Parkinson’s disease is a serious problem, especially as the life span increases in the general population, mostly because there is no cure and the current treatment options are far from being optimal. The disease is characterized by insufficient dopamine in the brain, a neurotransmitter involved in the motor function. Recent approaches recognize the need for personalized treatment, and one option is cell replacement therapy. This requires obtaining a large population of mature, dopaminergic neurons.

Our group has previously shown that SU-8-derived carbon surfaces (either flat carbon surfaces or carbon micropillars) induce spontaneous differentiation of human neural stem cells (hNSCs) into dopaminergic neurons¹. Here we report 3D carbon nanopillars, which show a high increase in the currents measured from dopamine exocytosis from hNSc-derived dopaminergic neurons. The carbon surfaces mentioned above can be employed as substrates for cell growth, but also as electrodes for monitoring dopamine release.

Fabrication of the nanopillars is done by colloidal lithography and pyrolysis. A silicon wafer with a 200 µm SiO₂layer on top is first spin-coated with 5 µm of SU-8 2005. Then, a monolayer of polystyrene beads is deposited on the SU-8 film and serves as etching mask during the plasma treatment. The diameter of the nanopillars can be tuned using beads of different sizes and the height can be controlled by adjusting the etching time. After etching, we use ultrasonication in isopropanol as lift-off technique to remove the beads. Finally, the samples are pyrolysed (1h at 900°C, with 2°C/min for both heating and cooling) to obtain the carbon nanopillar electrode chips. A schematic representation of the fabrication process is shown in figure 1A.

The nanopillars employed for the measurements described in this abstract were obtained by using 1µm beads and an etching time of 20 min, which leads to structures with a height of 1.2 µm and a diameter of 450 nm (before pyrolysis, figure 1B) and a height of 600 nm and a width of 200 nm after pyrolysis (figure 1C).

The carbon structures were employed as substrate for cell growth and differentiation (figure 1D shows a high-magnification SEM image of a cell growing on the nanopillars) after plasma treatment (to improve surface wettability) and poly-L-lysine modification (to improve cell adhesion). Even without adding differentiation factors to the system, differentiation into dopaminergic neurons was observed, with a yield of about 80% dopaminergic neurons. SEM imaging, immunocytochemistry and amperometry were employed as techniques to confirm this. The immunostaining was done for tyrosine hydroxylase (TH) and nuclei and shows that most cells are TH-positive and thus potentially dopaminergic. The dopaminergic phenotype was confirmed by amperometric monitoring of the dopamine released (exocytosis) from cells induced by KCl stimulation. Amperometric measurements were conducted on electrodes with nanopillars i) 48 h after cell seeding, in the absence of differentiation factors and ii) 10 days after cell seeding, in the presence of differentiation factors. For comparison, flat carbon surfaces and carbon micropillars were fabricated and tested in parallel.

Compared to flat carbon surfaces, both the micropillars and the nanopillars show increased currents when measuring released dopamine using amperometry. We calculated the charges for exocytosis on micropillars as being 4 times higher than in the case of flat carbon, although the surface area increase is of just 1.9 times. On nanopillars, the measured charges are even higher, 12 times larger than for the flat carbon and 3 times larger than for micropillars, although in this case, the surface area increase is of only 1.4 times compared to the flat carbon. This leads to an improved detection and quantification of released dopamine when using nanopillar electrodes. The electrode chips used in all 3 cases have the same basic footprint size and were seeded with the same number of cells, in the same conditions.

This work shows that carbon surfaces with nanopillars induce spontaneous differentiation of human neural stem cells into dopaminergic neurons and can be employed as electrodes for monitoring dopamine release from mature dopaminergic cells, with a much better electrochemical response compared to flat carbon surfaces or micropillar arrays.

 

1. L. Amato et. al., Pyrolysed 3D-Carbon Scaffolds Induce Spontaneous Differentiation of Human Neural Stem Cells and Facilitate Real Time Dopamine Detection, Advanced Functional Materials, 2014, Vol. 24, Issue 44, 7042-7052.