Corina Bot

Probing the membrane potential of living cells by dielectric spectroscopy

In this paper we demonstrate a quantitative way to measure the membrane potential of live cells by dielectric spectroscopy. We also show that the values of the membrane potential obtained using our technique are in good agreement with those obtained using traditional methods—voltage-sensitive dyes. The membrane potential is determined by fitting the experimental dielectric dispersion curves with the dispersion curves obtained from a theoretical model. Variations in the membrane potential were induced by modifying the concentration of potassium chloride in the solution of the cell suspension in the presence of valinomycin. For exemplification of the method, E. coli was chosen for our experiments.

Quantifying the membrane potential during E. coli growth stages.

The presence of the resting membrane potential has a strong effect on the dielectric behavior of cell suspensions. Using this observation and a well-established theoretical model, the low frequency dielectric dispersion curves of E. coli cell suspensions are de-convoluted to obtain the resting membrane potential of E. coli cells at various growth stages. Four regions of the exponential growth stage are investigated and the measurements indicate that the membrane depolarizes from -220mV in the early exponential phase to -140mV in the late exponential phase. The conductivity of the cell suspension is also found to decrease as the cells progress from the early to the late exponential phases.

New easy-to-use hybrid system for extracellular potential and impedance recordings.

The need for predictive, in vitro cardiac safety screening drives further development of automated, high-throughput–compatible drug evaluation based on cardiac cell preparations. Recently, pluripotent stem cells are evaluated as a new, more predictive model for cardiovascular risk assessment pertaining to in vitro assays. We present a new screening platform, the CardioExcyte 96, a hybrid instrument that combines impedance (cell contractility) with extracellular field potential (EFP) recordings. The electrophysiological measurements are noninvasive, label free and have a temporal resolution of 1 ms. This hybrid technology addresses the lack of easy-to-use high-throughput screening for in vitro assays and permits the reliable investigation of short- and long-term pharmacological effects. Several models of cardiomyocyte preparations were successfully validated for use with the CardioExcyte96. Furthermore, the pharmacological effects of a number of reference compounds were evaluated. Compound effects on cell monolayers of human-induced pluripotent stem cell–derived cardiomyocytes are evaluated using a quasi-simultaneous hybrid recording mode that combines impedance and EFP readouts. A specialized software package for rapid data handling and real-time analysis was developed, which allows for comprehensive investigation of the cellular beat signal. Combining impedance readouts of cell contractility and EFP (microelectrode array–like) recordings, the system opens up new possibilities in the field of in vitro cardiac safety assessment.

Correcting the polarization effect in very low frequency dielectric spectroscopy

Polarization impedance appears at the interface between electrodes and ionic solutions and is a major source of errors in dielectric spectroscopy measurements. This work presents a simple, robust and automated methodology for measuring and analysing the polarization impedance of non-dispersive electrolytes, with a focus on the very low frequency domain from 1 Hz and up. The accuracy of the method is demonstrated by comparing the corrected dielectric permittivity and conductivity of various electrolytes either with their nominal values or with measurements taken with other high precision measuring devices. The dependence of the polarization impedance on several parameters, such as ionic concentration, applied voltage and separation distance between the electrodes, is also presented. For colloidal suspensions, it is argued that a modified protocol of the substitution method is needed due to several shortcomings occurring only in the very low frequency domain. Such a protocol is presented and tested on suspensions of live E. coli cells. As opposed to most of the existing methodologies for polarization removal, the proposed protocol makes no assumptions on the behaviour of the polarization impedance. This could potentially lead to the quantitative resolution of the α-dispersion of live cells in suspension. (Some figures in this article are in colour only in the electronic version)

Safety pharmacology studies using EFP and impedance

INTRODUCTION While extracellular field potential (EFP) recordings using multi-electrode arrays (MEAs) are a well-established technique for monitoring changes in cardiac and neuronal function, impedance is a relatively unexploited technology. The combination of EFP, impedance and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has important implications for safety pharmacology as functional information about contraction and field potentials can be gleaned from human cardiomyocytes in a beating monolayer. The main objectives of this study were to demonstrate, using a range of different compounds, that drug effects on contraction and electrophysiology can be detected using a beating monolayer of hiPSC-CMs on the CardioExcyte 96. METHODS hiPSC-CMs were grown as a monolayer on NSP-96 plates for the CardioExcyte 96 (Nanion Technologies) and recordings were made in combined EFP and impedance mode at physiological temperature. The effect of the hERG blockers, E4031 and dofetilide, hERG trafficking inhibitor, pentamidine, β-adrenergic receptor agonist, isoproterenol, and calcium channel blocker, nifedipine, was tested on the EFP and impedance signals. RESULTS Combined impedance and EFP measurements were made from hiPSC-CMs using the CardioExcyte 96 (Nanion Technologies). E4031 and dofetilide, known to cause arrhythmia and Torsades de Pointes (TdP) in humans, decreased beat rate in impedance and EFP modes. Early afterdepolarization (EAD)-like events, an in vitro marker of TdP, could also be detected using this system. Isoproterenol and nifedipine caused an increase in beat rate. A long-term study (over 30h) of pentamidine, a hERG trafficking inhibitor, showed a concentration and time-dependent effect of pentamidine. DISCUSSION In the light of the new Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative to improve guidelines and standardize assays and protocols, the use of EFP and impedance measurements from hiPSCs may become critical in determining the proarrhythmic risk of potential drug candidates. The combination of EFP offering information about cardiac electrophysiology, and impedance, providing information about contractility from the same area of a synchronously beating monolayer of human cardiomyocytes in a 96-well plate format has important implications for future cardiac safety testing.

Scalable nano-bioprobes with sub-cellular resolution for cell detection.

Here we present a carbon nanotube based device to noninvasively and quickly detect mobile single cells with the potential to maintain a high degree of spatial resolution. The device utilizes standard complementary metal oxide semiconductor (CMOS) technologies for fabrication, allowing it to be easily scalable (down to a few nanometers). Nanotubes are deposited using electrophoresis after fabrication in order to maintain CMOS compatibility. The devices are spaced by 6 μm which is the same size or smaller than a single cell. To demonstrate its capability to detect cells, we performed impedance spectroscopy on mobile human embryonic kidney (HEK) cells, neurons cells from mice, and yeast cells (S. pombe). Measurements were performed with and without cells and with and without nanotubes. Nanotubes were found to be crucial to successfully detect the presence of cells. The devices are also able to distinguish between cells with different characteristics.

Cross - site comparison of excitation-contraction coupling using impedance and field potential recordings in hiPSC cardiomyocytes

INTRODUCTION Since 2005 the S7B and E14 guidances from ICH and FDA have been in place to assess a potential drug candidates ability to cause long QT syndrome. To refine these guidelines, the FDA proposed the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, where the assessment of drug effects on cardiac repolarization was one subject of investigation. Within the myocyte validation study, effects of pharmaceutical compounds on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were assessed and this article will focus on the evaluation of the proarrhythmic potential of 23 blinded drugs in four hiPSC-CM cell lines. METHODS Experiments were performed on the CardioExcyte 96 at different sites. A combined readout of contractility (via impedance) and electrophysiology endpoints (field potentials) was performed. RESULTS Our data demonstrates that hERG blockers such as dofetilide and further high risk categorized compounds prolong the field potential duration. Arrhythmia were detected in both impedance as well as field potential recordings. Intermediate risk compounds induced arrhythmia in almost all cases at the highest dose. In the case of low risk compounds, either a decrease in FPDmax was observed, or not a significant change from pre-addition control values. DISCUSSION With exceptions, hiPSC-CMs are sensitive and exhibit at least 10% delayed or shortened repolarization from pre-addition values and arrhythmia after drug application and thus can provide predictive cardiac electrophysiology data. The baseline electrophysiological parameters vary between iPS cells from different sources, therefore positive and negative control recordings are recommended.