April 02, 2020

Cell-culture microarchitectures for 3D neuronal networks

Neuronal cell-culture microarchitectures
Neuronal cell-culture microarchitectures, fabricated by Nanoscribe’s 3D printers, can be used to study phenomena of neuronal networks in detail. Image: Cornelius Fendler, Research Group Blick, Center for Hybrid Nanostructures, Universität Hamburg

A novel cell-culture microarchitecture enables cell attachment and neurite guidance of single neurons along 3D defined paths. With the use of a Nanoscribe Photonic Professional system, an interdisciplinary research team was able to design tailor-made neuronal networks on complex 3D printed scaffolds. Their findings pave the way for future customizable and more intricate 3D neuronal networks to study cellular behavior, information transfer and to monitor the entire network activity.

Networks of connected neurons help scientists to understand the brain functionality. Specifically, biological neuronal networks are studied to explore, for example, how large the processed data volumes are, how connectivity develops in learning processes or how diseased neuronal cells behave. In-vitro neuronal cell cultures are therefore valuable platforms to study neurons in low density networks on a cellular level. However, 2D in-vitro neuronal cultures are often unable to mimic the distinct 3D connections and extremely complex signal processing observed in the nervous system. With the advances in 3D Microfabrication, scientists have been able to develop a novel cell culture scaffold to guide neuronal cell outgrowth and study signal processing in 3D.

Customizable 3D microarchitectures for neurite guidance

Utilizing Nanoscribe’s 3D printer, scientists from the Center for Hybrid Nanostructures at the Universität Hamburg together with the Center for Molecular Neurobiology Hamburg - University Medical Center Hamburg-Eppendorf and the Institute of Physics at the Universität Greifswald fabricated a complex platform of interconnected 3D microstructures with pillars and tunnels. The scaffolds are 3D-printed with IP-Dip printing material and consist of pillars of varying heights with cavities at the top and free-standing tunnels that connect the cavities with each other.

The arrangement of pillars and tunnels can be freely designed in any spatial direction thanks to the design freedom of the 3D Microfabrication technology. Thus, the tunnels can act as customizable pathways for neurite outgrowth in 3D. The presented concept is one promising path for in-vitro investigation of neuronal networks with design-specific 3D complexity.

3D printed scaffold enables tailor-made neuronal networks

The scientists seeded the 3D microstructures with primary murine cerebellar granular cells. Surface coatings of Al2O3 and the polymer parylene-C promoted neuron adhesion and cell viability. The 3D microstructure geometry provided topological guidance while selective poly-D-lysine deposition contributed with chemical guidance. As a consequence, the resulting predefined paths thus guided in-vitro cell culture of neuronal networks for neurite outgrowth.

The cell-culture 3D microstructures simultaneously function as neurocages as well as neuroguides. The result is the formation of ordered neuronal networks: neuronal cells follow selective cell adhesion inside the cavities and neurite outgrow through the tunnels, connecting the cells with their neighboring partners. Moreover, patch clamp measurements revealed the electrophysiological activity of the cells after 10 days in vitro by verifying signal transmission.

3D Microfabrication for complex cell-compatible scaffolds

To mimic realistic microenvironments for cell studies, Nanoscribe’s 3D Microfabrication combined with IP-Visio printing material allows to print extremely intricate 3D microscaffolds. IP-Visio is a novel non-cytotoxic photoresin that is used to print cell-friendly scaffolds for life science applications. Furthermore, IP-Visio shows a very low autofluorescence. Thus, scientists can analyze cells by means of fluorescence microscopy without interference of the printed structures.

Read the scientific publication on Advanced Biosystems: Microscaffolds by Direct Laser Writing for Neurite Guidance Leading to Tailor‐Made Neuronal Networks

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