Applied_Biophysics_ECIS_Real_Time_Cell_Growth_Monitoring__Cell_Attachment_

One of the most direct ECIS measurements is that of the attachment and spreading behaviours of cells. These measurements allow one to study and quantify the interaction of cultured cells with extracellular matrix (ECM) proteins and other macromolecules continuously and in real time. Traditionally, cell attachment and spreading measurements are labour intensive, requiring many manipulations of the cultures for microscopic evaluation of cell behaviour. With ECIS, these same measurements can be made in an automated approach without opening the door of the incubator. Different cell line behaviour The graph shows the attachment and spreading behaviours of two different cell types -namely BSC1 (African Green Monkey Kidney) and NRK (Normal Rat Kidney) cells. Cells were inoculated in ECIS wells at time zero in sufficient number to form a confluent cell layer (100,000 cells per cm). The more rapid spreading dynamics of the BSC1 cells is clearly evident; however upon reaching confluence, these two lines have very similar final impedance values. Each cell line studies in this manner will have its own characteristic shape and final values. In these data, the electrodes were not treated in any special manner before the inoculation but are coated with proteins adsorbed from the fetal bovine serum in the culture medium.


Spreading on defined protein coats The gold film making up the ECIS electrodes is hydrophilic and can readily be coated with defined adsorbed protein layers for cell studies. Here we see data of WI-38/VA13 cells attaching and spreading on electrodes pre-coated with different proteins. These protein layers were established by simply placing a droplet of protein solution (100 micrograms per ml in 0.15M NaCl) on the active electrode, allowing 10 minutes for adsorption, and then rinsing the wells before the addition of the cell suspension. Since only the 250 µm diameter is observed in these measurement, exceedingly small amounts of solution (only a few microlitres) are needed for the protein coats – saving valuable protein solutions. Measurements made at 40,000 Hz The cell attachment data shown thus far was gathered using AC currents at 4000Hz. The impedance values obtained at this intermediate ECIS frequency depend not only on the fraction of the electrode area covered with the spreading cells but also on the height of the spaces between the electrode and the basal cell membrane. When the capacitive portion of the impedance is studied at 40,000Hz, the measurements essentially report only the fraction of the electrode covered with cells and so mimic the data obtained with normal microscopy. This is shown here with the attachment of MDCK cells to electrodes coated with different protein layers as indicated (J. Wegener, et al, Exp. Cell Res. 259, 2000).

In another example, the attachment and spreading behaviour of MDCK cells on fibronectin pre-coated electrodes is attenuated with the addition of RGDS tetrapeptides to the culture medium.