Jelena Žarković1,2,3, Ana Bedalov3,4, Boris Delipetar1,2, Tihomir Marjanović5, Milan Blašković5, Slaven Garaj5,6, Damir Kovačić1,2

1The Center of Research Excellence for Science and Technology Integrating Mediterranean region (STIM), University of Split, Croatia; 2Laboratory for Biophysics and Medical Neuroelectronics, Department of Physics, University of Split-Faculty of Science, Croatia; 3The doctoral program in Biophysics, University of Split-Faculty of Science, Croatia; 4Bedalov d.o.o for Research, Development and Consulting, Croatia; 5Centre for Advanced 2D Materials, National University of Singapore, Singapore; 6Department of Physics and Department of Biomedical Engineering, National University of Singapore, Singapore


A new generation of neural interfaces based on novel graphene-based materials is expected to address limitations of low spatial resolution and broad specificity of neuronal stimulation in current cochlear implant devices leading to poor pitch perception and significant variability in its efficacy. Graphene and other two-dimensional (2D) materials possess various properties, making them an attractive candidate for future cochlear implants. It was previously shown that graphene might support neural growth due to its unique electric properties, biocompatibility with neuronal cells, and ability to effectively interface with neuronal tissue. However, it is not known how graphene interacts with spiral ganglion neurons (SGN) morphology. In this study, three types of graphene-based substrates (single layer graphene – SLG, hydrogenated graphene – HG, and fluorinated graphene – FG) were used as a surface for in-vitro culturing of neonatal SGN extracted from rat pups. The cultures were immunocytochemically stained at eight days in vitro (8DIV), and the subsequent fluorescence images were analyzed with the custom-made machine learning-based image processing allowing automatic identification and segmentation of neurons. We compared morphological properties of neuronal cultures grown on graphene-based and single-layer h-BN surfaces with those grown on glass coverslips. We found that neonatal SGN neurons may grow on all four tested substrates with significantly altered morphological properties compared to standard glass coverslips. Analysis of the mixed effect model of different morphological parameters data revealed that HG and SLG substrates represented a more favorable environment for spiral ganglion neuron growth than those grown on control coverslips. These findings reveal substantial interaction between material properties of graphene-based substrates on in-vitro SGN cell cultures.

Acknowledgments: This research was partially supported from STIM – REI, Contract Number: KK., a project funded by the European Union through the European Regional Development Fund – the Operational Programme Competitiveness and Cohesion 2014-2020 (KK. BP has been fully supported by the “Young researchers’ career development project – training of doctoral students” of the Croatian Science Foundation funded by the European Union from the European Social Fund. S.G. acknowledges support National Research Foundation, Prime Minister’s Office, Singapore (Award No. NRF-CRP13-2014-03).

[1]   M. Mattotti, L. Micholt, D. Braeken, and D. Kovačić, “Characterization of spiral ganglion neurons cultured on silicon micro-pillar substrates for new auditory neuro-electronic interfaces,” Journal of Neural Engineering, vol. 12, no. 2, 2015.

[2]   V. Radotić, D. Braeken, P. Drviš, M. Mattotti, and D. Kovačić, “Advantageous environment of micro-patterned, high-density complementary metal-oxide-semiconductor electrode array for spiral ganglion neurons cultured in vitro,” Scientific Reports, vol. 8, no. 1, 2018.


Presenting author e-mail address: jelena.zarkovic@pmfst.hr