Intracellular Recording of Electroactive Cells Chip Based Automated Patch Clamp Recording

Fundamental cellular processes, such as neuronal signaling and the contraction of heart and skeletal muscle cells, are regulated by ion channels. A number of diseases which are caused by pathology of ion channels - so-called "channelopathies" [35] - originate from the loss or dysfunction of ion channels. Some well-known channelopathies affect the central nervous system (episodic ataxias, familial hemiplegic migraine and inherited epilepsies), skeletal muscles (myotonias, periodic paralyses, malignant hyperthermia) and the heart (long-OT syndromes, idiopathic ventricular fibrillation). In terms of effective therapies, ion channels represent excellent targets for drugs. Worldwide, the total revenue of drugs used to treat channelopathies amounts to six billion US$. Moreover, accompanied by the remarkable progress in molecular biology, the number of identified hereditary ion channel diseases is increasing constantly. Therefore, the screening for novel ion-modulating compounds that can be used as potential therapeutic drugs creates an enormous demand for novel HTS tools.

The patch-clamp technique, which was introduced by Neher and Sakmann in 1976 [36], is a traditional and outstanding method for the functional investigation of ion channel behavior on the cellular surface. Depending on the configuration, patch-clamping allows the direct recording of multiple or single ion channels. However, since the conventional patch-clamp technique is very slow, very laborious, and less cost-effective, this method is rather a method for basic research than for pharmaceutical testing, as the latter asks for HTS tools. At present, some promising chip-based patch-clamp arrays are available commercially, including PatchXpress and SealChip™ from Axon Industries, CYTOPATCH™ from Cytocentrics AG Reutlingen, and QPatch™ from Sophion Bioscience. When using planar chip devices, their fabrication can be achieved by using conventional semiconductor technologies, allowing individual chip designs and a cost-effective production. For the proper recording on a planar chip, the positioning of cells and the tight contact between cell and device is essential for a successful patch-clamping (giga seal formation). To overcome this problem, small micro-openings are generated, where cells can be placed and held by applying an appropriate negative pressure, as realized by the CYTOPATCH™ chip (Fig. 3.9). Here, one site of the CYTOPATCH™ chip is able to perform patch-clamp recordings on 200 cells per day.

Another chip-based automated patch clamp system has been developed by Molecular Devices Corp., and purchased by Axon Industries. This enables the simultaneous measurement of 16 mammalian cells, which potentiates the screening of 1000 components a week. The Qpatch™ system from Sophion Bioscience provides a so-called Qplate that is microfabricated by conventional photolithography techniques and consists of 16 individually controlled recording sites. An elaborated microfluidic channel system that is implemented in the silicon chip allows the controlled trapping of cells at the patch-clamping micro-opening site. In order to achieve a higher signal-to-noise ratio, the membrane at the patch aperture is disturbed by applying an acute and short suction pulse, which in turn transfers the patch-clamping from "on-cell" into "whole-cell" configuration. The first generation of automated chip-based patch-clamping systems (as described above) is well suited to distinct high-throughput approaches in pharmaceutical companies. In this context, it is possible to use these techniques at much earlier stages of functional drug discovery and drug testing processes and, therefore, to minimize development and preclinical evaluation costs. However, as the quality of chip- and non-chip-based automated patch-clamp platforms cannot yet be compared with the traditional patch-clamp technique, ongoing and further improvements of automated patch-clamping are necessary. Nevertheless, a number of studies have shown how automated patch-clamping can be improved

Fig. 3.9 The CYTOPATCH™ chip is characterized by two concentric openings a) formed by a focused ion beam in a 10 |m-thick silicon dioxide layer (SEM image; scale bar 2 |jm). Positioning and recording are carried out independently by a suction channel and contact channel. (b) A schematic drawing of automated cell-by-cell patch-clamping using the CYTOPATCH™ chip technique. (From Multi Channel Systems, Reutlingen, Germany).

Fig. 3.9 The CYTOPATCH™ chip is characterized by two concentric openings a) formed by a focused ion beam in a 10 |m-thick silicon dioxide layer (SEM image; scale bar 2 |jm). Positioning and recording are carried out independently by a suction channel and contact channel. (b) A schematic drawing of automated cell-by-cell patch-clamping using the CYTOPATCH™ chip technique. (From Multi Channel Systems, Reutlingen, Germany).

and optimized towards a real high-throughput application, especially in terms of the traditional patch-clamp quality [37-44].

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