*2.3. MEA-Based Analysis of Cell Behavior*

A common MEA system is composed of dot-like electrodes arranged in two-dimensional grids that measure the fluctuations in the extracellular field potential (FP) of an attached cell layer in respect to a reference electrode placed outside the grid (Figure 1). MEA is a non-invasive, label free methodology that has been initially applied to investigate neuronal activity [46]. However, in recent years an increasing number of studies have taken advantage of MEAs to particularly analyze compound-induced cardiac toxicity in iPSC-CMs [47–49]. Like optical recordings of the membrane potential, MEA systems allow non-invasive and cost-effective measurements at high throughput scale, and long-term observations [46,50]. On the other hand, Rynnännen et al. published data of a custom-made MEA platform for FP detection based on a single cell analysis [51]. In contrast to conventional MEA systems, this optimized device demonstrated a modified layout of larger electrodes, most suitable for observation of single iPSC-CMs. Similarly, agarose micro-chambers printed on MEA have been found to facilitate single cell detection of FPs in stem cell-derived CMs [52]. Moreover, electrophysiological assessment using MEAs is not only restricted to cell culture but can also be performed on the tissue level to better simulate in vivo conditions, as shown for murine and human heart tissue slices [53–55].

**Figure 1.** (**A**) Glass multi-/micro-electrode array (MEA) chip used to detect field potential (FP) of cells. (**B**) Cells seeded on an MEA surface, grown on top of the electrodes (black dots), Video S1.

An advantage of the MEA technology is its high flexibility as it can be combined with other detection methodologies to multiply the number of parameters describing cellular functions. The main parameter assessed is the FP of spontaneously beating CMs that can be correlated with certain elements of the AP pattern. Additionally, newly developed platforms provide the possibility to detect impedance of the attached cell layer [56,57]. Unlike the FP that reflects the electrical activity, impedance corresponds to the mechanical movement of the cell on the electrode. It is influenced by cell density, cell number and the extent of cell adhesion. Thus, measuring impedance helps to acquire valuable information about beating behavior, proliferation, cell death and viability [22,58].

A relationship between contraction parameters and electrophysiological activity has also been investigated by combination of MEA and high-speed video microscopy, followed by motion based image analysis of beating cells [59]. Likewise, fluorescence microscopy was used to correlate FP measurements with subcellular information [60]. However, the combined setup of MEA platforms with optical techniques requires certain structural features to achieve optimal visualization of target cells, such as transparent electrodes [51,60,61].

In another study, Siemenov et al. developed a combined scanning ion conductance microscopy–MEA system for simultaneous detection of cell surface morphology and FP in cardiomyocytes [62]. The platform reveals morpho-dynamic parameters, including maximum displacement and cell volume changes in a time-dependent manner. Together with the FP data obtained from MEA measurements, the authors were able to reconstruct 3-dimensional motion of the cell surface over a complete contraction-relaxation cycle [62].

In order to obtain reproducible and reliable experimental data, a number of points need to be considered when working with MEA systems that are particularly important for drug screening assays. Since individual iPSC-CMs show variations in AP waveforms [63], confluent monolayer cell sheets are preferred to reduce the variability of the acquired FP patterns. In this regard, cell density needs to be carefully addressed by the operator as it was found to influence electrical remodeling of CMs derived from human iPSCs [64].
