The European Human Brain Project funded within the Horizon 2020 framework is one of the Future Emerging Technologies Flagships of the EU with estimated total costs of more than one billion Euro. Understanding the (human) brain’s working principles is the first step to later allow for the preparation of artificial tailor-made 3D neuronal networks.
A brain can be regarded as a three-dimensional circuit and its inherent complex connectivity and signal processing differs strongly from the working principle in nowadays electronic circuits. Two-dimensional neuronal networks have been intensively studied in the past, but only recent technological progress in the development of nano-/microscale 3D structuring have led to novel preparation routes for artificial neuron culturing platforms which overcome the limitations of current 2D culture substrates.
We report on in vitro 3D culturing of neuronal networks determining predefined pathways through topological and chemical neurite guiding. Tailor-made biocompatible 3D culturing substrates of micro-towers of different heights connected by ascending and descending channels were fabricated using direct laser writing by two-photon polymerization and atomic layer deposition. The targeted design allows for site-specific cell attachment inside the tower cavities and directed outgrowth of single neurites along defined 3D paths, to build tailor-made neuronal networks with a controlled cell density. Murine neurons cultured in the scaffolds show characteristic electrophysiological properties of vital cells after 6-10 days in vitro. Active neuronal networks within the tailor-made substrate are revealed by calcium imaging after stimulating a single cell in the network.
The herein presented route offers a promising concept for future complex neuronal network studies on pre-defined 3D neuronal circuits with a tailor-made design for specific neurite connections. Such manufactured neuronal structures supported by a scaffold may give further insights and a deeper understanding of artificial neuronal processes not specifically targeted within the Human Brain Project.