Microscaffolds by Direct Laser Writing for Neurite Guidance Leading to Tailor‐Made Neuronal Networks

Abstract

cone. [7,8] Topological cues such as stiffness, [4] hydrophobicity, [9] and modulation of the surrounding surface, for example, through variations in roughness [10,11] or through guiding barriers [12][13][14] and channels, [15][16][17][18][19][20] have also been found to be effective methods to direct the neurite outgrowth on artificial substrates.A significant drawback of classical 2D neuronal in vitro cultures is their inability to sufficiently mimic the distinct 3D connections in a nervous system. [21] Most of the standard lithography techniques are only available in 2D or 2.5D. [22] However, recent advances in micro-and nanoscale fabrication, especially direct laser writing by two-photon polymerization (2PP-DLW), now enable the construction of complex resist-based 3D substrates with feature sizes down to ≈150 nm. [23] Using 2PP-DLW, nearly any mechanically stable structure can be realized as real 3D culturing substrate consisting of a biocompatible polymer within only a few hours. 2PP-DLW has already been utilized for neuronal growth studies. [24][25][26][27][28][29] Note, while most of these applications try to mimic the natural brain environment to a certain structural degree, the adhesions spots of the cell somata and/or the direction of neurite outgrowth on 3D substrates have been statistical so far. As a first step toward 2PP-DLW application for neurite guidance, Turunen et al. used DLW in 2014 to print walls on planar glass substrates to form cavities and channels, and applied laminin with a microinjection system inside the neurocages. [27] The design lacked efficient cell confinement properties, but potential function of printed channels as neurite guides was shown.Other approaches, which enable in vitro neuronal network growth in a 3D environment in different ways, such as synthetic hydrogel platforms, [30][31][32] network growth on micro beads, [21,33] polymer scaffolds fabricated by gaseous salt leaching, [34,35] or microfibrous scaffolds [36] feature only statistically oriented network growth.Here, we present an approach for guided neuronal network growth that features single neurites specifically grown through rectangular tubes in a 3D scaffold. The design we show herein combines the tunability and the precision of a 3D design utilizing 2PP-DLW manufacturing with topologically and chemically predefined adhesion areas as well as guidance paths for While modern day integrated electronic circuits are essentially designed in a 2D fashion, the brain can be regarded as a 3D circuit. The thus enhanced connectivity enables much more complex signal processing as compared to conventional 2D circuits. Recent technological advances in the development of nano/microscale 3D structuring have led to the development of artificial neuron culturing platforms, which surpass the possibilities of classical 2D cultures. In this work, in vitro culturing of neuronal networks is demonstrated by determining predefined pathways through topological and chemical neurite guiding. Tailor-made culturing substrates of microtowers ...

Toward Brain-on-a-Chip: Human Induced Pluripotent Stem Cell-Derived Guided Neuronal Networks in Tailor-Made 3D Nanoprinted Microscaffolds

“…Technical advances in the conceptionally different technology of direct laser writing (DLW) by two-photon polymerization (2PP) now introduce previously impossible approaches to take the step from an 2D BoC toward a more natural 3D environment using CAD-designed scaffolds with feature sizes down to 150 nm . To date, DLW has already been used to create growth guiding neurocages in 2D as well as 3D scaffolds, such as microtowers and woodpile structures without guiding − or scaffolds with controlled cell adhesion or neurite guidance. , Hence, 2PP-DLW is a promising candidate to fabricate scaffolds for BoCs with 3D network topologies.…”

Rapid Prototyping of 3D Biochips for Cell Motility Studies Using Two-Photon Polymerization

“…Given the intrinsically invasive nature of glioma and neuroblastoma, it can be assumed that neuronal neoplastic cells must possess the ability to infiltrate dense tissue. Although the NG108-15 cell line has been previously employed in the past for cell migration studies (Kilian et al, 2008;Jacobshagen et al, 2014;Abe et al, 2018;Fendler et al, 2019), it is not known yet how they would react to migration through strict confinement. Specifically, it has not been shown whether the whole cell body has the ability to squeeze through very tight spaces, or whether this can be achieved only by its dendritic protrusions.…”

“…Studies on the stiffness of the scaffold material demonstrate that also this parameter could influence cellular response to the structure, showing for instance that tumoral cells prefer "softer" materials. Scaffolds with a fully custom 3D geometry, with empty pillars for cell adhesion and microtubes as connections between them, were used by Fendler et al (2019) to guide the growth of neuronal cells and of their axons into a pre-determined neuronal circuit, opening the possibility to singularly investigate the 3D connection between neurons with patch-clamp experiments. A different evolution of the technique is linked to the composition of the employed photoresist.…”

Neurite guidance and neuro-caging on steps and grooves in 2.5 dimensions

“…Parylene C (ParC) and alumina (Al 2 O 3 ) surface coatings are applied by chemical vapor deposition (CVD) and atomic layer deposition (ALD), respectively, before culturing for improved cell adhesion and viability. [40][41][42][43][44][45][46][47] Furthermore, a spatially localized poly-D-lysine (PDL) coating is utilized to improve the selectivity of soma adhesion and neurite outgrowth paths. 3,48,49 Moreover, additional laminin coating promotes neurite outgrowth and neuron viability.…”