Design, implementation, and functional validation of a new generation of microneedle 3D high-density CMOS multi-electrode array for brain tissue and spheroids

In the last decades, planar multi-electrode arrays (MEAs) have been widely used to record activity from in vitro neuronal cell cultures and tissue slices. Though successful, this technique bears some limitations, particularly relevant when applied to three-dimensional (3D) tissue, such as brain slices, spheroids or organoids. For example, planar MEAs signals are informative on just one side of a 3D-organized structure. This limits the interpretation of the results in terms of network functions in a complex structured and hyperconnected brain tissue. Moreover, the side in contact with the MEAs often shows lower oxygenation rates and related vitality issues. To overcome these problems, we empowered a CMOS high-density multi-electrode array (HD-MEA) with thousands of microneedles (μneedles) of 65-90 μm height, able to penetrate and record in-tissue signals, providing for the first time a 3D HD-MEA chip. We propose a CMOS-compatible fabrication process to produce arrays of μneedles of different widths mounted on large pedestals to create microchannels underneath the tissue. By using cerebellar and cortico-hippocampal slices as a model, we show that the μneedles efficiently penetrate the 3D tissue while the microchannels allow the flowing of maintenance solutions to increase tissue vitality in the recording sites. These improvements are reflected by the increase in electrodes sensing capabilities, the number of sampled neuronal units (compared to matched planar technology), and the efficiency of compound effects. Importantly, each electrode can also be used to stimulate the tissue with optimal efficiency due to the 3D structure. Furthermore, we demonstrate how the 3D HD-MEA can efficiently penetrate and get outstanding signals from in vitro 3D cellular models as brain spheroids. In conclusion, we describe a new recording device characterized by the highest spatio-temporal resolution reported for a 3D MEA and significant improvements in the quality of recordings, with a high signal-to-noise ratio and improved tissue vitality. The applications of this game-changing technique are countless, opening unprecedented possibilities in the neuroscience field and beyond. ### Competing Interest Statement Mariateresa Tedesco, Giacomo Sciacca, and Chiara Battaglia are employees of 3Brain AG. Mauro Gandolfo, Kilian Imfeld, and Alessandro Maccione are shareholders of 3Brain AG. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.