Extracellular Recording of Electrically Excitable Cells Multiple Site Recording of Field Potentials by MEAs

In general, for extracellular recording of field potentials or low-frequency potentials from electrogenic cells, glass and silicon-based MMEAs with embedded passive metal electrodes [45, 46] or integrated FETs are commonly used [7, 29, 47]. Although, in general, intracellular recording techniques are more sensitive than extracellular recordings, the latter - when carried out with MEAs - have several remarkable advantages. For example, cells cultured on substrate-integrated electrodes allow the noninvasive and simultaneous recording of extracellular potentials on multiple recording sites [48]. Based on these noninvasive recordings, the same cells can be repeatedly monitored, allowing true long-term experiments in parallel. Over the years, a number of companies (Multi Channel Systems, Reutlingen, Germany; Ayanda Biosytems, Lausanne, Switzerland; Alpha MED Sciences (Panasonic), Osaka, Japan; UNT Center for Network Neuroscience, Denton, USA) have offered customer-friendly and fully equipped systems that can be used without time-consuming staff training. A large number of publications relating to various applications can be found on the homepages of these companies.

In general, cell- or tissue-based MEAs are placed in a heatable amplifier which allows an adequate signal amplification and simultaneous stimulation and recording of multiple microelectrodes. For data acquisition and processing, the MEA and the amplifier are connected to an analogue/digital (A/D) converter and computer. Typically, MEAs can be used for almost all electrically active cells derived either from heart, muscle, or neural tissues. Although several studies using neuronal cells and tissues in combination with MEAs have revealed promising applications for the pharmaceutical industry [2, 3, 5, 8, 9, 46, 49-55], here we focus only on the use of cardiomyocytes for extracellular recordings.

In contrast to neuronal cell cultures, heart muscle cells show several advantages for HTS as they possess a spontaneous electric activity, are stable in culture even over long periods of time, and provide feasible and reliable extracellular recordings. For instance, Robitzki and co-workers used MEA-based extracellular recordings to identify extremely low concentrations of positive chronotropic compounds (range of 10-11 M), as shown for example by the application of angiotensin II to rat cardiomyocytes [56]. Moreover, by improving their functional cardiomyocyte-based sensor, these authors were able to detect low concentrations of autoimmune antibodies in sera from pregnant women suffering from pre-eclampsia [1]. The existence ofthese autoimmune antibodies has been demonstrated by an automated counting of extracellular field potentials after application of pre-eclamptic sera. Due to the binding of antibodies to the angiotensin II type 1 receptors, an increase in contraction frequency was observed.

With a view to its use as a diagnostic and/or drug screening tool, this MEA platform may need to be implemented in an automated work station for liquid handling, chip positioning, cell growth, and maintenance. The work station allows the screening of 10 to 20 chips per hour, with each chip being divided into four application chambers, each containing six to ten electrodes that can be recorded simultaneously. Thus, between 40 and 80 compounds per hour - that is, 960 to 1920 per day - can be screened. Since the work station is placed in a CO2 incubator, long-term experiments are possible by repeated measurement of the same chips under cell culture conditions (over a period of 28 days). The ability of cardiomyocyte-based MEAs to detect chronotropic and arrhythmic effects of drugs and compounds has been demonstrated in several previous reports.

MEAs have also been shown applicable for the screening of QT prolongation [6, 9, 57, 58]. For example, Multi Channel Systems GmbH, Reutlingen, Germany has developed a 96-well QTplate™ for HTS of drug-induced QT prolongation in primary cardiomyocytes (Fig. 3.10). Each well of the QT-plate consists of a round recording electrode and an octagonal reference electrode; this allows the simultaneous extracellular recording of field potentials from all 96 wells. A combination of the QT-Screen system (amplifier and recording unit) with an automated liquid-handling system permits the daily screening of approximately 6000 data points or compounds [57, 58]. Based on the low fabrication costs, the average cost of each data point is US$ 0.20.

Fig. 3.10 QTplate™ from Multi Channel Systems enables HTS of QT prolongation of cardiomyocytes in a 96-well plate (a). For real-time recording of extracellular field potentials, microplates are connected to an amplifier board and computer. Cells can be cultured directly on the recording and reference electrodes implemented on the bottom of each well (b).

Fig. 3.10 QTplate™ from Multi Channel Systems enables HTS of QT prolongation of cardiomyocytes in a 96-well plate (a). For real-time recording of extracellular field potentials, microplates are connected to an amplifier board and computer. Cells can be cultured directly on the recording and reference electrodes implemented on the bottom of each well (b).

Another application of MEAs is the quantification of neurotoxins and drugs on neurons and/or neuronal networks. Nerve cells must express electrical activity depending on the molecular expression pattern as a part of their physiological function. Any interference with these patterns of electrophysiology - created, for example, by toxic agents or drugs - can generate alterations or malfunctions that might be classified as a reaction and could be monitored in real-time by microelectrodes. Therefore, spontaneously active neuronal monolayer networks in vitro can be cultured on thin film microelectrode arrays as a screening platform to determine the activities of drugs and other compounds under test. Such a screening module has been developed as an experimental platform by Gramowski et al. [59] to measure the acute neurotoxicological effects of trimethyltin (TMT). Murine spinal cord and auditory cortex both exhibited dose-dependent changes of their electrophysiological activity after treatment with TMT. However, microscopic inspection showed no acute cytotoxic effects on the neurons, such as beading of axons, retraction ofdendrites, shrinking or swelling of somata, up to ranges which were 10-fold higher than doses of TMT that interfered with spontaneous activity. An ongoing effort to validate a cellular test system based on cultured networks by a simultaneous multichannel chart recording based on sputtered indium tin oxide (ITO) plates, spin-insulated with polysiloxane and electrolytically gold-plated at the electrode tips, has also been reported [59].

The measurement of different cellular parameters under noninvasive and realtime conditions on a single chip has been realized by Wolfet al., with the fabrication of a multiparametric sensor chip [16]. Here, tumor cells were cultured directly onto the sensor chip that comprises interdigital electrode structures (IDES) to record electric current-associated changes in cellular membrane properties, ionsensitive effect transistors (ISFETs) to analyze the extracellular pH of the culture medium, and an amperometric oxygen electrode to detect oxygen consumption. Although the chip has been designed primarily for monitoring the efficiency of chemotherapeutic drugs, it can also be applied to almost all other cell types.

Bionas GmbH (Rostock, Germany) has developed a similar multifunctional system to monitor various cellular parameters on a single device. Different chip-integrated sensors allow HCS of cell metabolism-dependent parameters such as acidification, respiration, cell adhesion, and membrane integrity. Based on these properties, this cell-based sensor represents a promising tool for functional drug and cytotoxicity screening.

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