Maccione, A. | Search bioRxiv

bioRxiv2023

Human-derived cortical neurospheroids coupled to passive, high-density and 3D MEAs: a valid platform for functional tests

With the advent of human-induced pluripotent stem cells (hiPSCs) differentiation protocols, different methods to create in-vitro human-derived neuronal networks have been proposed. Although monolayer cultures represent a valid model, adding three-dimensionality would make them more representative of an in-vivo environment. Thus, human-derived neurospheroids and brain-organoids are becoming increasingly used for in-vitro disease modeling. Achieving control over the final cell composition and investigating the exhibited electrophysiological activity is still a challenge. Thence, platforms capable of measuring and characterizing the functional aspects of these samples are needed. Here, we propose a method to rapid generate neurospheroids of human origin with control over cell composition that can be used for functional investigations. We show a characterization of the electrophysiological activity exhibited by the neurospheroids by presenting for the first-time results from the main micro-electrodes arrays (MEAs) types available on the market (passive electrode, C-MOS electrodes, 3D electrodes). Neurospheroids grown in free culture and transferred on MEA exhibited functional activity that can be chemically and electrically modulated. Our results indicates that this model holds great potential for in-depth study of signal transmission to drug screening, disease modeling and offers a reproducible and stable platform for in-vitro functional testing.

bioengineering↗

bioRxiv2022

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 (needles) of 65-90 m 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 needles 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 needles 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.

neuroscience↗