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

摘要

Objective. In this work we adapted a protocol for the fast generation of human neurons to build 3Dneuronal networks with controlled structure and cell composition suitable for systematicelectrophysiological investigations. Approach. We used biocompatible chitosan microbeads asscaffold to build 3D networks and to ensure nutrients-medium exchange from the core of thestructure to the external environment. We used excitatory neurons derived from human-inducedpluripotent stem cells (hiPSCs) co-cultured with astrocytes. By adapting the well-established NgN2differentiation 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 microelectrode arrays (MEAs) to monitor and characterize their electrophysiological development. Inparallel, we generated two-dimensional neuronal networks cultured on chitosan to compare theresults of the two models. Main results. We sustained samples until 60 d in vitro (DIV) and 3Dcultures were healthy and functional. From the structural point of view, the hiPSC derived neuronswere able to adhere to chitosan microbeads and to form a stable 3D assembly thanks to theconnections among cells. From a functional point of view, neuronal networks showed spontaneousactivity after a couple of weeks. Significance. We presented a particular method to generate 3Dengineered cultures for the first time with human-derived neurons coupled to MEAs, overcomingsome of the limitations related to 2D and 3D neuronal networks and thus increasing thetherapeutic target potential of these models for biomedical applications.