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-à-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–blocking 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’s 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.