Electrochemically Driven Polymerization of pH Sensitive Polymers on High Density Microelectrode Arrays for Neural Patterning In Vitro

In-vitro neuroscience makes use of dissociated neural networks of iPSC derived or animal origin, grown on the surface of microelectrode arrays (MEA) for electrical access. Upon exposure of neurons to a MEA surface, neurons adhere and connect to each other at random, creating highly complex systems. The complexity of neural networks can be reduced by various surface patterning methods, which render part of the surface cell adhesive and other parts cell repellant [1]. Alternatively, the use of biocompatible microstructures is a popular method to confine the neural adhesion and guide the growth of their axons. In the past, polydimethylsiloxane-based microstructures were used with great success to pattern precise neural networks<i> in vitro </i>on low-density glass MEAs with an electrode pitch of 500 µm [2,3] and more recently on high density complementary metal-oxide-semiconductor (CMOS) MEAs [4], that offer up to 26’400 electrodes at a pitch of 17.5 µm.<br/>Here, we demonstrate an alternative patterning method that aims to establish neural guidance structures directly on the surface of the CMOS MEA with a spatial resolution only limited by the electrode pitch of the MEA. By inducing hydrolysis locally on user-defined electrode sets, the pH can be selectively altered in the vicinity of the selected electrodes. This mechanism allows for the local formation of polymers that show a pH-dependent formation, such as transglutaminase crosslinked hydrogel which assembles close to neutral pH [5] or the nature-inspired biopolymer polydopamine that forms in slightly basic (pH &gt; 8) environments [6]. We demonstrate that we can alter the pH with single-electrode precision and can quantify the induced pH change optically using the pH reporter 5-(and-6)-carboxy SNARF-1. Using CMOS MEAs with platinum black coated microelectrodes, we are able to inject large enough currents to shift the local pH of the precursor solutions precisely from 6.6 to 8.4 for dopamine polymerization and 5.8 to 7.5 to form hydrogel. We observe constant pH elevations without precision loss for up to 35 min, which is enough time for local polymer formation. Finally, we provide preliminary results that show that the formed hydrogel guides the growth of primary rat cortical neurons and that the observed effects are dependent on the polymerization time of the hydrogel.<br/><br/>[1] Aebersold et al. “Brains on a chip”: Towards engineered neural networks. Trends in Analytical Chemistry (2016)<br/>[2] Ihle et al. An experimental paradigm to investigate stimulation dependent activity in topologically constrained neuronal networks. Biosensors and Bioelectronics (2022)<br/>[3] Girardin et al. Topologically controlled circuits of human iPSC-derived neurons for electrophysiology recordings. Lab on a Chip (2022)<br/>[4] Duru et al. Engineered Biological Neural Networks on High Density CMOS Microelectrode Arrays. Frontiers in Neuroscience (2022)<br/>[5] Milleret et al. Electrochemical Control of the Enzymatic Polymerization of PEG Hydrogels: Formation of Spatially Controlled Biological Microenvironments. Advanced Healthcare Materials (2013)<br/>[6] Lee et al. Mussel-Inspired Surface Chemistry for Multifunctional Coatings, Science (2007)