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

Abstract

Directed guidance of neurites is a pre-requisite for tailor-made designs of interfaces between cells and semiconducting components. Grayscale lithography, reactive ion etching, and ultraviolet nanoimprint lithography are potent semiconductor industry-compatible techniques for a cost- and time-effective fabrication of modulated surfaces. In this work, neurite outgrowth of murine cerebellar neurons on 2.5D pathways produced with these methods is studied. Structures of micron-sized steps and grooves serve as cell culture platforms. The effects of contact guidance through topography and chemical guidance through selective poly-d-lysine coating on these platforms are analyzed. As a consequence, the herein presented fabrication approach can be utilized to cultivate and to study low-density neuronal networks in 2.5D configuration with a high degree of order.

Fig. 1. (a–c) CLSM images of MCGCs cultured on ParC coated step structures produced by GSL at 4 DIV. Brightness of the steps correlates with the height: light and dark correspond to top steps and bottom pits, respectively. Red arrows point to neurites guided along edges. (d) Circular histogram of the normed distribution of neurite orientations in (a) relative to the step edges. (e–i) False-colored scanning electron microscopy (SEM) images of neurons on step-structures after 4 DIV.

Fig. 2. CLSM images of MCGCs at 10–11 DIV on steps of varied height (h = 3 μm, 6 μm and 9 μm) and width (w = 12 μm and 32 μm) produced by RIE. All substrates have been coated with ParC and PDL.

Fig. 3. False-colored SEM images and a CLSM image of MCGCs on chessboard structures produced by GSL after 6 DIV. (a) Two connected neurons. Three magnified areas of interest show (1) guidance along the bottom of a channel, (2) guidance on the sidewall of a channel, and (3) a neurite pathway exiting a channel. (b) Red arrows indicate spots, where neurites leave the channels. Yellow arrows indicate neurites, that are not guided. Blue arrows indicate re-entering of neurites into channels. Neurites originating from a cell cluster and somata that are settled on top of channels. (c–h) Examples of neurites exiting, entering, and crossing channels.

Fig. 4. CLSM images and orientation analysis of neurons cultured on PDL circles with diameters of approx. 36 μm printed on an Al₂O₃ surface (7 DIV). (a–c) Circles printed in a pattern of 90 μm periodicity and an average distance between two circles of 54 μm. (d) Color-coded confocal image of neurons cultured on PDL circles in a pattern of 90 μm. Coloration of neurites corresponds to the neurite direction relative to the pattern. (e) Circular histogram of the normed distribution of neurite orientations on the pattern. (f) Percentage share of neurite pathways between nearest (first), second nearest and third-nearest circles. (g–i) Circles printed in a pattern of 190 μm and an average distance between two circles of 154 μm.

Fig. 5. CLSM images of MCGCs at 6–8 DIV on chessboard structures produced by RIE and UV-NIL. (a) Cavities of 10 μm depth and steps in channels of 5 μm height coated with PDL with a materials printer. (b–e) Cavities of 14 μm depth and steps in channels of 4 μm height. PDL coated by a material printer (b) and with a micromanipulator (c–e). Red arrows indicate neurites guided at the top edge of cavities. Blue arrows indicate neurites at the bottom of cavities.