Three-dimensional Functional Neuronal Networks in Free-Standing Bioprinted Hydrogel Constructs.

The composition, elasticity and organization of the extracellular matrix within the central nervous system contribute to the architecture and function of the brain. From an in vitro modelling perspective, soft biomaterials are needed to mimic the three-dimensional (3D) neural microenvironments. While many studies have investigated 3D culture and neural network formation in bulk hydrogel systems, these approaches have limited ability to position cells to mimic sophisticated brain architectures. In this study, cortical neurons and astrocytes acutely isolated from the brains of rats are bioprinted in a hydrogel to form 3D neuronal constructs. Successful bioprinting of cellular and acellular strands in a multi-bioink approach allow the subsequent formation of gray- and white-matter tracts reminiscent of cortical structures. Immunohistochemistry shows the formation of dense, 3D axon networks. Calcium signalling and extracellular electrophysiology in these 3D neuronal networks confirm spontaneous activity in addition to evoked activities under pharmacological and electrical stimulation. Our system and bioprinting approaches are capable of fabricating soft, free-standing neuronal structures of different bioink and cell types with high resolution and throughput, which provides a promising platform for understanding fundamental questions of neural networks, engineering neuromorphic circuits and for in vitro drug screening. This article is protected by copyright. All rights reserved.