Channelrhodopsins (ChRs) are light-gated proteins that control phototaxis in protists and are commonly used in optogenetics to manipulate neuron activity. ChR2-NaV1.5, a fusion of ChR2 with cardiac-specific Na+ channel, modulates sodium channel kinetics and produces photosensitive inward currents.Using automated low and high throughput patch clamp (APC), we found that blue-light intensity directly correlates with ChR2 channel activity, with higher intensities resulting in larger peak amplitudes under constant light exposure durations. We also found that light could be used to activate ChR2, depolarizing the membrane sufficiently in current clamp mode to elicit action potentials.This is demonstrated on both the Port-a-Patch, a semi-automated patch clamp device recording from a single cell at a time, and the high throughput fully automated device, the SyncroPatch 384. The SyncroPatch 384 could also be used to investigate different ChR2 variants. Several different constructs could be activated using blue light on a single NPC-384 chip. In this study we used HEK cells stably expressed ChR2-NaV1.5 but the technology could be extended to hiPSC-neurons or other stem cells or primary cells which express light activated ion channels.Our findings underscore the utility of APC combined with optogenetics in enhancing assay throughput, consistency, and mechanistic resolution. These advanced platforms could support early-stage safety screening and contribute to more predictive, human-relevant models for drug-induced excitability disorders and off-target toxicities.

Optical in-vitro platforms will be of particular relevance in the early stages drug discovery processes. We show recordings on impedance and extracellular field potential (EFP)-based devices with induced pluripotent stem cell (iPSC)-derived cardiomyocytes as well as automated patch clamp data. Optogenetic stimulation and the recording of electrophysiological and contractile parameters of ChR2 (channelrhodopsin 2) transfected iPS Cor.4U cardiomyocytes were performed in a new assay approach, which allows a parallel investigation of impedance and EFP signals. This allowed a mechanistic understanding of cardiomyocyte cell physiology, which has been investigated over a physiological frequency range (60-180 ppm). Frequency dependent effects on cell physiology with reference compounds such as Ranolazine and Mexiletine will be presented. Furthermore, automated patch clamp investigations in the voltage-and current-clamp mode on blue-light activated ChR2 (channelrhodopsin 2) transfected cells will be presented and discussed in association with impedance/EFP results.

Introduction: Chip-based automated patch clamp systems are widely used in drug development and safety pharmacology, allowing for high quality, high throughput screening at standardized experimental conditions. The merits of automation generally come at the cost of large amounts of cells needed, since cells are not targeted individually, but randomly positioned onto the chip aperture from cells in suspension. While cell usage is of little concern when using standard cell lines such as CHO or HEK cells, it becomes a crucial constraint with cells of limited availability, such as primary or otherwise rare and expensive cells, like induced pluripotent stem (IPS) cell-derived cardiomyocytes or neurons. Methods: We established application protocols for CHO cells, IPS cell-derived neurons (iCell (R) Neurons, Cellular Dynamics International), cardiomyocytes (Cor.4U (R), Axiogenesis) and pancreatic islet cells, minimizing cell usage for automated patch clamp recordings on Nanion's Patchliner. Use of 5 mu l cell suspension per well for densities between 55,000 cells/ml and 400,000 cells/ml depending on cell type resulted in good cell capture. Results: We present a new cell application procedure optimized for the Patchliner achieving > 80% success rates for using as little as 300 to 2000 cells per well depending on cell type. We demonstrate that this protocol works for standard cell lines, as well as for stem cell-derived neurons and cardiomyocytes, and for primary pancreatic islet cells. We present recordings for these cell types, demonstrating that high data quality is not compromised by altered cell application. Discussion: Our new cell application procedure achieves high success rates with unprecedentedly low cell numbers. Compared to other standard automated patch clamp systems we reduced the average amount of cells needed by more than 150 times. Reduced cell usage crucially improves cost efficiency for expensive cells and opens up automated patch clamp for primary cells of limited availability. (C) 2013 Elsevier Inc. All rights reserved.