Table 3.

Summary of in vitro cytological studies on the effects of graphene and graphene-based materials on nerve cells

Year of publication	Cell type	Nerve cell sources	Culture method	Detection method	Main results	Significance
Chen et al., 2012	Human embryonic stem cells	Wicell (Madison, WI, USA; passages 32 to 55)	A neuron induction medium consisting of F12/DMEM, N2 supplement, and FGF2 (20 ng/mL)	Immunocytochemistry and fluorescence measurement; scanning electron microscopy	The results demonstrated that cells on silk-CNT scaffolds have a higher β-III tubulin and nestin expression, suggesting augmented neuronal differentiation.	The silk-CNT composite scaffolds developed here can serve as efficient supporting matrices for stem cell-derived neuronal transplants.
Solanki et al., 2013	Neural stem cells	Unclear	Proliferated in culture media containing basic fi broblast growth factor (bFGF, 20 ng/mL) and epidermal growth factor (EGF, 20 ng/mL)	Quantitative RT-PCR; scanning electron microscopy; immunostaining	This work has demonstrated that the graphene-nanoparticle hybrid structures lead to enhanced neuronal differentiation of hNSCs along with significant axonal alignment.	The hybrid nanostructures have tremendous implications for the potential use of GO as an ECM component especially in the field of neurobiology.
Tu et al., 2013	Primary rat hippocampal neurons	Prepared from postnatal Sprague-Dawley rat pups (aged 1 to 3 d)	The DMEM medium supplemented with 10% fetal calf serum, 0.5 mM L-glutamine, 0.03 mM glucose, 100 U/mL penicillin, and 0.1 mg/mL streptomycin	Scanning electron microscopy; Immunocytochemistry; western blotting	These biomimetic choline-like GO composites can significantly boost neurite sprouting and outgrowth.	All results demonstrate the potential of DMAEMA- and MPC-modified GO composites as biomimetic materials for neural interfacing.
González-Mayorga et al., 2017	Embryonic neural progenitor cells	Obtained from cerebral cortices of E18 Wistar rat embryos	500 μL of complete neurobasal media containing B-27 supplement (2%), streptomycin (100 U/mL), penicillin (100 U/mL), and L-glutamine (1 mM)	Scanning electron microscopy; confocal laser scanning microscopy; transmission electron microscopy	These microfibers behave as supportive substrates of highly interconnected cultures composed of neurons and glial cells for up to 21 days, and the colonization by meningeal fibroblasts is dramatically hindered by N-cadherin coating.	These positive findings boost further investigation at longer implantation time and enhancing repair in the damaged central neural tissue including the injured spinal cord.
Min et al., 2017	Human neuroblastoma cells	American type culture collection (Manassas, VA, USA)	The DMEM medium supplemented with 10% heat-activated fetal bovine serum and 1% antibiotics (penicillin and streptomycin)	Fluorescence imaging; confocal imaging; Raman spectroscopy	The study developed a novel MF-driven GO hybrid pattern that was highly effective for controlling synaptogenesis.	This work provides treatment and modeling of brain diseases and spinal cord injuries.
Serrano et al., 2018	Embryonic neural progenitor cells	Obtained from cerebral cortices of E18 Wistar rat embryos	500 μL of complete neurobasal media containing B-27 supplement (2%), streptomycin (100 U/mL), penicillin (100 U/mL), and L-glutamine (1 mM)	Confocal laser scanning microscopy; inflammatory cytokine detection; flow cytometry	The capacity of rGO microfibers to inhibit the proliferation of RAW-264.7 macrophages, without affecting their viability and cell cycle profiles.	These findings encourage further investigation of these microfibers as attractive biomaterials to interact with neural cells.
Pan et al., 2019	Neural stem cells	Isolated from the cerebral cortex of embryonic mice (E11.5)	The growth media contained neurobasal media, B27 neural supplement, 100 ng/mL penicillin-streptomycin, 20 ng/mL epidermal growth factor and 20 ng/mL basic fibroblast growth factor.	MTT assay; immunofluorescence; quantitative real-time PCR analysis	PLGA/GO nanofibres loaded with IGF-1 and BDNF not only protected NSCs from oxidative stress induced by H2O2 but also enhanced NSC proliferation and neuronal differentiation in vitro.	The study indicated that immobilization of IGF-1 and BDNF onto PLGA/GO nanofibres has a great potential as a nerve implant for spinal cord injury applications.
Domínguez-Bajo et al., 2020	Embryonic neural progenitor cells	Isolated from cerebral cortices of gravid Wistar rats	Samples were covered with 500 L of complete neurobasal media containing B-27 supplement (2%), streptomycin (100 U/mL), penicillin (100 U/mL), and glutamine (1 mM).	Immunofluorescence studies by confocal laser scanning microscopy	This work confirmed the capacity of rGO-MFs to support the growth of ENPCs in vitro.	In this work, the regenerative potential of rGO-MFs when chronically interfaced with a cervical spinal cord injury was investigated for the first time.
Li et al., 2020	Primary rat Schwann cells	The spinal nerves of Sprague-Dawley rats (newborn to postnatal d 4–5)	The cells were cultured with laminin coated culture plate or bottle in DMEM/F12 containing 15% FBS and 1% penicillin-streptomycin.	Scanning electron microscopy; western blotting; MTT assay; immunofluorescence	The results of cell experiments indicated the better adhesion and higher expression of neural proteins in rat Schwann cells (RSCs) on PDA/CGO/PPy-PLLA films.	These results indicated that PDA/CGO/PPy-PLLA films were promising to be applied for the ES therapy of peripheral nerve repair in vivo.

CGO: Carboxylic graphene oxide; CNTs: carbon nanotubes; DMAEMA: dimethylaminoethyl methacrylate; DMEM/F12: Dulbecco’s modified Eagle media: nutrient mixture F-12; ECM: cell-extracellular matrix; EGF: epidermal growth factor; ENPCs: embryonic neural progenitor cells; FBS: fetal bovine serum; FGF: fibroblast growth factor; MF: magnetic force; MPC: 2-methacryloyloxyethyl phosphorylcholine; NSCs: neural stem cells; PDA: polydopamine; PLLA: ply-l-lactic acid; PPy: polypyrrole; TH: L-theanine.