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Label-Free and Dynamic Monitoring of Cell-Based Assays

Yama Abassi
ACEA Biosciences, San Diego, USA

Introduction

The xCELLigence system from Roche Applied Science, originally invented by the US company ACEA Biosciences, allows for label-free and dynamic monitoring of cell-based assays. The xCELLigence system utilizes impedance readout to non-invasively quantify cellular status in real-time [1]. The cells are seeded in 96-well microtiter E-Plates with integrated microelectronic sensor arrays at the bottom of each well. Application of a low-voltage (20 mV) AC current generates an ionic environment inside the wells of the E-Plates that is a reflection of the number of cells in the well, the morphology of the cells, and the strength of cell attachment (Figure 1). A number of cell-based applications have been developed for the ­xCELLigence system including:

  • Cellular quality control
  • Cell proliferation
  • Compound-, cell- and virus-mediated cytotoxicity
  • Barrier function
  • Cell adhesion and spreading
  • Receptor-mediated signaling

To demonstrate the utility of the ­xCELLigence system for cell-based assays, a subset of these applications are highlighted in this article.

Applications of the xCELLigence System

Most assay formats for cell-based assays are end-point assays which provide a “snapshot” of the experiment and often involve labeling and destruction of the cells. This is a major limitation of the current assays since cells are living entities and biological and cellular processes are not static but dynamic. Therefore, in order to fully understand and measure biological and cellular processes, it is pertinent 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 application of cell-sensor impedance technology for cell-based assays provides several fundamental advantages. First and foremost, cell-sensor impedance allows the non-invasive monitoring of cells during the entire experiment, including cell attachment, proliferation, and confluence. Overall, the real-time monitoring provides excellent quality control for the cells in the same assay and between different assays. Moreover, because of the real-time continuous display of the acquired data, manipulations and treatments of the cells can be performed with more confidence than by presuming that the cells are at the appropriate stage for treatment. Because of the unique nature of impedance readout combined with real-time data acquisition and display, each treatment leads to unique response profiles which were proven to be predictive in terms of mechanism. Finally, because the readout is non-invasive, traditional end-point assays can still be used in conjunction with impedance readout to determine the optimal time point for performing measurements with traditional end-point assays.

Dynamic monitoring of cell proliferation and cytotoxicity

Cell proliferation assays using various cancer cell line models are among the basic assays used to assess the efficacy and potency of different anti-cancer compounds. The ­xCELLigence system can be used to quantitatively and dynamically monitor cell proliferation and cytotoxicity. Each cell displays a unique proliferation curve depending on the seeding density (Figure 2a). This characteristic growth signature curve can be used as a measure of quality control for the cell line of interest to ensure consistency in the same experiment and between different experiments (Figure 2a) [2].

In addition, the real-time proliferation data can help guide and determine the optimal window of treatment for each specific cell line. To demonstrate the utility of the ­xCELLigence system for cytotoxicity assays, H460 cells were seeded in the wells of E-Plates and continuously monitored using the ­xCELLigence system. At about 30 hours after initial seeding the cells were treated with increasing doses of the cytotoxic compound MG132, which is a well-known proteasome inhibitor. The cells were continuously monitored for an additional 45 hours. As shown in Figure 2b, the real-time data obtained by the ­xCELLigence system allow the assessment of important parameters such as rate and onset of cytotoxicity as well as calculation of time-dependent IC50s. As an example, IC50 values were calculated at three specific time points throughout the MG132-mediated cytotoxicity assay (Figure 2c), which clearly shows that IC50 can change with time. These results suggest that one should exercise caution not only when carefully optimizing the time of compound treatment but also when determining the optimal time for assessing compound effect. In addition to compound-mediated cytotoxicity, applications for cell-mediated cytotoxicity as well as virus-mediated cytotoxicity have also been developed on the ­xCELLigence platform [3,4].

Functional monitoring of receptor activity

G-protein-coupled receptors (GPCRs) are important targets for pharmaceutical drug development, and about 50% of the current drugs on the market are targeted against GPCRs. GPCRs have been shown to modulate the actin cytoskeleton, and hence it is possible to harness this information as a functional and biologically relevant readout for GPCR activity [5]. As shown in Figure 3a, CHO-K1 cells expressing the human H1 histamine receptor were seeded in the wells of the E-Plates and stimulated with histamine. Histamine induced a transient cellular response which correlates with actin cytoskeletal reorganization (Figure 3b). Plotting the log histamine concentration versus the maximal cellular response allows the generation of dose-response curves with an EC-50 value that is comparable to standard assays (Figure 3c). The important contribution that the xCELLigence system offers for GPCR cell-based assays is severalfold: 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 the 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 xCELLigence system. Traditionally, several different instrumentations would be required to carry out assays for GPCRs coupled to different signaling pathways. Third and most important, cells expressing endogenous GPCRs including primary cells can also be used with the xCELLigence system, precluding the need of over-expression of exogenous GPCRs or engineering the cells to express promiscuous G-proteins [5]. 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 can also be monitored in functional cell-based assays on the xCELLigence system [6].

Cell adhesion and spreading

Cell adhesion is an important component of multiple physiological and pathophysiological processes such as wound healing, inflammation, angiogenesis and cancer. Cellular interaction with and adhesion on different biological surfaces is a dynamic and integrated process that requires the participation of specialized cell surface receptors, structural proteins, signaling proteins and the cellular cytoskeleton. To demonstrate the utility of the xCELLigence system in dynamic monitoring of cell adhesion, the wells of the E-Plates were coated with increasing concentrations of the extracellular matrix protein, fibronectin. As a control, the wells were coated with bovine serum albumin (BSA). COS-7 cells were serum-starved, gently trypsinized, and applied to the coated wells, and cell attachment and spreading were continuously monitored using the xCELLigence system (Figure 4). Increasing coated fibronectin concentration leads to increasing cell index, while BSA coating does not support cell attachment and spreading. The xCELLigence system can be used to assess the effect of cytoskeletal drugs, enzyme and signaling protein inhibitors, and agents which physically disrupt or block cell attachment and spreading or intervene with downstream signaling pathways [7].

Conclusions

In summary, some key and fundamental observations have been provided which demonstrate the utility of the xCELLigence system as a quantitative, non-invasive and real-time assay system for conducting cell-based assays. The convergence of label-free technology coupled to a non-invasive readout and kinetics culminates in obtaining information-rich and high-content data allowing users to make well-informed decisions regarding the quality of their assay as well as the quality of the data obtained from their assay. The time resolution provided by the xCEL­Ligence technology can be used as an essential tool to better refine molecular-based assays such as gene-expression profiling, and we envision future integration of these technologies.

References

1. Atienza JM et al. (2006) Assay Drug Dev Technol 4:597–607

2. Kirstein SL et al. (2006) Assay Drug Dev Technol 4:545–553

3. Zhu J et al. (2006) J Immunol Methods 309:25–33

4. Glamann J, Hansen AJ (2006) Assay Drug Dev Technol 4:555–563

5. Yu N et al. (2006) Anal Chem 78:35–43

6. Atienza JM et al. (2006) J Biomol Screen 11:634–643

7. Atienza JM et al. (2005) J Biomol Screen 10:795–805

This article was originally published in Biochemica 2/2008, pages 8-11. ©Springer Medizin Verlag 2008

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