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

Development of an on-chip model for cardiac arrhythmia and pacing

Cardiovascular diseases are accountable for almost 50 % of the mortality number in Europe which is why research on its causes is highly prioritised. For practical and ethical reasons, in vitro study models are of high importance to enable research on cardiac abnormal behaviours. To meet this issue, researchers from Imec, Belgium, developed Neuray-II, the second generation of a biocompatible chip with micrometer sized electrodes as a platform for in vitro studies of cardiac arrhythmia and pacing. For this study, an HL-1 cell line was used and their action potential waves were visualised with fluorescent imaging. Low and high frequency protocols were applied to perform electrical stimulation with the chip-electrodes to induce pacing and blocking, respectively, of the action potentials. Macroscopic image analysis of time-lapses capturing the stimulation, with a custom developed Matlab-script, did not show any large scale change in action potential propagation, induction, or termination of re-entrant spirals. The local effect of stimulation on a cellular level was analysed with another script developed for this purpose. The single-cell analysis showed that cells were stimulated only in the near vicinity of the selected electrode. The results from the frequency analysis of the cellular signals did not prove that pacing could be performed. In the future, the analysis method needs higher accuracy to provide a valid conclusion. Analysis of the cellular signals from high frequency protocols indicate that local blocking of action might be possible but also needs further experiments for confirmation. Attempts were also made to alter the propagation of action potential waves by structural patterning. This was done by microcontact printing and application of a silicone insert, both with a non successful outcome. In conclusion the Neuray-II could possibly be used as an in vitro model for studies of cardiac arrhythmia and pacing since the basic tools and knowledge have been provided with this project. Further development is needed for completion of a functional model

A modular multi electrode array system for electrogenic cell characterisation and cardiotoxicity applications

Multi electrode array (MEA) systems have evolved from custom-made experimental tools, exploited for neural research, into commercially available systems that are used throughout non-invasive electrophysiological study. MEA systems are used in conjunction with cells and tissues from a number of differing organisms (e.g. mice, monkeys, chickens, plants). The development of MEA systems has been incremental over the past 30 years due to constantly changing specific bioscientific requirements in research. As the application of MEA systems continues to diversify contemporary commercial systems are requiring increased levels of sophistication and greater throughput capabilities. [Continues.

Complexity analysis of experimental cardiac arrhythmia

International audienceTo study the cardiac arrhythmia, an in vitro experimental model and Multielectrodes Array (MEA) are used. This platform serves as an intermediary of the electrical activities of cardiac cells and the signal processing / dynamics analysis. Through it the extracellular potential of cardiac cells is acquired, allowing a real-time monitoring / analyzing. Since MEA has 60 electrodes / channels dispatched in a rectangular region, it allows real-time monitoring and signal acquisition on multiple sites. The in vitro experimental model (cardiomyocytes cultures from new-born rats' heart) is directly prepared on the MEA. This carefully prepared culture has similar parameters as cell of human's heart. In order to discriminate the cardiac arrhythmia, complexity analysis methods (Approximate Entropy, ApEn and Sample Entropy, SampEn) are used especially taking into account noise. The results showed that, in case of arrhythmia, the ApEn and SampEn are reduced to about 50\% of the original entropies. Both parameters could be used as factors to discriminate arrhythmia. Moreover, from a point of view of biophysics this decrease 50\% of Entropy coincides with the bifurcation (periods, attractors etc.) in case of arrhythmia which have been reported previously. It supports once more the hypothesis that in case of cardiac arrhythmia, the heart entered into chaos which helps to better understand the mechanism of atrial fibrillation

Patterned Cell Cultures For High Throughput Studies Of Cell Electrophysiology And Drug Screening Applications

Over the last decade, the field of tissue and bio-engineering has seen an increase in the development of in vitro high-throughput hybrid systems that can be used to understand cell function and behavior at the cellular and tissue levels. These tools would have a wide array of applications including for implants, drug discovery, and toxicology, as well as for studying cell developmental behavior and as disease models. Currently, there are a limited number of efficient, functional drug screening assays in the pharmacology industry and studies of cell-surface interactions are complicated and invasive. Most cell physiology studies are performed using conventional patch-clamp techniques or random networks cultured on silicon devices such as Microelectrode Arrays (MEAs) and Field Effect transistors (FETs). The objective of this study was to develop high-throughput in vitro platforms that could be used to analyze cell function and their response to various stimuli. Our hypothesis was that by utilizing surface modification to provide external guidance cues for various cell types and by controlling the cell environment in terms of culture conditions, we could develop an in vitro hybrid platform for sensing and testing applications. Such a system would not only give information regarding the surface effects on the growth and behavior of cells for implant development applications, but also allow for the study of vital cell physiology parameters like conduction velocity in cardiomyocytes and synaptic plasticity in neuronal networks. This study outlines the development of these in vitro high throughput systems that have varied applications ranging from tissue engineering to drug development. We have developed a simple and relatively high-throughput method in order to test the physiological effects of varying iii chemical environments on rat embryonic cardiac myocytes in order to model the degradation effects of polymer scaffolds. Our results, using our simple test system, are in agreement with earlier observations that utilized a complex 3D biodegradable scaffold. Thus, surface functionalization with self-assembled monolayers combined with histological/physiological testing could be a relatively high throughput method for biocompatibility studies and for the optimization of the material/tissue interface in tissue engineering. Traditional multielectrode extracellular recording methods were combined with surface patterning of cardiac myocyte monolayers to enhance the information content of the method; for example, to enable the measurement of conduction velocity, refractory period after action potentials or to create a functional reentry model. Two drugs, 1-Heptanol, a gap junction blocker, and Sparfloxacin, a fluoroquinone antibiotic, were tested in this system. 1-Heptanol administration resulted in a marked reduction in conduction velocity, whereas Sparfloxacin caused rapid, irregular and unsynchronized activity, indicating fibrillation. As shown in these experiments, the patterning of cardiac myocyte monolayers increased the information content of traditional multielectrode measurements. Patterning techniques with self-assembled monolayers on microelectrode arrays were also used to study the physiological properties of hippocampal networks with functional unidirectional connectivity, developed to study the mono-synaptic connections found in the dentate gyrus. Results indicate that changes in synaptic connectivity and strength were chemically induced in these patterned hippocampal networks. This method is currently being used for studying long term potentiation at the cellular level. For this purpose, two cell patterns were optimized for cell migration onto the pattern as demonstrated by time lapse studies, and for iv supporting the best pattern formation and cell survival on these networks. The networks formed mature interconnected spiking neurons. In conclusion, this study demonstrates the development and testing of in vitro highthroughput systems that have applications in drug development, understanding disease models and tissue engineering. It can be further developed for use with human cells to have a more predictive value than existing complex, expensive and time consuming methods

Development of a Plasmonic On-Chip System to Characterize Changes from External Perturbations in Cardiomyocytes

Today’s heart-on-a-chip devices are hoped to be the state-of-the-art cell and tissue characterizing tool, in clinically applicable regenerative medicine and cardiac tissue engineering. Due to the coupled electromechanical activity of cardiomyocytes (CM), a comprehensive heart-on-a-chip device as a cell characterizing tool must encompass the capability to quantify cellular contractility, conductivity, excitability, and rhythmicity. This dissertation focuses on developing a successful and statistically relevant surface plasmon resonance (SPR) biosensor for simultaneous recording of neonatal rat cardiomyocytes’ electrophysiological profile and mechanical motion under normal and perturbed conditions. The surface plasmon resonance technique can quantify (1) molecular binding onto a metal film, (2) bulk refractive index changes of the medium near (nm) the metal film, and (3) dielectric property changes of the metal film. We used thin gold metal films (also called chips) as our plasmonic sensor and obtained a periodic signal from spontaneously contracting CMs on the chip. Furthermore, we took advantage of a microfluidic module for controlled drug delivery to CMs on-chip, inhibiting and promoting their signaling pathways under dynamic flow. We identified that ionic channel activity of each contraction period of a live CM syncytium on a gold metal sensor would account for the non-specific ion adsorption onto the metal surface in a periodic manner. Moreover, the contraction of cardiomyocytes following their ion channel activity displaces the medium, changing its bulk refractive index near the metal surface. Hence, the real-time electromechanical activity of CMs using SPR sensors may be extracted as a time series we call the Plasmonic Cardio-Eukaryography Signal (P-CeG). The P-CeG signal render opportunities, where state-of-the-art heart-on-a-chip device complexities may subside to a simpler, faster and cheaper platform for label-free, non-invasive, and high throughput cellular characterization

MEMS-based Lab-on-chip platform with integrated 3D and planar microelectrodes for organotypic and cell cultures

La presente tesis doctoral se centra en el desarrollo y la validación de plataformas lab on chip (LOC) para su aplicación en el campo de la Biología, la Medicina y la Biomedicina, particularmente relacionados con el cultivo de células y tejidos, así como su tratamiento mediante electroestimulación y su actividad eléctrica. Actualmente, las investigaciones centradas en el desarrollo de LOCs han experimentado un crecimiento considerable, gracias, en gran medida, a la versatilidad que ofrecen. Dicha versatilidad se traduce en numerosas aplicaciones, de las cuales, aquellas relacionadas con la Biología y la Medicina, están alcanzando especial relevancia. La integración de sensores, actuadores, circuitos microfluídicos y circuitos electrónicos en la misma plataforma, permite fabricar sistemas con múltiples aplicaciones. Esta tesis se centra fundamentalmente en el desarrollo de plataformas para el cultivo in vitro de tejidos y células, así como para la monitorización y la interacción con dicho cultivo. Los cultivos in vitro resultan de vital importancia para realizar estudios en células o tejidos, experimentar con medicamentos o estudiar su proliferación y morfología. De esta manera, se cubriría la creciente necesidad de encontrar una alternativa para replicar modelos humanos de enfermedades in vitro para poder desarrollar nuevos fármacos y avanzar en la medicina personalizada. Por tanto, la posibilidad de realizar cultivos de media o larga duración en plataformas que no precisen de un equipamiento costoso como las incubadoras de CO2 y que puedan ser monitorizadas mediante aplicaciones ópticas, supone un salto cualitativo en el desarrollo de los cultivos in vitro. En este contexto, se presenta el trabajo relacionado con esta tesis que ha sido desarrollada dentro del grupo de Microsistemas de la Escuela Superior de Ingeniería de la Universidad de Sevilla. La tesis está estructurada de manera que a lo largo de este escrito se da respuesta a los distintos aspectos anteriormente descritos. En primer lugar, se hace una breve introducción a la tecnología MEMS y a los principios básicos de la microfluídica. Dado que este trabajo se ha desarrollado en un ambiente multidisciplinar, esta sección resulta necesaria para dar una visión general a aquellos no familiarizados con esta disciplina. Tras esa introducción se realiza una descripción del estado del arte en el que se encuadra este trabajo, incluyendo los sistemas LoCs, y sus principales aplicaciones en el campo de la Biología, Medicina y Biomedicina, prestando especial atención a las aplicaciones de los LoCs relacionadas con cultivos organotípicos y de células. Tras la introducción y el estado del arte en el que se enmarca la tesis, se explican los resultados obtenidos durante este trabajo. Durante la primera parte, se describe el desarrollo, fabricación y caracterización de un sistema autónomo para el cultivo y electroestimulación de tejidos que integra un lab on PCB (LOP) formado por un array de microelectrodos en 3D (MEA) formado por hilos de oro de 25 µm en sustrato transparente, sensores y actuadores, junto con una plataforma microfluídica fabricada en metacrilato (PMMA) y polidimetilsiloxano (PDMS). El LOP permite mantener las condiciones de temperatura idóneas para llevar a cabo cultivos de media-larga duración sin necesidad de usar incubadoras deCO2 , así como su seguimiento de forma continua a través de un microscopio, gracias al uso de materiales transparentes. Este sistema también incluye una electrónica suplementaria y un programa que permite la monitorización del sistema y la electrostimulación de la muestra biológica. Tras explicar detalladamente el diseño y el novedoso proceso de fabricación del LOP, se presentan los resultados experimentales. En primer lugar, se ha demostrado que es posible desarrollar cultivos organotípicos de retinas de ratón durante más de 7 días, obteniendo resultados muy similares a los conseguidos para las mismas muestras biológicas, pero mediante métodos de cultivo tradicionales. Además, se ha logrado la neuro-protección mediante la electroestimulación de retinas de ratón con la enfermedad de la retinosis pigmentaria, logrando de esta manera ralentizar la degeneración de la muestra debido a la enfermedad. Otra de las aplicaciones que se quiere conseguir con el desarrollo del LOP anteriormente descrito se centra en la adquisición de señales eléctricas procedentes de las muestras biológicas cultivadas en el dispositivo, así como extrapolar su uso a cultivos celulares. Para la adquisición de señales procedentes del cultivo, la impedancia de los electrodos fabricados con hilos de oro de 25 µm ha resultado ser demasiado alta como para discernir entre el ruido base y la actividad eléctrica del cultivo. Por ello, la segunda parte de esta tesis doctoral se centra en la mejora de la MEA para la adquisición de actividad eléctrica. Dado el objetivo marcado en esta segunda parte, durante esta tesis se ha realizado una estancia en la Universidad de Bath. En dicha estancia, se ha caracterizado la actividad eléctrica de células del cáncer de próstata (PC-3), que fueron cultivadas en chips de silicio con electrodos de oro. La experiencia obtenida durante la estancia ha permitido avanzar en el desarrollo y la fabricación de nuevas MEAs para la adquisción de señales eléctricas de cultivos celulares. La primera aproximación para mejorar la MEA se ha realizado sobre PCB. Se trata de un dispositivo compuesto por pilares de oro en 3D fabricados mediante la técnica de Resumen XXV electroplating. Estos electrodos tienen 100 µm de diámetro y una altura de 25 µm que aseguran el contacto en el caso de cultivos de tejidos. Se ha demostrado una mejora significativa, traducida tanto en una impedancia más baja, como en una línea base de ruido menor con respecto a la MEA con hilos de oro. Asimismo, se han obtenido patrones de actividad eléctrica en las células PC-3 muy similares a los obtenidos con el chip de silicio y oro empleado en la estancia. Como mejora de la MEA 3D se ha cambiado el sustrato por otro transparente, como vidrio o PMMA, para permitir su uso en aplicaciones ópticas. Dichas MEAs integran electrodos planares fabricados mediante la técnica de sputtering de oro sobre su superficie. Estas MEAs están en una fase preliminar de desarrollo, y se está probando en primer lugar su biocompatibilidad y viabilidad para el desarrollo de cultivos celulares. Para finalizar, se exponen las conclusiones de esta tesis doctoral, entre las que destacan: el proceso de fabricación del LOP con electrodos de oro y la aplicación del sistema completo para desarrollar cultivos organotípicos, monitorizarlos y aplicar electroestimulación, logrando la neuro-protección de retinas de ratón con la retinosis pigmentaria; la transición hacia el desarrollo de una plataforma para cultivos celulares mejorando la MEA y su fabricación usando diferentes sustratos; la caracterización de la actividad eléctrica de las células PC-3. También se incluyen las líneas de investigación abiertas para continuar lo que se ha empezado en esta tesis doctoral. Para facilitar la comprensión del lector, se adjuntan los apéndices complementarios a esta tesis doctoral.The presented thesis is focused on the development and validation of lab on chip (LOC) platforms for their application on Biology, Medicine and Biomedicine, particularly those related with cells and tissues cultures, as well as their treatment through electrostimulation and their electrical behavior. Nowadays, research works focused on the development of LOCs have significantly increased, mostly thanks to its high versatility, which involves countless applications. Among all this applications, those related with Biology and Medicine are becoming more and more important. The integration of sensors, actuators, microfluidic circuits and electronic circuits in the same platform allows the fabrication of systems with lots of applications. This thesis is focused on the development of platforms for in vitro cultures of cells and tissues, to monitor their behavior and interact with the biological samples. The importance of in vitro cultures lies on the study of cells and tissues proliferation and morphology or performing drug delivery experiments. In this respect, through LOC technologies, it would be possible to model human diseases in vitro, in order to improve the development of new drugs and advance personalized medicine. Thus, the possibility of carrying out medium-long term cultures on platforms without the need of any expensive equipment, such as CO2 incubators, with software and monitoring, implies a qualitative step forward in the development of in vitro cultures. Within this framework, the work related to this thesis is presented. This PhD has been undertaken in the Microsystem group of the High School Engineering of the University of Seville. The structure of this thesis is organized in such a way that, all along the text, the different aspects previously described are explained in detail. Firstly, a brief introduction about MEMS technology and the basic principles of Microfluidics is presented. Due to this work has been developed in a multidisciplinary environment, this section becomes necessary in order to give a wide view to those non XXVII XXVIII Abstract directly familiarized with these fields. Subsequently, a description of the state of the art is presented, including LOC systems and their applications in Biology, Medicine and Biomedicine, taking special attention to those applications related to organotypic and cell cultures. After the introduction and the state of the art of the framework of this thesis, the results obtained are presented. In the first part of this PhD, the development, fabrication and characterization of the autonomous system for culture and electrostimulation of tissues is described. This system includes a lab on PCB (LOP) composed of a 3D microelectrode array (MEA), with gold wires of 25 µm on transparent substrate, sensors and actuators, together with a microfluidic platform made of PMMA and PDMS. This LOP allows to maintain the appropriate temperature conditions to carry out medium-long term cultures without using a CO2 incubator, as well as its continuous monitoring through an inverted microscope, thanks to the transparent materials used for its fabrication. This system is connected to an external electronic circuit and a software to control the whole system, including the electrostimulation of the biological sample. After explaining the design and the innovative fabrication process of the LOP, the experimental results are presented. Firstly, it has been demonstrated the suitability of this system to perform organotypic cultures of mice retinas for longer than 7 days, obtaining similar results to the same samples, but cultured through traditional methods. In addition, it has been provided neuroprotection to mice retinal explants with the retinitis pigmentosa (RP) disease through the electrostimulation of the samples, being able to slowdown the degeneration of the retinas caused by RP

Effects of Dip-coated films on the Properties of Implantable Intracortical Microelectrodes

The successful clinical use of implantable intracortical microelectrodes (ICMs) to treat certain types of deafness, blindness, and paralysis is limited by a reactive tissue response (RTR) of the brain. This RTR culminates in the formation of a tight glial scar and a loss of neuronal density around implanted ICMs, and is accompanied by a decrease in signal to noise ratio and an increase in impedance. While no comprehensive mechanistic understanding of the underlying biology is currently agreed upon in the field, a general consensus exists around a highly volatile acute RTR phase. During this acute phase, the electrical properties of ICMs do not always coincide with cellular responses, and the extent of initial injury appears to greatly influence the degree of the chronic RTR. While many electrode modifications and treatments are effective in the short term, the chronic RTR appears impervious to most interventions. To better understand the acute phase of the RTR, this dissertation aims to investigate the effects of various dip-coated biomolecules on the electrical properties of ICMs and cellular responses to microscale ICM-like foreign bodies. We first present an examination of silica sol-gel thin films as a potential biomolecule delivery platform which does not adversely affect the electrical properties of ICMs. The second study shows that adsorbed proteins, thought to play an important role in modulating the RTR, cause significant increases in electrode impedance. In contrast to prevalent electrical models of the electrode tissue interface which assume purely resistive impedance changes due to adsorbed proteins, our results show both resistive and capacitive changes. We also show that increases in impedance related to protein adsorption can be prevented by dip coating ICMs in an aqueous solution of high molecular weight polyethylene glycol (PEG). We then describe a method to clean electrode sites using direct current (DC) biasing, showing that DC biasing is capable of restoring electrode impedance following exposure to enzymatic cleaning solutions, proteins, phantom brains, and actual brain tissue. The final study in an in vitro mixed primary cortical cell culture model shows that lipopolysaccharide (LPS), a well-known ligand to toll-like 4 (TL4) receptors, dip-coated onto segments of metal microwire, can simulate localized inflammation around an implanted ICM. We observe elevated activation of glial cells in interface regions, and extending into more distant regions. This elevation in glial responses is not accompanied by a decrease in neuronal density. We additionally show that microwire dip-coated with a mixture of LPS and PEG exhibits significantly lower microglial and astrocyte responses. These findings highlight the importance of adsorbed proteins, some of which are implicated in aggravating the reactive tissue response, but which we show can result in significant increases in electrode impedance before the RTR even begins. These impedance changes can be prevented through the use of dip-coated PEG. Our cell culture data presents further evidence for the attractiveness of TL4 receptors as a target for intervention, and suggests that the loss of neuronal density observed in vivo is better explained by other mechanisms following device insertion than pure glial activation

A combined experimental and computational approach to investigate emergent network dynamics based on large-scale neuronal recordings

Sviluppo di un approccio integrato computazionale-sperimentale per lo studio di reti neuronali mediante registrazioni elettrofisiologich

Optical mapping and optogenetics in cardiac electrophysiology research and therapy:a state-of-the-art review

State-of-the-art innovations in optical cardiac electrophysiology are significantly enhancing cardiac research. A potential leap into patient care is now on the horizon. Optical mapping, using fluorescent probes and high-speed cameras, offers detailed insights into cardiac activity and arrhythmias by analysing electrical signals, calcium dynamics, and metabolism. Optogenetics utilizes light-sensitive ion channels and pumps to realize contactless, cell-selective cardiac actuation for modelling arrhythmia, restoring sinus rhythm, and probing complex cell–cell interactions. The merging of optogenetics and optical mapping techniques for ‘all-optical’ electrophysiology marks a significant step forward. This combination allows for the contactless actuation and sensing of cardiac electrophysiology, offering unprecedented spatial–temporal resolution and control. Recent studies have performed all-optical imaging ex vivo and achieved reliable optogenetic pacing in vivo, narrowing the gap for clinical use. Progress in optical electrophysiology continues at pace. Advances in motion tracking methods are removing the necessity of motion uncoupling, a key limitation of optical mapping. Innovations in optoelectronics, including miniaturized, biocompatible illumination and circuitry, are enabling the creation of implantable cardiac pacemakers and defibrillators with optoelectrical closed-loop systems. Computational modelling and machine learning are emerging as pivotal tools in enhancing optical techniques, offering new avenues for analysing complex data and optimizing therapeutic strategies. However, key challenges remain including opsin delivery, real-time data processing, longevity, and chronic effects of optoelectronic devices. This review provides a comprehensive overview of recent advances in optical mapping and optogenetics and outlines the promising future of optics in reshaping cardiac electrophysiology and therapeutic strategies