Growth and Follow-Up of Primary Cortical Neuron Cells on Nonfunctionalized Graphene Nanosheet Film

Abstract

Background Conductive biomaterials are an ideal biosubstrate for modifying cellular behaviors by conducting either internal or external electrical signals. In this study, based on a simple-preparation graphite exfoliation method in organic reagent, a nonfunctionalized graphene nanosheet film (NGNF) with high conductivity and large size was simply fabricated through spraying coating. The biocompatibility of the NGNF was carefully tested with primary cortical neuron cells, and its biocompatibility properties were compared with a chemical vapor deposition (CVD) graphene film. Methods Nonfunctionalized graphene nanosheet (NGN) was first exfoliated from graphite with a flat-tip ultrasonicator probe, and then spray-coated onto glass slide substrate to form the film. The morphology of NGNF was observed with light microscopy and SEM. The morphology and neuronal network formation of primary cortical neuron cells onto NGNF, as shown by DAPI and Alexa Fluor® 488 staining, were observed with fluorescent microscopy. Cell viability and proliferation were measured with MTT. Results NGNF had better cell biocompatibility than CVD graphene film. MTT test showed that NGNF exhibited no cytotoxicity. According to neuronal network formation at 7 days of cell culture, primary neuron cells aggregated into 50-μm “nuclei”; the average neurite number and length were 3 and 100 μm, respectively. However, these values were almost doubled after 14 days of cell culture. Conclusions These results may improve the use of NGNF as a conductive scaffold for nerve regeneration.