Development of a Novel Platform for in vitro Electrophysiological Recording

The accurate monitoring of cell electrical activity is of fundamental importance for pharmaceutical research and pre-clinical trials that impose to check the cardiotoxicity of all new drugs. Traditional methods for preclinical evaluation of drug cardiotoxicity exploit animal models, which tend to be expensive, low throughput, and exhibit species-specific differences in cardiac physiology (Mercola, Colas and Willems, 2013). Alternative approaches use heterologous expression of cardiac ion channels in non-cardiac cells transfected with genetic material. However, the use of these constructs and the inhibition of specific ionic currents alone is not predictive of cardiotoxicity. Drug toxicity evaluation based on the human ether-\ue0-go-go-related gene (hERG) channel, for example, leads to a high rate of false-positive cardiotoxic compounds, increasing drug attrition at the preclinical stage. Consequently, from 2013, the Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative focused on experimental methods that identify cardiotoxic drugs and to improve upon prior models that have largely used alterations in the hERG potassium ion channel. The most predictive models for drug cardiotoxicity must recapitulate the complex spatial distribution of the physiologically distinct myocytes of the intact adult human heart. However, intact human heart preparations are inherently too costly, difficult to maintain, and, hence, too low throughput to be implemented early in the drug development pipeline. For these reasons the optimization of methodologies to differentiate human induced Pluripotent Stem Cells (hiPSCs) into cardiomyocytes (CMs) enabled human CMs to be mass-produced in vitro for cardiovascular disease modeling and drug screening (Sharma, Wu and Wu, 2013). These hiPSC-CMs functionally express most of the ion channels and sarcomeric proteins found in adult human CMs and can spontaneously contract. Recent results from the CiPA initiative have confirmed that, if utilized appropriately, the hiPSC-CM platform can serve as a reliable alternative to existing hERG assays for evaluating arrhythmogenic compounds and can sensitively detect the action potential repolarization effects associated with ion channel\u2013blocking drugs (Millard et al., 2018). Data on drug-induced toxicity in hiPSC-CMs have already been successfully collected by using several functional readouts, such as field potential traces using multi-electrode array (MEA) technology (Clements, 2016), action potentials via voltage-sensitive dyes (VSD) (Blinova et al., 2017) and cellular impedance (Scott et al., 2014). Despite still under discussion, scientists reached a consensus on the value of using electrophysiological data from hiPSC-CM for predicting cardiotoxicity and how it\u2019s possible to further optimize hiPSC-CM-based in vitro assays for acute and chronic cardiotoxicity assessment. In line with CiPA, therefore, the use of hiPSC coupled with MEA technology has been selected as promising readout for these kind of experiments. These platforms are used as an experimental model for studying the cardiac Action Potentials (APs) dynamics and for understanding some fundamental principles about the APs propagation and synchronization in healthy heart tissue. MEA technology utilizes recordings from an array of electrodes embedded in the culture surface of a well. When cardiomyocytes are grown on these surfaces, spontaneous action potentials from a cluster of cardiomyocytes, the so called functional syncytium, can be detected as fluctuations in the extracellular field potential (FP). MEA measures the change in FP as the action potential propagates through the cell monolayer relative to the recording electrode, neverthless FP in the MEA do not allows to recapitualte properly the action potential features. It is clear, therefore, that a MEA technology itself is not enough to implement cardiotoxicity assays on hIPSCs-CMs. Under this issue, researchers spread in the world started to think about solutions to achieve a platform able to works both at the same time as a standard MEA and as a patch clamp, allowing the recording of extracellular signals as usual, with the opportunity to switch to intracellular-like signals from the cytosol. This strong interest stimulated the development of methods for intracellular recording of action potentials. Currently, the most promising results are represented by multi-electrode arrays (MEA) decorated with 3D nanostructures that were introduced in pioneering papers (Robinson et al., 2012; Xie et al., 2012), culminating with the recent work from the group of H. Park (Abbott et al., 2017) and of F. De Angelis (Dipalo et al., 2017). In these articles, they show intracellular recordings on electrodes refined with 3D nanopillars after electroporation and laser optoporation from different kind of cells. However, the requirement of 3D nanostructures set strong limitations to the practical spreading of these techniques. Thus, despite pioneering results have been obtained exploiting laser optoporation, these technologies neither been applied to practical cases nor reached the commercial phase. This PhD thesis introduces the concept of meta-electrodes coupled with laser optoporation for high quality intracellular signals from hiPSCs-CM. These signals can be recorded on high-density commercial CMOS-MEAs from 3Brain characterized by thousands of electrode covered by a thin film of porous Platinum without any rework of the devices, 3D nanostructures or circuitry for electroporation7. Subsequently, I attempted to translate these unique features of low invasiveness and reliability to other commercial MEA platforms, in order to develop a new tool for cardiac electrophysiological accurate recordings. The whole thesis is organized in three main sections: a first single chapters that will go deeper in the scientific and technological background, including an explanation of the cell biology of hiPSCs-CM followed by a full overview of MEA technology and devices. Then, I will move on state-of-the-art approaches of intracellular recording, discussing many works from the scientific literature. A second chapter will describe the main objectives of the whole work, and a last chapter with the main results of the activity. A final chapter will resume and recapitulate the conclusion of the work

Investigating computational properties of a neurorobotic closed loop system

This work arises as an attempt to increase and deepen the knowledge of the encoding method of the information by the nervous system. In particular, this study focuses on computational properties of neuronal cultures grown in vitro. Through a neuro-robotic close-loop system composed of either cortical or hippocampal cultures (plated on micro-electrode arrays) on one side and of a robot controlled by the cultures on the other side, it has been possible to analyze experimental dataopenEmbargo per motivi di segretezza e/o di proprietà dei risultati e/o informazioni sensibil

System for Dynamic Measurements of Membrane Capacitance in Intact Epithelial Monolayers

AbstractDynamic measurements of exocytosis have been difficult to perform in intact epithelial monolayers. We have designed a system that estimates with ±1% accuracy (99% confidence) the total membrane capacitance of monolayers represented by a lumped model. This impedance measurement and analysis system operates through a conventional transepithelial electrophysiology clamp, performing all signal measurements as frequently as every 5s. Total membrane capacitance (the series combination of apical and basolateral membranes) is the inverse of one of three unique coefficients that describe the monolayer impedance. These coefficients are estimated using a weighted, nonlinear, least-squares algorithm. Using the estimated coefficients, solution ranges for individual membrane parameters are calculated, frequently providing results within ±20% of true values without additional electrophysiological measurements. We determined the measurement system specifications and statistical significance of estimated parameters using 1) analytical testing with circuit simulation software and equation-generated data; 2) a system noise analysis combined with Monte Carlo simulations; and 3) analog model circuits for calibration of the electronic system and to check equation-generated results. Finally, the time course of capacitance changes associated with purinergically stimulated mucin exocytosis are quantified in monolayers of the colonic goblet cell-like cell line HT29-CI.16E

The potential of microelectrode arrays and microelectronics for biomedical research and diagnostics

Planar microelectrode arrays (MEAs) are devices that can be used in biomedical and basic in vitro research to provide extracellular electrophysiological information about biological systems at high spatial and temporal resolution. Complementary metal oxide semiconductor (CMOS) is a technology with which MEAs can be produced on a microscale featuring high spatial resolution and excellent signal-to-noise characteristics. CMOS MEAs are specialized for the analysis of complete electrogenic cellular networks at the cellular or subcellular level in dissociated cultures, organotypic cultures, and acute tissue slices; they can also function as biosensors to detect biochemical events. Models of disease or the response of cellular networks to pharmacological compounds can be studied in vitro, allowing one to investigate pathologies, such as cardiac arrhythmias, memory impairment due to Alzheimer's disease, or vision impairment caused by ganglion cell degeneration in the retin

Modulation of cardiac Kv currents by Kvbeta2 and pyridine nucleotides.

Myocardial voltage-gated potassium (Kv) channels regulate the resting membrane potential and the repolarization phase of the action potential. Members of the Kv1 and Kv4 family associate with ancillary subunits, such as the Kvβ proteins, that modify channel kinetics, gating and trafficking. Previous investigation into the function of cardiac β subunits demonstrated that Kvβ1 regulates Ito and IK,slow currents in the heart, but the role of Kvβ2 in the myocardium remains unknown. In heterologous expression systems, Kvβ2 increases surface expression of Kv1 channels, shifts the activation potential of Kv1 channels to more polarized voltages, and increases the inactivation of Kv1 channels. Accordingly, the electrophysiological phenotype in Kvβ2-/- mice was examined to uncover its role. To investigate the effects of the loss of Kvβ2 on cardiac repolarization, we performed whole-cell electrophysiology on primary cardiac myocytes. We found Kv current density was reduced and action potential duration prolonged in myocytes lacking Kvβ2. To isolate the molecular interactions by which Kvβ2 was affecting Kv currents, we show that Kvβ2 co-immunoprecipitates with Kv1.4 and Kv1.5 in heart lysates. To measure if surface expression of these Kv channels was reduced with the loss of Kvβ2, we performed immunofluorescent confocal microscopy of isolated cardiac myocytes. We found that the surface expression of Kv1.5 was reduced in Kvβ2-/- myocytes. We also performed a membrane fractionation technique to demonstrate that the proportion of total cellular Kv1.5 at the membrane was reduced in Kvβ2-/-. Together, these findings support our hypothesis that Kvβ2 plays a role in the generation of functional Kv currents in the myocardium by interacting with members of the Kv family. The pyridine nucleotides, NAD[P](H), are ubiquitous cofactors utilized as electron donors and acceptors by over 250 cellular oxidoreductases. Work out of our laboratory has shown that the Kvβ proteins are functional enzymes of the aldo-keto reductase family, that utilize NAD[P]H to catalyze the reduction of substrates. Furthermore, follow up work has shown that the redox status of bound pyridine nucleotide (PN) modifies the gating of Kvα-Kvβ channel complexes in heterologous expression systems. To examine a physiological role for PN in cardiac repolarization, whole-cell and single channel cardiac myocyte currents were recorded under the exposure to various PN redox states. We found that the inactivation rates and open probabilities of Kv currents in isolated myocytes are sensitive to the redox status of PN, and that surface action potentials in an isolated heart model are prolonged by treatment with factors that increase intracellular NADH concentration

Nonlinear Dynamic Modeling, Simulation And Characterization Of The Mesoscale Neuron-electrode Interface

Extracellular neuroelectronic interfacing has important applications in the fields of neural prosthetics, biological computation and whole-cell biosensing for drug screening and toxin detection. While the field of neuroelectronic interfacing holds great promise, the recording of high-fidelity signals from extracellular devices has long suffered from the problem of low signal-to-noise ratios and changes in signal shapes due to the presence of highly dispersive dielectric medium in the neuron-microelectrode cleft. This has made it difficult to correlate the extracellularly recorded signals with the intracellular signals recorded using conventional patch-clamp electrophysiology. For bringing about an improvement in the signalto-noise ratio of the signals recorded on the extracellular microelectrodes and to explore strategies for engineering the neuron-electrode interface there exists a need to model, simulate and characterize the cell-sensor interface to better understand the mechanism of signal transduction across the interface. Efforts to date for modeling the neuron-electrode interface have primarily focused on the use of point or area contact linear equivalent circuit models for a description of the interface with an assumption of passive linearity for the dynamics of the interfacial medium in the cell-electrode cleft. In this dissertation, results are presented from a nonlinear dynamic characterization of the neuroelectronic junction based on Volterra-Wiener modeling which showed that the process of signal transduction at the interface may have nonlinear contributions from the interfacial medium. An optimization based study of linear equivalent circuit models for representing signals recorded at the neuron-electrode interface subsequently iv proved conclusively that the process of signal transduction across the interface is indeed nonlinear. Following this a theoretical framework for the extraction of the complex nonlinear material parameters of the interfacial medium like the dielectric permittivity, conductivity and diffusivity tensors based on dynamic nonlinear Volterra-Wiener modeling was developed. Within this framework, the use of Gaussian bandlimited white noise for nonlinear impedance spectroscopy was shown to offer considerable advantages over the use of sinusoidal inputs for nonlinear harmonic analysis currently employed in impedance characterization of nonlinear electrochemical systems. Signal transduction at the neuron-microelectrode interface is mediated by the interfacial medium confined to a thin cleft with thickness on the scale of 20-110 nm giving rise to Knudsen numbers (ratio of mean free path to characteristic system length) in the range of 0.015 and 0.003 for ionic electrodiffusion. At these Knudsen numbers, the continuum assumptions made in the use of Poisson-Nernst-Planck system of equations for modeling ionic electrodiffusion are not valid. Therefore, a lattice Boltzmann method (LBM) based multiphysics solver suitable for modeling ionic electrodiffusion at the mesoscale neuron-microelectrode interface was developed. Additionally, a molecular speed dependent relaxation time was proposed for use in the lattice Boltzmann equation. Such a relaxation time holds promise for enhancing the numerical stability of lattice Boltzmann algorithms as it helped recover a physically correct description of microscopic phenomena related to particle collisions governed by their local density on the lattice. Next, using this multiphysics solver simulations were carried out for the charge relaxation dynamics of an electrolytic nanocapacitor with the intention of ultimately employing it for a simulation of the capacitive coupling between the neuron and the v planar microelectrode on a microelectrode array (MEA). Simulations of the charge relaxation dynamics for a step potential applied at t = 0 to the capacitor electrodes were carried out for varying conditions of electric double layer (EDL) overlap, solvent viscosity, electrode spacing and ratio of cation to anion diffusivity. For a large EDL overlap, an anomalous plasma-like collective behavior of oscillating ions at a frequency much lower than the plasma frequency of the electrolyte was observed and as such it appears to be purely an effect of nanoscale confinement. Results from these simulations are then discussed in the context of the dynamics of the interfacial medium in the neuron-microelectrode cleft. In conclusion, a synergistic approach to engineering the neuron-microelectrode interface is outlined through a use of the nonlinear dynamic modeling, simulation and characterization tools developed as part of this dissertation research

Acclimatization to High-Altitude, Long-Term Hypoxia Alters BK Channel Structure and Function

We examined the major possible mechanisms for the left shift of the BK channel I-V relationship in native basilar artery myocytes from the two LTH groups. These mechanisms included: differential expression of the accessary BK -1 subunit; differential phosphorylation of the BK subunit; and splice variation of the BK subunit. Using molecular cloning, heterologous expression, and patch-clamp electrophysiology techniques, we elucidated a mechanism that, at least in part, contributes to the differences we observed between channels from native normoxic and LTH myocytes

Novel miniaturised and highly versatile biomechatronic platforms for the characterisation of melanoma cancer cells

There has been an increasing demand to acquire highly sensitive devices that are able to detect and characterize cancer at a single cell level. Despite the moderate progress in this field, the majority of approaches failed to reach cell characterization with optimal sensitivity and specificity. Accordingly, in this study highly sensitive, miniaturized-biomechatronic platforms have been modeled, designed, optimized, microfabricated, and characterized, which can be used to detect and differentiate various stages of melanoma cancer cells. The melanoma cell has been chosen as a legitimate cancer model, where electrophysiological and analytical expression of cell-membrane potential have been derived, and cellular contractile force has been obtained through a correlation with micromechanical deflections of a miniaturized cantilever beam. The main objectives of this study are in fourfold: (1) to quantify cell-membrane potential, (2) correlate cellular biophysics to respective contractile force of a cell in association with various stages of the melanoma disease, (3) examine the morphology of each stage of melanoma, and (4) arrive at a relation that would interrelate stage of the disease, cellular contractile force, and cellular electrophysiology based on conducted in vitro experimental findings. Various well-characterized melanoma cancer cell lines, with varying degrees of genetic complexities have been utilized. In this study, two-miniaturized-versatile-biomechatronic platforms have been developed to extract the electrophysiology of cells, and cellular mechanics (mechanobiology). The former platform consists of a microfluidic module, and stimulating and recording array of electrodes patterned on a glass substrate, forming multi-electrode arrays (MEAs), whereas the latter system consists of a microcantilever-based biosensor with an embedded Wheatstone bridge, and a microfluidic module. Furthermore, in support of this work main objectives, dedicated microelectronics together with customized software have been attained to functionalize, and empower the two-biomechatronic platforms. The bio-mechatronic system performance has been tested throughout a sufficient number of in vitro experiments.Open Acces

A chemical biology approach to understanding the basis of voltage-gated sodium channel modulation

The voltage-gated sodium channel, Nav1.7, is involved in the propagation of pain signals from the peripheral nervous system. Genomic data from individuals with non-functional Nav1.7 expression strongly suggest it has potential to be the target of novel analgesics; loss of Nav1.7 function completely abolishes pain sensations in otherwise healthy phenotypes. The focus of this thesis is the development of chemical tools to elucidate mechanisms of Nav1.7 modulation in the cell. The design, synthesis, characterisation and potency data of photocrosslinking probes that target two distinct Nav1.7 domains is reported. Domain II is targeted by photoprobes derived from the spider venom inhibitory cystine knot peptide Huwentoxin-IV. Moreover, a photoprobe based on the novel family of Nav1.7-selective aryl sulfonamide inhibitors targets domain IV of Nav1.7. Determining the binding sites that lead to modulation of gating was firstly attempted in bacterial/hNav1.7 chimeric proteins that have been purified and used for crystallographic and biophysical studies. According to gel shift assays, certain photoprobes exhibited efficient photocrosslinking capabilities and were taken forward to proteomic mass spectrometry analysis in pursuit of photocrosslinking sites. Additionally, a series of approaches were explored in order to optimise the identification of Nav1.7 by proteomic mass spectrometry in an engineered cell line. Finally, the maturation of induced pluripotent stem cells from patients that carry a Nav1.7 mutation was followed by quantitative proteomics as an initial approach to understand Nav1.7-related mechanisms in a disease model.Open Acces

Integration of single-cell electropermeabilization together with electrochemical measurement of quantal exocytosis on microchips

An electrochemical microelectrode located immediately adjacent to a single neuroendocrine cell can record spikes of amperometric current that result from quantal exocytosis of oxidizable transmitter from individual vesicles. Using electroporation we have developed an efficient method where the same electrochemical microelectrode is used to electropermeabilize an adjacent chromaffin cell and then measure the consequent quantal catecholamine release using amperometry. Trains of voltage pulses, 5-7 V in amplitude and 0.1-0.2 ms in duration can reliably trigger release from cells using gold electrodes. Amperometric spikes induced by electropermeabilization have similar areas, peak heights and durations as amperometric spikes elicited by depolarizing high K+ solutions. Uptake of trypan blue stain into cells demonstrated that the plasma membrane is permeabilized by the voltage stimulus. Robust quantal release is elicited upon electroporation in 0 Ca2+/5 mM EGTA in the bath solution. Electropermeabilization-induced transmitter release requires Cl- in the bath solution--bracketed experiments demonstrate a steep dependence of the rate of electropermeabilization-induced transmitter release on [Cl- ] between 2 and 32 mM. Using the same electrochemical electrode to electroporate and record quantal release of catecholamines from an individual chromaffin cell allows precise timing of the stimulus, stimulation of a single cell at a time, and can be used to load membrane impermeant substances into a cell.Includes bibliographical references (pages 110-128)