maestro Z, Pro, and Edge systems offers impedance-based cell analysis for real-time, continuous, label-free monitoring of cytopathic effects (CPEs). Continuous data reveals the kinetics of in vitro viral infections for more complete dataset without the time- and cost-intensive process of repeating multiple endpoint assays.

Example Data

 

Screen antiviral drugs for SARS-CoV-2 (COVID-19)

COVID-19 is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2, SARS-CoV-2. While early symptoms may present as relatively mild, they can develop into acute respiratory distress syndrome (ARDS) as the virus damages and disrupts healthy lung tissue. Axion BioSystems' Maestro Z, Pro, and Edge systems makes it quick and easy to evaluate virus-induced cytopathic effects (CPE).

Anti-viral drug resulted in increased cell death of infected cells.
Anti-viral drug resulted in increased cell death of infected cells.
Anti-viral drug resulted in increased cell death of infected cells.
Anti-viral drug resulted in increased cell death of infected cells.
At their highest concentrations the drug was toxic to the cells.

Vero E6 cells were seeded into the CytoView-Z 96-plate and impedance was continuously monitored on the Maestro Z system. After 24 hours, test compounds and SARS-CoV-2 were added. Antiviral drugs resulted in a dose dependent decrease in cell death in the presence of virus with the exception of Drugs 3 and 4 at their highest concentrations, likely due to their own toxicity. Infected and uninfected wells show low and high impedance values, respectively.

 

Remdesivir prevents cell death from SARS-CoV-2 (COVID-19)

After 24 hours Remdesivir prevent cell death from SARS-CoV-2
Anti-viral drug treatment with remdesivir

After 24 hours growth, Vero E6 cell death was inhibited starting at 1.38 µM remdesivir. At 8.33 and 50.0 µM, remdesivir completely prevented cell death from SARS-CoV-2 as compared to uninfected cells (teal). Data courtesy of Drs. Alex Jureka and Chris Basler at Georgia State University.

Assay Steps

Anti-viral drug resulted in increased cell death of infected cells.

Getting started with Maestro Z, Pro, and Edge couldn't be easier. Culture your cells in an Axion multiwell CytoView-Z plate (Day 0). Load this plate into the Maestro system and allow the environmental chamber to automatically equilibrate. Observe cells adhering to the plate and proliferating as changes in the recorded impedance signal (Hour 0 up to 96). Add virus and antivirals, and track cytopathic effects (CPE) label free and in real time with the Impedance Module software.

 

Key Features

 

Maestro Z user

 

The advantages of measuring the effects of antiviral drugs on the Maestro Z, Pro, and Edge platforms:

  • Continuous cell monitoring – 96 simultaneous live recordings from your cells. Unlike plaque assays, now you can track cytopathic effects (CPEs) in real-time, even when you are out of the lab.

  • Analyze cell activity label-free – Perform noninvasive electrical measurements from the cultured cell population, circumventing the use of dyes/reporters that can perturb your cell model and confound results.

  • Precise assay environment – No need for an additional cell culture incubator, saving valuable lab space and money. The smart environmental chamber finely controls heat and CO₂ while rejecting electrical noise and mechanical vibrations.

  • See your cells – Sometimes you just want to look at your cells under a microscope. The CytoView-Z 96-well plates have a viewing window in each well which allows cell visualization.

  • Probe cell models in the same plate they were cultured in – Other higher throughput platforms (e.g. flow cytometry) often require cell samples to be transferred into a single-cell suspension before testing. In the case of adherent cells this is not ideal since they exist as a functional network of interlinked cells.

  • Smart phone App for your assay – You can't always be in the lab. But cytopathic effects (CPEs) seldom occur at convenient time points. The Maestro Z App allows you see results and system status.

  • It’s easy – With effortless assay setup and intuitive analysis software designed for quick export of figures and results, you can now focus on the science.

Technology

Impedance Technology

Impedance - General

 

Impedance: For real-time cell analysis

Impedance-based cell analysis is a well-established technique for monitoring the presence, morphology, and behavior of cells in culture. Impedance describes the obstruction to alternating current flow. To measure impedance, small electrical currents are delivered to electrodes embedded in a cell culture substrate. The opposition to current flow from one electrode to another defines the impedance of the cell-electrode interface. When cells are present and attached to the substrate, they block these electrical currents and are detected as an increase in impedance.

Impedance is sensitive to many aspects of cell behavior: attachment, spreading, shape,  cell-cell connections (e.g. tight junctions), and death. Even small transient changes, such as swelling or signaling, are detectable by impedance. Because impedance is noninvasive and label free, the dynamics of these changes can be monitored in real time over minutes, hours, or even days without disturbing the biology.

Interdigitated electrodes embedded in the cell culture substrate at the bottom of each well detect small changes in the impedance of current flow caused by cell presence, attachment, and behavior.

Interdigitated electrodes embedded in the cell culture substrate at the bottom of each well detect small changes in the impedance of current flow caused by cell presence, attachment, and behavior.

In the example below, the electrodes are initially uncovered before cells are added. The electrical current passes easily and the impedance is low. When cells begin to attach and cover the electrodes, less electrical current passes and the impedance is high. After dosing with a cytotoxic agent, cells die or detach, and the impedance decreases back towards baseline.

Cells on electrode
Dosing cells and recording impedance

Impedance measures how much electrical signal (orange arrows) is blocked by the cell-electrode interface. Impedance increases as cells cover the electrode and decreases back to baseline due to cell death.

 

Continuous cell monitoring

Many cell-based assays are endpoint assays, limited to a single snapshot in time. Repeating these assays at multiple time points can be labor intensive, time consuming, and costly. Key time points can be easily missed. Impedance-based cell analysis is nondestructive and label free, meaning that cellular dynamics can be monitored continuously.

The impedance assay can be used to characterize dynamic cell profiles, revealing how cells grow, attach, and interact over time. Each cell type exhibits a different cell profile, or “fingerprint”, of dynamic cell behavior. These profiles are sensitive to cell type, density, purity, and environmental factors. In this example, the Maestro Z impedance assay readily distinguished cell profiles across different cell densities and cell types.

HeLa cells were seeded on a CytoView-Z plate at varying densities and the impedance was continuously monitored by the Maestro Z
Impedance scaled proportionally with cell density and readily distinguished different densities of the same cell type.
Maestro monitored the growth of three cell types, HeLa, A549, and Calu-3, and readily distinguishes their distinct cell profiles over time.

(A, B) HeLa cells were seeded on a CytoView-Z plate at varying densities and the impedance was continuously monitored by the Maestro Z. Impedance scaled proportionally with cell density and readily distinguished different densities of the same cell type. (C) Maestro monitored the growth of three cell types, HeLa, A549, and Calu-3, and readily distinguishes their distinct cell profiles over time.

 

 

The Maestro Z impedance assay can also be used to capture the kinetics of cell responses to drugs or immune cell therapies. The kinetics, which cannot be captured by endpoint assays, often provide key insights into the efficacy of novel therapies. In the example below, the Maestro Z impedance assay was used to quantify the kinetics of cytotoxicity of chemotherapy agents.

A549 were dosed with Doxorubicin, vehicle (DMSO), or Tergazyme. Wells dosed with Tergazyme showed an immediate decrease in impedance, reflecting complete cell death.
Cells dosed with 1 uM doxorubicin reached 50% cytolysis at 31 hrs.
Higher doses of Doxorubicin resulted in a slower decrease in impedance and cell death

A549 cells were dosed with doxorubicin, vehicle (DMSO), or tergazyme. Wells dosed with tergazyme showed an immediate decrease in impedance, reflecting complete cell death. Higher doses of doxorubicin resulted in a slower decrease in impedance and cell death. Cells dosed with 1 μM doxorubicin reached 50% cytolysis at 31 hrs.

 

Different frequencies reveal cell properties

Impedance varies with frequency, such that different frequencies reveal different aspects of cell biology. The small currents used to measure impedance will always take the path of least resistance. At low frequencies, such as 1 kHz, the impedance of the cell membrane is relatively high, forcing the current to flow under and between the cells. Low frequencies provide details about barrier integrity, the presence of gap junctions, and transepithelial or transendothelial resistance (TEER).

At high frequencies, such as 41.5 kHz, the impedance (and capacitive reactance) of the cell membrane is relatively low. Thus, most of the current couples capacitively through the cell membranes, providing information about the cell layer such as confluency and coverage.

In other words, low frequencies are sensitive to “what” cells are there, whereas high frequencies are sensitive to “how many” cells are there. The Maestro Z impedance assay uses multiple frequencies to provide the most information about the cells, simultaneously, continuously, and in real time.

Multiple frequencies were used to simultaneously and continuously monitory the coverage and barrier function
TEER, measured at 1 kHz, reveals that only Calu-3 cells form a strong barrier, as they express tight junctions to block flow between neighboring cells.

Multiple frequencies were used to simultaneously and continuously monitor the coverage and barrier function (TEER) of Calu-3 and A549 cells. Coverage, measured as resistance at 41.5 kHz, increases over time for both cell types. TEER, measured at 1 kHz, reveals that only Calu-3 cells form a strong barrier, as they express tight junctions to block flow between neighboring cells.