Modification of Glial Attachment by Surface Nanostructuring of SU-8 Thin Films ^†

Abstract

Various methods are currently under development to enhance the biocompatibility of neural electrodes and to minimize the reactive gliosis around the implant surface. As cells in their native microenvironment interact with 3D nanoscale topographies of the extracellular matrix, physical modification of implant surfaces may provide an alternative solution to the negative tissue response by imitating the structure of the extracellular matrix, and therefore affecting the attachment and behavior of neurons and glial cells. The attachment of primary mouse astrocytes on nanostructured SU8 polymer surfaces fabricated by e-beam lithography was investigated in our study. We found that attachment of primary mouse astrocytes on silicon-SU8 surfaces is strongly influenced by the surface topography.

1. Introduction

The long-term application of implanted CNS sensors and stimulators is currently limited by the immuneresponse of the brain tissue, affecting both the performance of the device and the integrity of the neural network. Implants are required to limit the activation of astrocytes, which would ultimately lead to insulation from the surrounding tissue [1]. Topographical modification of implant surfaces may serve as a possible solution, since imitating the structure of the extracellular matrix has an influence on both attachment and behavior of neural cells [1,2].

Various groups showed the impact of surface topography on cell attachment and development: the fill factor of micropillars influences cell development [3]; cells sense the depth of micro gratings [4]; the distance between a micropattern and soma of a neuron affects the neurite outgrowth [5]. The neurites length, spreading, branching were studied to get more information about the interaction between the artificial surface and the cells [6].

2. Materials and Methods

2.1. Surface Patterning

To create our surface pattern, we used the epoxy-based negative photoresist, SU-8. This material is an epoxy-based negative photoresist, which is as biocompatible as medical steel [7].

In our work, SU-8 structures were patterned by standard photolithography and electron beam lithography depending on the desired feature size. SU-8 2000.5 was used to form the 2 microns high micro- and nanostructures. The applied dose in the Raith 150 E-beam Litography system was 100 mJ/cm². The post exposure bake was performed at 95 °C for 10 min.

The optimal irradiation depends on the sensitivity of the photoresist type, its thickness and the electron energy. These parameters were investigated preliminarily to form nanostructures with the desired geometry. To produce structures at the sub-micron scale, O₂ plasma etching was used. Representative features are shown in Figure 1 and Figure 2.

2.2. Cell Culture

Primary astrocytes were prepared postnatally from 3-day-old mouse pups according to the method described in [8]. Cultures were maintained in HDMEM (Sigma) with 10% FCS (Gibco), 2 mM glutamine (Sigma, Kawasaki, Japan), 40 μg/mL gentamicin (Hungaropharma, Budapest, Hungary) and 2.5 μg/mL amphotericin B (Sigma-Aldrich Co. Ltd., Gillingham, United Kingdom). Cells were allowed to proliferate and passaged 1× with 0.05% trypsin – 0.02% EDTA (Sigma-Aldrich Co. Ltd., Gillingham, United Kingdom) before being seeded onto the test surfaces. Test surfaces were sterilized at 180 °C for 4 h then placed in 24-well culture plates. No additional surface treatment was applied. Astrocytes were seeded at starting densities of 210 cells/mm². Cultures were kept at 37 °C in a 5% CO₂ atmosphere and fixed after 24 or 48 h.

3. Results and Discussion

In vitro test chips were designed to investigate the cell-nanostructure interaction on different SU-8 surfaces using scanning electron microscopy. We found that shape and density of SU-8 nanostructures strongly affect the attachment of primary mouse astrocytes, opening up the way to control glial attachment by local nanoscale surface modification of polymer thin films. Representative results are shown in Figure 3.