Most cell-based assays are single-point tests, which provide a "snapshot" of the experiment and often involve labeling and destruction of the cells. This is a major limitation, as cells are living entities and biological and cellular processes are dynamic. In order to fully understand, appreciate and measure biological and cellular processes, it is necessary to use a system that is non-invasive and provides kinetic data regarding the dynamic nature of cellular response to certain challenges such as drug treatment or stimulation with a growth factor.

     The RT-CES (Real-Time Cell Electronic Sensing) system (ACEA Biosciences, San Diego, CA; www.aceabio.com) allows for label-free and dynamic monitoring of cell-based assays (Figure 1A). The RT-CES system utilizes an electronic readout of impedance to non-invasively quantify cellular status in real-time.^(1-3) Cells are seeded in E-Plate microtiter plates (ACEA Biosciences) which are integrated with microelectronic sensor arrays (Figure 1B). The interaction of cells with the microelectrode surface leads to the generation of a cell-electrode impedance response, which indicates the status of the cells in terms of morphology, quality of adhesion and number. In this article we'll provide some brief examples of the utility of the RT-CES system in different cell-based assays and, where appropriate, we'll highlight the advantages offered by RT-CES system over existing technology.

Applications

Figure 1a. ACEA Biosciences RT-CES system.

In order to observe the kinetics of drug interaction with target cells using the RT-CES system, H460 lung cancer cells were seeded in E-Plates and continuously monitored until the cells reached the log growth phase at which point different concentrations of an anti-mitotic drug were added to the cells at the indicated final concentration. As shown in Figure 2, the drug at the highest concentration initially induces a cytotoxic effect which is mainly due to cell death as judged by Annexin V staining (data not shown). Remarkably, the cells recover from the initial cytotoxic effect of the drug and start to re-proliferate. While it remains to be determined if this phenomenon is due to metabolism and inactivation of the drug or due to the emergence of a drug-resistant subpopulation, this experiment points out the advantages of real-time measurement offered by the RT-CES system and allows the user to the opportunity to observe and assess the entire history of drug interaction with the target cells. The phenomenon observed in Figure 2 would have been easily missed by traditional single-point assays such as MTT.

Figure 1b. ACEA Biosciences proprietary 96x and 16x E-Plate devices.

     G-protein coupled receptors (GPCRs) are an important target for pharmaceutical drug development and about 50% of drugs currently on the market are targeted against GPCRs.⁽⁴⁾. In order to determine if the RT-CES system can be used to dynamically monitor the functional activation of GPCRs, CHO-K1 cells expressing the human H1 histamine receptor were seeded on ACEA E-Plates and stimulated with the indicated final concentrations of histamine (Figures 3A and 3B). Histamine induced a transient and dose-dependent cellular response. Plotting the log histamine concentration versus the maximal cellular response allows for the generation of a dose-response curve. The contributions RT-CES offers for GPCR cell-based assays are several fold. First, since the readout is non-invasive, the cells can be stimulated multiple times in order to assess desensitization or cross-talk with other receptor types. Second, in addition to histamine receptor, which is coupled to the Gq subfamily of G-proteins, cells expressing other receptors coupled to the Gs and Gi families can also be monitored on the RT-CES system. Traditionally, several different instruments would be required to carry out assays for GPCRs coupled to different signaling pathways.⁽⁴⁾ Third and most importantly, cells expressing endogenous GPCRs, including primary cells, can also be used with the RT-CES system precluding the need for overexpression of exogenous GPCRs or engineering the cells to express promiscuous G-proteins. This allows the evaluation of the receptors in physiologically appropriate cell types. In addition to GPCRs, other receptor types that are important drug targets such as receptor tyrosine kinases,⁽⁵⁾ can also be monitored in functional cell-based assays on the RT-CES system (Figures 4A and 4B).

     Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells have been implicated in the recognition and annihilation of tumor cells.⁽⁶⁾ Both CTL and NK assays are another area in which the RT-CES system offers an alternative with significant contributions in ease of use and quality of data. Traditionally, these assays are conducted by radio-labeling the target cells and then adding the effector cells, such as CTLs or NKs. The extent of CTL and NK activity towards target cells is assayed by the release of radioactivity by the target cells upon cytolysis. These assays are cumbersome not only due to the use of radioactivity but also due to high background because of the tendency of the label to permeate out of the target cells. In order to demonstrate the utility of the RT-CES system in NK-mediated cytolysis experiments, A549 lung carcinoma cells were seeded on E-Plates (Figure 5). At the indicated time, the cells were incubated with NK-92 cells at different effector to target (E/T) ratios. The cytolysis induced by the NK-92 cells can be dynamically monitored using the RT-CES system. As shown in Figure 5, higher E/T ratios resulted in enhanced cytolysis of target cells. The lysis of target cells leads to loss of interaction with the microelectrodes at the bottom of the well and hence decreases the impedance signal.

click the image to enlarge Figure 2. Dynamic monitoring of H460 response to paclitaxol on the system.	click the image to enlarge Figure 3a. Dynamic monitoring of CHO-K1 response to histamine on the RT-CES system.
click the image to enlarge Figure 3b. Dose response curve of CHO-K1 cells at varying concentrations of histamine	click the image to enlarge Figure 4a. Dynamic monitoring of COS7 cellular response to EGF and Insulin on the RT-CES system.
click the image to enlarge Figure 4b. Dose response curve of COS7 cells at varying concentrations of insulin.	click the image to enlarge Figure 5. NK-92 mediated cytolysis of A549 cells at different effector/target ratios on the RT-CES system.

Summary
The convergence of label-free technology coupled with non-invasive readout offered by the RT-CES system culminates in information rich, high-content data. Instrument applications include cell-based assays for proliferation, cytotoxicity, receptor-ligand interaction, CTL and NK.

About the author
Yama Abassi, PhD is the Associate Director of Cell Biology and Application Development at ACEA Biosciences. More information is available from Jerome David (email: jdavid@aceabio.com). Dr. Abassi acknowledges the contributions of ACEA's management and scientific staff.bagey

References
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