A novel Three-Dimensional Micro-Electrode Array for in-vitro electrophysiological applications

Microelectrode arrays (MEAs) represent a powerful and popular tool to study in vitro neuronal networks and acute brain slices. The research standard for MEAs is planar or 2D-MEAs, which have been in existence for over 30 years and used for extracellular recording and stimulation from cultured neuronal cells and tissue slices. However, planar MEAs suffer from rapid data attenuation in the z-direction when stimulating/recording from 3D in-vitro neuronal cultures or brain slices. The existing proposed 3D in-vitro neuronal models allow to record the electrophysiological activity of the 3D network only from the bottom layer (i.e. the one directly coupled to the planar MEAs). Thus, to further develop and optimize such 3D neuronal network systems and to study and understand how the 3D neuronal network dynamics changes in different layers of the 3D structure, new three-dimensional microelectrodes arrays (3D-MEAs) are required. Early attempts in this field resulted in interesting integrated approaches toward protruding or spiked 3D-MEAs. Although these first prototypes could be successfully employed with brain slices, the limited heights of the electrodes (up to max 70 μm) and the peculiar shape of the recording areas made them not an ideal solution for 3D neuronal cultures. Moreover, a convenient and versatile method for the fabrication of multilevel 3D microelectrode arrays has yet to be obtained, due to the usually complicated and expensive designs and a lack of a full compatibility with standard MEAs both in terms of materials and recording area dimensions. To overcome the afore-mentioned challenges, in this work, I present the design, microfabrication, and characterization of a new 3D-MEA composed of pillar-shaped gold 3D structures with heights of more than 100 μm that can be used, in principle, on every kind of MEA, both custom-made and commercial. I successfully demonstrate the capability and ability of such 3D-MEA to record electrophysiological spontaneous activity from 3D engineered in-vitro neuronal networks and both 4-AP-induced epileptiform-like and electrically-evoked activity from mouse acute brain slices. I also demonstrate how the developed 3D-MEA allows better recording and stimulating conditions while interfacing with acute brain slices as compared to planar electrode arrays and previously reported 3D MEA technologies.