# Graphene, graphene-based nanomaterials (GBNs), and carbon nanotubes (CNTs) are being investigated

Graphene, graphene-based nanomaterials (GBNs), and carbon nanotubes (CNTs) are being investigated as potential substrates for the growth of neural cells. and oligodendrocytes were more branched. In contrast, neurons growing on PVDF membranes had reduced neurite branching, and on MWCNTs-loaded membranes oligodendrocytes were lower in numbers than in controls. Overall, these findings indicate that uncoated TRG may be biocompatible with the generation, differentiation, and maturation of aOBSC-derived neurons and glial cells, implying a potential use for TRG to study functional neuronal networks. (Li et al., 2011, 2013; Park et al., 2011; Akhavan and Ghaderi, 2013a,b, 2014; Lorenzoni et al., 2013; Solanki et al., 2013; Tang et al., 2013; Akhavan et al., 2014, 2015; Shah et al., 2014). In these previous studies, cells were either seeded on graphene or on GBNs coated with proteins such as laminin SB269652 manufacture and synthetic polymers such as poly-lysine, substances which are known to promote cell adhesion and neurite outgrowth (Vicario et al., 1993; Calof et al., 1994; Otaegi et al., 2007; Nishimune et al., 2008). In addition, cells were RNF23 plated on graphene composites, graphene oxides, or on SB269652 manufacture reduced graphene oxides with different surface charges and degree of electrical, photo, and laser stimulation (Akhavan and Ghaderi, 2013a,b, 2014; Tu et al., 2013a, 2014; Akhavan et al., 2014, 2015; Guo et al., 2016a). Similarly, both uncoated and coated functionalized single-walled CNTs (SWCNTs) and multi-walled CNTs (MWCNTs) as well as aligned CNTs and nanofibers have been reported to permit and stimulate neuronal growth and the formation of active synaptic contacts (Jan and Kotov, 2007; Malarkey et al., 2009; Cellot et al., 2011; Jin et al., 2011; Fabbro et al., 2013; Gupta et al., 2015; Vicentini et al., 2015). In spite of these potential applications, other studies have reported that GBNs can cause cytotoxic and genotoxic effects on cell lines (PC12, neuroblastoma, and A549 cells), mesenchymal stem cells (Zhang et al., 2010; Chang et al., 2011b; Akhavan et al., 2012; Lv et al., 2012; Bianco, 2013; Tu et al., 2013b), and neurons (Bramini et al., 2016). CNTs, particularly if used as produced materials, can also induce toxic effects on neural cells in part due to the presence of CNT aggregates, impurities such as amorphous carbon and metallic nanoparticles (Jakubek et al., 2009; Cellot et al., 2010; Wu et al., 2012; Chen et al., 2013; Meng et al., 2013; Bussy et al., 2015). However, recent studies indicate that chemical functionalization can reduce toxicity while preserving the highly conductive SB269652 manufacture character of CNTs (John et al., 2015; Oliveira et al., 2015; Marchesan et al., 2016). To the best of our knowledge, no studies reporting the biocompatibility of uncoated graphene with adult NSCs (aNSCs) have yet been published. Moreover, very few works have addressed the effect of uncoated graphene on the growth of neurons and glial cells. They reported that neurons can develop on graphene but their attachment was reduced compared to when the neurons were grown on poly-d-Lysine and laminin (Bendali et al., 2013; Sahni et al., 2013), that graphene stimulated neurite length compared to a glass substrate (Lee et al., 2015), or that pristine graphene and graphene-based substrates were permissive for neuronal outgrowth (Veliev et al., 2016) and synapse formation and function (Fabbro et al., 2016). In the present study, we have investigated the effects of uncoated thermally reduced graphene (TRG) (Defterali et al., 2016) on the proliferation and differentiation potential of cultured adult mouse olfactory bulbs (aOBSCs), a population SB269652 manufacture of previously characterized aNSCs (Verga?o-Vera et al., 2009; Nieto-Estvez et al., 2013) as well as on neuronal and glial survival and maturation. Since membranes are being used to make biocompatible neural scaffolds (see above), the differentiation of aOBSCs was also tested on pristine poly(vinylidene fluoride) (PVDF) membranes and on PVDF membranes loaded with MWCNTs. Our findings indicate that uncoated TRG is a permissive material that allows for the multi-lineage differentiation of cultured aOBSCs into neurons, astrocytes, and oligodendrocytes and the synaptic maturation of aOBSC-derived neurons. They also show that TRG supports the morphological differentiation of aOBSC-derived oligodendrocytes. In contrast, the morphological differentiation of aOBSC-derived neurons and oligodendrocyte survival were reduced when seeded on the PVDF membranes. Materials and Methods Animals All animal care and handling was carried out in accordance with European Union guidelines (directive 2010/63/EU) and Spanish.