ELECTROPHYSIOLOGY - Impedance Based Cellular Assay - ECIS

APPLICATIONS

• Attachment and spreading
  Traditional "counting attached cells assays" canonly quantify the number of cells attached to anyECM coating. ECIS® assays give feedback on thestrength of the attachment of the cells to the ECM.
• Barrier function
  Epithelial cells and endothelial cells regulate thepassage of molecules across cell layers. Diseases,especially vascular disease, occur when this functionis impaired.
• Cell migration
  ECIS instruments include an elevated field modeallowing for electroporation and wounding. TheECIS® wound is precisely defined, as it includes onlythose cells on the electrode. Additionally, with ECIS®the ECM protein coating is not scraped off and isunaffected by the current.
• Cell proliferation
  As cells proliferate two factors act to change theimpedance: cell number and cell morphology. Inmost instances the cells grow asynchronously andthe impedance gradually increases until a maximumwhen cells become confluent. The impedancechange is approximately linear with cell numberwhile the cells are sub-confluent.
• Signal transduction
  ECIS® is especially useful to monitor the signaltransduction pathways activated by G protein cou-pled receptors (GPCR). GPCR activation, regardlessof the second messenger, results in alterations ofthe cell’s cytoskeletal elements, causing morpholog-ical changes.
• Inflammation
  ECIS recovery-after-wounding assays allow for thediscovery of molecules which aid in the process oftissue repair. ECIS barrier function assays specificallymeasure the response of epithelial and endothelialcells to secreted cytokines and can give indirectinformation about the binding of immune cells tothe epithelium or endothelium.
• Differentiation and stem cell biology
  When cells differentiate they change their behaviorallowing ECIS® to follow the events of cell differen-tiation. While most tools available to characterizestem cells preclude their further use, the label-freenon-invasive nature of ECIS® allows for subsequentuse of characterized stem cells.
• Cell invasion
  ECIS® can distinguish between transmigrationmechanisms that leave the monolayer intact fromthose that disrupt the cell layer. Published examplesinclude metastatic cell and leukocyte trans-endothe-lial migration, as well as the migration of pathogenssuch as yeast, anthrax, streptococcus, plasmodium,trypanosomes, and spirochetes.
• Teer
  Continuous long-term measurement of TEER fromunder 10 to 10,000 ohm cm2 in up to 24 wellsusing commercially available 6mm membraneinserts. Fast barrier function dynamics can beaccurately monitored.
• Cell toxicity
  The ECIS® system has been used specifically toassess the cytotoxicity of a variety of toxicants.ECIS-based toxicity tests are far superior to simplecell death assays, because cell function is alsomonitored.

How ECIS works

Cell function modulates cellmorphology. ECIS® is capableof detecting and quantifyingmorphology changes in thesub-nanometer to micrometerrange. In ECIS® a small alternatingcurrent (I) is applied across theelectrode pattern at the bottomof the ECIS® arrays (direct currentcannot be used). This results in apotential (V) across the electrodeswhich is measured by the ECIS®instrument.

The impedance (Z) is determinedby Ohm’s law Z = V/I. When cellsare added to the ECIS® Arrays andattach to the electrodes, theyact as insulators increasing theimpedance. As cells grow andcover the electrodes, the currentis impeded in a manner related tothe number of cells covering theelectrode, the morphology of thecells and the nature of the cellattachment.

When cells are stimulated tochange their function, theaccompanying changes in cellmorphology alter the impedance.The data generated is impedanceversus time.

ECIS Data

The instrument can also use a range of AC frequencies from 100-100kHz and complex impedance measurement to determine different cell morphology parameters including barrier function, close contacts, and membrane capacitance.

How Frequencies Reveal Cell Behavior

To understand why AC frequency is important in ECIS® we have to consider how frequency affects the current paths of cell-covered electrodes. (Note: the total current is maintained constant and voltage changes are measured.) At relatively low frequencies (< 2,000Hz) most of the current flows in the solution channels under and between adjacent cells (red lines). At higher frequencies (> 40,000 Hz) more current now capacitively couples directly through the insulating cell membranes (green lines). The high frequency impedance is more affected by cell-cover-age, whereas the low frequency responds more strongly to changes in the spaces under and between the cells. With the more advanced Z instrument, where the impedance is broken down into its components (resistance and capacitance), quantitative information about the cells can be obtained by modeling (Giaever and Keese PNAS 1991). Using impedance data at multiple AC frequencies the ECIS® model calculates time course changes in:

• The barrier function (permeability) of the cell layer
• The degree of constricted flow of current under the cells
• The cell membrane capacitance

How Electrode Designs Reveal Aspects of Cell Behavior

Small Electrodes
Small electrodes (1E, 10E, 10E+ type arrays) and their layoutwithin the wells ensure that all current passes through the cellmonolayer. This allows the ability to analyze data with the ECIS®modeling software to determine barrier function, cell membranecapacitance as well as the spacing between the cell basal membraneand electrode.Keeping the total surface area of the electrodes small alsoallows for a relatively low AC current to generate the large electricfield necessary to either electroporate or kill the cells in migrationexperiments.Small electrodes also provide the ability to monitor theuncorrelated nano-scale morphological changes of individual orsmall populations of cells (<100), while larger or multiple elec-trodes provide the averaged morphological response of many cells(1000+).

Large Electrodes
Some experimental protocols, such as cell proliferation,require sparse inoculations leading to a variance of cell densityat the bottom of the well. Large electrodes (CP Array) or a largecollection of small electrodes (10E+ Array) increases the samplingsize resulting in less variability.