Barrier Function

Barrier application
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In the body, cellular barriers line many surfaces to protect the tissue within and control the passage of material in and out. These barriers are primarily composed of endothelial and epithelial cells. Biological events such as inflammation, infection, cancer metastases, leukocyte migration and many “normal” cues (e.g. GPCR) can alter the permeability of the barrier.

Axion BioSystems' Maestro Z, Pro, and Edge systems offers impedance-based cell analysis for real-time, continuous, label-free monitoring of your cells. Continuous data reveals the full time course of barrier disruption for a more complete picture without the time- and cost-intensive process of repeating multiple endpoint assays. 

 

Track barrier integrity in real time

Transepithelial electrical resistance (TEER) is commonly used to measure barrier integrity. The Maestro impedance platform can measure this resistance and expands on it with barrier index. By simultaneously measuring at different frequencies, the Maestro impedance platform calculates barrier index by normalizing TEER values for confluence, providing a sensitive and less variable measure to small, transient disruptions in barrier function. Barrier permeability can be altered by many common drugs and signaling molecules. Here, TEER was monitored continuously for Calu-3 cells in a 96-well CytoView-Z plate on the Maestro Z system. After reaching confluence, cells were dosed with cytochalasin D and ethanol. Cytochalasin D inhibits actin polymerization to increase tight junction permeability, as reflected by the rapid decrease in resistance. Ethanol acts more slowly on ZO-1 and claudin-1, two key tight junction proteins. The rapid disruption, as well as the distinct dynamics, are captured by TEER measurements on the Maestro Z.

TEER can be measured continuously.
Monolayers cultured for 14 days and treated with Ethanol which significantly reduced TEER

(A) Barrier permeability can be measured continuously from proliferation to confluence. (B) Calu-3 monolayers were cultured for 14 days in the CytoView-Z 96-well plate and then treated with cytochalasin D and ethanol, both of which significantly reduced the TEER.

 

Evaluate barrier function in disease-in-a-dish model

Barrier properties serve critical functions throughout the human body and may be disrupted by diseases, such as cystic fibrosis. Calu-3 is an immortalized cell line that produces tight junctions and expresses functional cystic fibrosis transmembrane conductance regulator (CFTR), which allows ion transport across the cell membrane upon β-adrenergic stimulation [Shen et al., 1994]. TEER measurements capture the function of CFTR, which opens conducting ion channels to facilitate mucus secretion, a process disrupted in cystic fibrosis.

Addition of isproterenol significantly reduced TEER
Measurements cconfirming CFTR as the mechanism for action for the change in TEER

(C) Addition of isoproterenol (teal, 100 nM) and forskolin (orange, 10 µM) significantly reduced TEER of the Calu-3 cells. CFTR(inh)-172 was then added to the plate at 30 and 300 µM causing a rapid dose-dependent reversal of the isoproterenol and forskolin response. (D) 5 minutes after CFTR(inh)-172 addition isoproterenol and forskolin responses are inhibited in a dose-dependent manner.

Barrier protocol assay steps

Getting started with Maestro Z, Pro, and Edge couldn't be easier. Culture your cells in an Axion multiwell CytoView-Z plate (Hour 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 to 24-72). Add test compounds as required. Track changes in barrier integrity in the CytoView-Z plate label-free and in real-time with the Impedance Module software

 

 

Maestro MEA user

 

The advantages of measuring barrier integrity on the Maestro Z, Pro, and Edge platforms:

  • Continuous cell monitoring – 96 simultaneous live recordings from your cells. Now you can track barrier integrity 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 changes in barrier integrity seldom occur at convenient time points. The Maestro Z App allows you to see live 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.

TEER Technology

TEER

 

TEER Assay: Barrier function on the Maestro Z

Endothelial and epithelial cells form barriers throughout the body that serve to protect and compartmentalize, regulating what gets in and out of the tissue. Endothelial cells line the inside surfaces of the body, such as the inner layer of blood vessels, while epithelial cells line the outside surfaces, such as skin and most organs. Both cell types express tight junctions, allowing them to link tightly with neighboring cells to form a selectively permeable barrier.

These important barriers can be disrupted by disease, injury, infection, or drugs. Thus, measuring barrier integrity and permeability is vital for understanding disease and predicting drug behavior.

The Maestro Z can continuously monitor barrier integrity via measurements of electrical resistance across the cell layer, a technique known as transepithelial or transendothelial resistance (TEER).

 

How does TEER work on the Maestro Z?

The Maestro Z uses impedance technology to measure TEER, which is used to assess the barrier integrity of cells. TEER quantifies how much of an electrical signal is blocked by a cell layer when a small AC current is passed from one electrode to another. Since impedance is noninvasive and label free, barrier integrity can be continuously monitored for minutes, hours, or even days without disturbing the cellular biology.

Traditionally, TEER is measured by placing two chopstick-style electrodes on either side of a transwell insert with a confluent cell layer. Manual methods, where the electrodes are placed well-by-well, are highly labor intensive. Even automated TEER methods are typically constrained to lower throughputs, like 24-well plates. In contrast, TEER on the Maestro Z is high throughput and hands free, allowing measurements across 96 wells simultaneously.

How TEER is measured on a plate

TEER on Maestro Z. Electrodes embedded in the cell culture substrate at the bottom of each well detect small changes in the impedance of current flow. Barriers, such as tight junctions between cells, resist current flow, leading to an increase in TEER measurements of barrier function.

 

Measure coverage and TEER in the same assay

Traditional TEER measurements require a fully confluent cell layer to accurately measure barrier function. In contrast, the Maestro Z tracks coverage and TEER simultaneously and continuously by measuring impedance at multiple frequencies.

Measuring impedance at low frequency is highly sensitive to the intercellular barrier formed by tight junctions and the paracellular barrier formed by cell membranes. On the other hand, measurements at higher frequency can be used to quantify coverage of the well bottom. In other words, low frequencies are sensitive to “what” cells are there, whereas high frequencies are sensitive to “how many” cells are there. By calculating the ratio of resistance at these two frequencies, the Impedance Module can normalize TEER to the cell coverage and give the result as barrier index.

In this example, Maestro Z resistance measurements were used to continuously and simultaneously monitor cell coverage and barrier function of two human lung epithelial cell lines. Both cell lines reach full coverage within 24 hours. However, only the Calu-3 cells, which express tight junctions, produce a significant low-frequency TEER signal, indicating a strong cellular barrier

Measurement of TEER or impedance as cell coverage increases over time
TEER increases with the formation of tight junctions.
Barrier index

(A) Coverage, measured as resistance at 41.5 kHz, increases over time for both Calu-3 and A549 cells. (B) TEER, measured at 1 kHz, reveals that only Calu-3 cells form a strong barrier though, as they express tight junctions to block flow between neighboring cells. (C) Barrier Index both reveal that only Calu-3 cells form a strong barrier though, as they express tight junctions to block flow between neighboring cells.

 

Real-time detection of rapid barrier disruption

In addition to long-term monitoring, TEER on the Maestro Z is sensitive to small, transient disruptions to barrier function. Many signaling molecules in the body can alter barrier permeability. TEER on the Maestro Z can capture these disruptions in real time without being limited to a single endpoint measurement.

Here, TEER was monitored continuously for Calu-3 cells in a 96-well CytoView-Z 96-well plate on the Maestro Z. After reaching confluence, cells were dosed with cytochalasin D and ethanol. Cytochalasin D inhibits actin polymerization to increase tight junction permeability, as reflected by the rapid decrease in TEER. Ethanol acts more slowly on ZO-1 and Claudin-1, two key tight junction proteins. The rapid disruption, as well as the mechanistically distinct dynamics, are captured by TEER measurements on the Maestro Z.

Teer increases as confluence is reached and then dips when media is changed.
TEER is highly sensitive to transient drug-induced changes

(A) TEER can be measured over the course of proliferation and barrier formation. (B) TEER is highly sensitive to transient drug-induced changes in barrier permeability, such as those induced by cytochalasin D and ethanol.

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.