Studying Cell–Surface Interactions In Vitro: A Survey of Experimental Approaches and Techniques - Tissue Engineering: Cell-Surface Interactions for Tissue Culture - page 51

4.3.1 Monitoring Cell Spreading Kinetics with High Time Resolution
The AC frequency used for ECIS recordings is an extremely important parameter
as it determines the current pathway across the cell layer and, thus, the processes
that are mirrored in the time-resolved signal. For the electrode size discussed here,
the presence of the cells on the electrode surface alters the impedance of the
electrode in the frequency range between 10 Hz and 100 kHz. Within this
frequency range the current can flow along two different current pathways: (1)
around the cell bodies via the cell-surface junctions and the cell-cell junctions
into the bulk (paracellular pathway) or (2) across the plasma membranes and
directly through the cell bodies (transcellular pathway). The first case describes
approximately the flow of AC current for frequencies f \ 10 kHz whereas the
latter case describes the current pathway for frequencies f [ 10 kHz. Thus,
selecting the frequency determines where the current flows and as a consequence
which part of the cell or which cellular processes are actually probed. A rule of
thumb says that whenever morphological changes of the cells are the focus of
interest, the measurement should be made sensitive for changes in the paracellular
current pathway (f \10 kHz). When coverage of the electrode is of interest—as in
spreading and migration experiments—the measurement should be performed in
the transcellular frequency regime (f [ 10 kHz).
According to this rule of thumb, cell attachment and spreading is usually
recorded in the high-frequency regime ([10 kHz). At these frequencies, the main
part of the current passes capacitively through the cells, passing the basal and the
apical cell membrane. For a more detailed analysis of cell spreading kinetics, not
the impedance but the capacitive part of the complex impedance (cf. Fig. 9 )is
followed at a sampling frequency of 40 kHz. When the dielectric cell bodies attach
and spread on the electrode surface, they decrease the equivalent capacitance of
the electrode at 40 kHz proportionally to the fraction of the area they cover [ 46 ].
Measuring the capacitance of the system at 40 kHz as a function of time is
therefore the most direct approach to monitor the coverage of the electrode surface
with time, thus providing the spreading kinetics.
The following examples illustrate the analytical performance of the device.
Figure 10 a shows the kinetics of cell spreading for epithelial MDCK (Madin-
Darby canine kidney) cells seeded on ECIS electrodes that were pre-coated with
different ECM proteins [ 46 ]. The time courses of the individual electrode capac-
itances at a sampling frequency of 40 kHz show clear differences in the time to
confluence on these different ECM proteins. The electrode capacitance decreases
as the cells spread out on the electrode surface. Whereas cell attachment and
spreading is fastest on a fibronectin-coated electrode, spreading on the
non-adhesive serum albumin (BSA) takes significantly longer. Thus, the individual
spreading kinetics provide quantitative information on the interaction of the cells
under study with this particular protein coating.
Two parameters can be extracted to describe the adhesion and spreading
kinetics on the different proteins quantitatively: the parameter t 1/2 provides the
time required for half-maximal cell spreading and the parameter s stands for the