Advancing three-dimensional electrophysiology: Development and evaluation of versatile platform for tailored 3D microelectrode array fabrication

Microelectrode arrays (MEAs) are widely used tools to investigate in vitro neuronal networks and acute brain slices. Planar or 2D MEAs have been the conventional standard for decades, enabling the extracellular recording and stimulation of cultured neuronal cells and tissue slices. However, the effectiveness of planar MEAs diminishes when stimulating or recording from 3D in vitro neuronal cultures or brain slices owing to rapid data attenuation in the z-direction. Existing 3D in vitro neuronal models only permit recording electrophysiological activity from the bottom layer directly connected to planar MEAs. Consequently, to advance and optimize 3D neuronal network systems and comprehensively study the dynamics of neuronal networks across different layers of 3D structures, the development of new three-dimensional microelectrode arrays (3D MEAs) is necessary. Over the last three decades, numerous approaches for developing 3D MEAs have been reported; however, most reported technologies can develop quasi-3D MEAs, that is, 3D MEAs with uniform electrode heights. To fully exploit the potential of 3D neuronal constructs, advanced technological platforms capable of developing versatile 3D MEAs consisting of variable-height electrodes, customizing the array topography on demand, and compatibility with existing readout platforms are required. This thesis introduces a novel technological platform for fabricating diverse 3D MEA architectures using established micromachining techniques such as lithography and templated-assisted electrodeposition. The key to this platform is an innovative approach that enables selective electrodeposition of 3D electrodes within an array. This technological platform utilizes an advanced method to enhance the deposition rate and uniformity of electrodeposited microstructures, which is crucial for precise control over the electrode height in 3D MEAs. Using template-assisted electrodeposition, this platform facilitated the transformation of planar MEAs into 3D configurations with varying electrode heights. Notably, the platform enables the development of 3D MEAs with up to four different electrode heights using a single layout definition mask, ensuring cost-effectiveness and scalability. This precise control over electrode height unlocks the potential for tailoring 3D MEAs to specific research needs. A unique multi-height 3D MEA was created to demonstrate the versatility of the proposed platform. This MEA features uniformly distributed electrodes of four distinct heights across the array, enabling the probing of 3D neuronal constructs from the surface to multiple depths. This unique design approach has the potential to provide insights into cellular activities. The performance of the multi-height 3D MEA was validated through rigorous testing involving the recording of electrophysiological activities from a neurospheroid and comparison with a commercially available 3D MEA with similar electrode layouts. This comparative analysis demonstrates the proposed platform's superiority and adaptability. This thesis is divided into seven chapters. Chapter 1 lays the groundwork by explaining the significance of electrophysiological investigations in understanding brain function and highlighting the limitations of traditional electrophysiological techniques designed for 2D cultures or tissue slices. This chapter sets the stage for the core focus of this thesis, that is, the development of advanced 3D MEAs consisting of electrodes with variable heights. It outlines the morphological and functional advantages offered by 3D neuronal constructs and introduces the escalating demand for innovative MEAs capable of navigating the complexities of these environments. Furthermore, this chapter discusses state-of-the-art approaches for developing 3D MEAs and identifies the limitations of existing technologies. Chapter 2 presents a comprehensive overview of the current knowledge landscape in the field of MEA technology in the context of both traditional 2D MEA technology and recent advancements in 3D MEA technology. This chapter delves into the intricacies of each aspect, including design considerations, fabrication techniques, electrode materials, integration and interface electronics, cell-loading methods, recording electrophysiological activities, applications, challenges, and future directions associated with each of these areas. The thorough review presented in this chapter serves as the theoretical foundation for subsequent empirical investigation. Chapter 3 provides an in-depth analysis of the design philosophy and inception of the MEA fabrication platform, shedding light on the motivations, design objectives, and methodology employed in developing this innovative technology. This chapter is a crucial building block for subsequent discussions on the fabrication processes and outcomes. Chapter 4 introduces the conceptualized platform by developing multilevel 3D MEAs using a conceptualized platform consisting of electrodes with three distinct heights. This chapter describes the device layout at the wafer scale and individual MEA levels. This chapter details the layout and functioning of custom circuitry to enable the selective electrodeposition of 3D electrodes. Finally, this chapter highlights the platform's advantages, shortcomings, and potential solutions. Chapter 5 explores the cutting-edge application of ultrasonic vibrations in template-assisted electrodeposition to enhance the fabrication of three-dimensional (3D) microelectrode arrays (MEAs). Building on the insights from Chapter 4, which highlighted the development of multilevel 3D MEAs using the proposed platform, this study aimed to overcome the limitations of conventional electrodeposition methods. By incorporating ultrasonic vibrations into the electrodeposition process, this chapter seeks to increase the deposition rate and improve the uniformity of microstructures, such as micropillars, within MEAs. Chapter 6 details the development of a multi-height 3D MEA comprising four electrode heights. This was achieved using optimized layouts and an enhanced electrodeposition process incorporating ultrasonic vibrations. This marks the culmination of extensive research. This chapter presents the empirical results of electrophysiological recordings, including their interpretation and comparison with existing state-of-the-art commercial 3D MEA. Finally, Chapter 7 offers a reflective synthesis. It summarizes the research findings, accentuates their significance, and underscores their contributions to the field. Importantly, this chapter sets the stage for future inquiries, identifying unresolved questions and proposing avenues for further research and technological advancements. This thesis embarks on a multifaceted exploration of seamlessly weaving theoretical frameworks, experimental methodologies, and empirical findings. Through a meticulously structured journey, it endeavors to contribute to the advancement of 3D MEA technology, bridging the gap between traditional electrophysiological tools and the intricacies of 3D neuronal cultures by focusing on the development and application of advanced 3D MEAs for electrophysiological recordings in 3D neuronal cultures. It delves into the principles of 3D MEA technology, discusses the challenges and solutions to its implementation, and highlights its significance in advancing neuroscientific research.