Rapid generation of functional engineered 3D human neuronal assemblies: network dynamics evaluated by micro-electrodes arrays

Abstract

<i>Objective.</i>In this work we adapted a protocol for the fast generation of human neurons to build 3D neuronal networks with controlled structure and cell composition suitable for systematic electrophysiological investigations.<i>Approach.</i>We used biocompatible chitosan microbeads as scaffold to build 3D networks and to ensure nutrients-medium exchange from the core of the structure to the external environment. We used excitatory neurons derived from human-induced pluripotent stem cells (hiPSCs) co-cultured with astrocytes. By adapting the well-established NgN2 differentiation protocol, we obtained 3D engineered networks with good control over cell density, volume and cell composition. We coupled the 3D neuronal networks to 60-channel micro electrode arrays (MEAs) to monitor and characterize their electrophysiological development. In parallel, we generated two-dimensional neuronal networks cultured on chitosan to compare the results of the two models.<i>Main results.</i>We sustained samples until 60 d<i>in vitro</i>(DIV) and 3D cultures were healthy and functional. From the structural point of view, the hiPSC derived neurons were able to adhere to chitosan microbeads and to form a stable 3D assembly thanks to the connections among cells. From a functional point of view, neuronal networks showed spontaneous activity after a couple of weeks.<i>Significance.</i>We presented a particular method to generate 3D engineered cultures for the first time with human-derived neurons coupled to MEAs, overcoming some of the limitations related to 2D and 3D neuronal networks and thus increasing the therapeutic target potential of these models for biomedical applications.