Monitoring the Barrier Function of Cell Monolayers using ECIS®


The ECIS Method

Introduction

In vivo, barriers are provided by monolayers of epithelial or endothelial cells. These cell layers play a key role regulating the free movement of molecules between different tissues and/or interstitial compartments. In many diseases as well as in inflammation, these barriers become compromised, and hence, measuring their permeability is of considerable interest to cell biologists and the health community in general.

Most epithelial and endothelial cells types can be cultured in vitro to form confluent monolayers where it is possible to measure the barrier function afforded by these cell layers. In addition, with the right tools, dynamic changes of the layers can be followed when the cellular environment is altered by exposure to compounds or physical changes such as shear stress.

Measurements
We offer four different real-time approaches for monitoring the barrier function (permeability) of cell monolayers using the ECIS® instrumentation.

Two of these use standard ECIS arrays and two use commercially available filter supports that fit into the ECIS trans-Filter Adapter. The selection of the method of choice will depend upon the magnitude of the barrier function and differences in the behavior of cells upon solid versus filter supports.

Let's first consider the importance of the AC frequency used in the ECIS constant current measurement. The cartoon below shows a cross section of a confluent cells layer where the path of the current is indicated by the arrows. The red arrows represent ion current flow coming from the electrode interface, moving in the solution spaces between the electrode and the basal plasma membrane and then moving through the paracellular passage between the cells (the barrier function). The green arrows on the other hand indicate a path of current that is possible as ECIS is an AC measurement, and current can couple capacitively through the cell membranes.

Current will always follow the path of least resistance. At high AC frequency (40,000 Hz) the impedance (capacitive reactance) of the membrane is relatively small, and current mainly capacitively couples through the insulating cell membranes with little current passing through the paracellular path. At low AC frequency on the other hand, the membrane impedance is high, and most of the current now flows under the cells and through the tight spaces between the cells (the solution path).

As a result of this difference, high frequency impedance can be used to monitor the establishment of a confluent layer, and low frequency impedance can be used to monitor the solution paths about the cells and hence the layer's barrier function.

Epithelial cells with tight junctions

The data below report the changes of impedance in two duplicate wells following inoculation of MDCK II cells (canine kidney epithelium). The micrographs show that the cell layer is in place and confluent about 3 hours after inoculation, and this is conveyed by the plateau in the impedance at 40,000 Hz. Measuring the same wells at 400 Hz we see the formation of the barrier function

is not completed until about 10 hours after inoculation. This is also confirmed microscopically using stains for the junctional proteins E-cadherin and ZO-1 (zona occludens protein). (Data courtesy of Professor Joachim Wegener, Univ. of Regensburg) For tight epithelial cells, impedance at low AC frequencies provides a very effective measure of the layer's barrier function.

Endothelial cells

With endothelial cells the barrier function is relatively low compared with epithelial cells with tight junctions, and the resistive portion of the ECIS impedance is commonly used as a measure of barrier function changes. This assay for endothelium was introduced in 1992 (Tiruppathi et.al.) and relied on the use of complex impedance measurements of endothelial cells grown upon ECIS electrodes. In this first study, the response of bovine pulmonary microvascular endothelial cells exposed to thrombin was evaluated (see figure).

Since this first study, the resistive portion of the impedance at 4000 Hz has been used to monitor real-time changes in endothelial permeability in several laboratories, and the assay appears in many peer-reviewed publications. ECIS used in this manner has increasingly become an attractive alternative to techniques involving the use of membrane inserts and Ussing chambers. In addition to its convenience in gathering barrier function data with a minimum of labor, the measurement requires no tagged compounds and associated sampling/measuring techniques.

Flow arrays measurements:

Another important feature of ECIS system is its ability to follow the barrier function of endothelial cell monolayers as they are exposed to flow conditions. This is accomplished using ECIS flow arrays that permit measurements of cell layers subjected to shear stress as high as those experienced in vivo.

Traditionally, in vitro barriers are quantified by growing confluent cell layers upon filter supports, where medium on both the apical and basal side of the cell layer can be altered. The filters commonly used for this application are porated with pores etched in plastic films running vertically between the faces of the filter. This type of filter is generally preferred by cell biologists as not only can the pore size and density be tightly controlled, but, unlike most filters, the material is transparent making microscopic observation of the cells possible.

To make filter measurements with the ECIS instrumentation, a trans-Filter Adapter is used. This device (shown in the figures) fits into the standard 16 well station and converts it into a multiple well, filter-based device for real-time measurements of barrier function. Filter inserts are placed in the wells and a common dipping electrode contacts solution in the small inner wells above the filter. At the base of the wells are large electrodes that can be individually addressed by the instrumentation. Special software converts ECIS impedance measurements made with the device into time course changes in TEER (trans epithelial electrical resistance) values.

Data collected with the ECIS instrumentation from a Corning Costar filter (# 3413) with 0.4 micrometer diameter pores at a density of ~108 pores/cm2 is shown. At time zero a high inoculum of MDCK II epithelial cells was added to the inner well and the TEER monitored over time. The trans-Filter Adapter will accept all commercial filter inserts manufactured for a standard 24 well plate, and so a variety of pore sizes and densities can be chosen.

Below is a 3D plot of the first 20 hours of the experiment showing data from the blue trace (1.0X inoculation). Notice how the four lowest frequencies (circled) are in good agreement with curves peaking around 370 ohm-cm2.

When using either the ECIS solid substrate array or measurements with porated filters, there is a component to the solution-path impedance other than the paracellular path - namely the narrow solution passageway sandwiched beneath the cell and its substrate. (Lo et.al.).

This is illustrated in the figure showing a cross section of an epithelial cell with tight junctions to its neighboring cells. In this case we illustrate a porated filter as the cell substrate, but one could just as well have an ECIS solid substrate beneath the cells. In either situation there is a constricted space beneath the cell (shaded in blue) that must be first traversed before current (or tracers) can pass though the paracellular space (the barrier of interest shaded in red).

For many epithelial cell layers, the contribution of this passage under the cell is small relative to the tight paracellular passage and probably does not play a major role in the measurement of barrier function. However, with cells having weaker barrier functions (e.g. most endothelial layers), this may present a complication in making a clean measurement of the paracellular path (link to table)

Fortunately, using the ECIS mathematical modelling software (Giaever and Keese), it is possible to separate these two aspects of the measurement. With this feature, time course changes in both the barrier function (Rb) and the passage beneath the cells (alpha) can be independently presented. The modelling software comes standard with the ZTheta instrumentation.

An example is shown of modeled data software, where time course changes of Rb are reported for microvascular endothelial cells exposed to thrombin. The value of alpha remains essentially constant throughout the measurements and is also plotted for comparison.

There is another means to eliminate the contribution of the spaces beneath the cells and measure only the paracellular space; this is accomplished using tortuous-path rather than porated filters.

As shown in the figure, without the vertical pores, current can now flow with little hindrance directly to the cell-cell junctions - essentially eliminating this space (alpha) from the measurement.

Tortuous-path filters for 24 well plates are available from several vendors and can be used with the ECIS trans-Filter Adapter and the special filter software.

 

Key References

Tiruppathi, C., Malik, A.B., Del Vecchio, P.J., Keese, C.R., and Giaever,I., Electrical method for detection of endothelial cell shape change in real time, PNAS USA 89, 7919-7923 (1992).

Lo, C.M., Keese, C.R., Giaever, I., "Cell-substrate contact: Another factor may influence transepithelial electrical resistance of cell layers cultured on permeable filters", Experimental Cell Research, 250 (2): 576-580 (1999).

Giaever, I. and Keese,C.R., PNAS USA 88, 7896 (1991).

 

 

Barrier Function of Cell Layers
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Sandoval, R., A. B. Malik., R. D. Minshall, P. Kouklis, C. A. Ellis, and C. Tiruppathi. Ca 2+ signaling and PKC? activate increased endothelial permeability by disassembly of VE-cadherin junctions, J. Physiol. (London).533:433-445, (2001).

Minnear FL. Patil S, Bell D, Gainor JP, Morton CA. Related Articles Platelet lipid(s) bound to albumin increases endothelial electrical resistance: mimicked by LPA, Am J Physiol Lung Cell Mol Physiol. 281(6):L1337-44, (2001).

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Lo, C.M., Keese, C.R., Giaever, I. , Cell-substrate contact: Another factor may influence transepithelial electrical resistance of cell layers cultured on permeable filters, Experimental Cell Research, 250 (2): 576-580 (1999).

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Tiruppathi, C., Malik, A.B., Del Vecchio, P.J., Keese, C.R., and Giaever,I., Electrical method for detection of endothelial cell shape change in real time, PNAS USA 89, 7919-7923 (1992).