Synthesis and characterization of 3D-GFs

3D-GFs were synthesized by chemical vapor deposition method using Ni foam template5. Scanning electron microscopy (SEM) observation (Fig. 1a and 1b) shows that 3D-GFs exhibited a monolith of continuous and porous structure, which copied and inherited the interconnected 3D structure of the Ni foam template. The porosity of 3D-GFs was determined to be 99.5 ± 0.2% and had a pore size of 100–300 μm, while the width of the graphene skeleton was about 100–200 μm. 3D-GF surface was covered with many ripples and wrinkles at micro- and nano-scale (Supplementary Fig. S1). Furthermore, the absence of D band in Raman spectrum (Fig. 1c) indicated that 3D-GFs were of high quality with few defects, while the shape of 2D band and the intensity ratio between 2D and G band proved that 3D-GFs were constructed by few-layer or multi-layer graphene sheets15. Although the graphene layers were extremely thin, the network possesed excellent mechanical strength and flexibility and can stand alone. Addtionally, compared with the structure of 3D-GFs prepared by freeze-drying or template assembly method, the monolith of 3D-GF network by chemical vapor deposition ensured a high conductivity owing to the lack of defects and inter-sheet junction contact resistance16.

Figure 1 Characterization of 3D-GF scaffold. SEM micrographs of 3D-GFs at low (a) and high (b) magnification, the inset shows an enlarged view of 3D-GF skeleton surface. (c) Typical Raman spectrum of 3D-GFs. (d) High-resolution C (1s) XPS spectra of 3D-GFs. (e) High-resolution N (1s) XPS of 3D-GFs with pre- and post- laminin treatment. Full size image

The surface chemistry of 3D-GFs was further characterized by the X-ray photoelectron spectroscopy (XPS). In Fig. 1d, two obvious components were observed in the C1s XPS spectrum of pure 3D-GFs. The main peak at 284.6 eV corresponds to the non-oxygenated ring C, while a small peak reflects C in C–O bonds, indicating a relative inert surface chemistry. The removal of trace amounts of the catalyst and etching agents was also verified by XPS and re-validated by energy dispersive X-ray spectrum (Supplementary Fig. S2). In addition, lots of protocols for facile chemical modification using natural and synthetic bioactive molecules have been well developed for graphene, which facilitates NSC scaffold functionalization11,17. In this work, laminin coating was adapted to enhance cell adhesion. As shown in Fig. 1e, after immersing 3D-GFs in a laminin solution for more than 4 h, there were obviously N 1s peaks arising in the high-resolution N (1s) XPS spectrum of 3D-GFs, indicating the success of laminin modification (N contained in the laminin molecule). Also, morphology of laminin-coated 3D-GFs was almost identical with that of pure 3D-GFs and no obvious difference between them was observed (Supplementary Fig. S3).

NSC adhesion on 3D-GFs

NSC adhesion on 3D-GFs was firstly investigated. For no more than 10 hours after cell seeding, almost no free-floating cell could be found in the culture medium (data not shown), indicating a rapid cell attachment on the 3D-GFs. After another 5 days of culture, the interaction between NSCs and 3D-GFs was examined by SEM. NSCs cultured on 3D-GFs formed a well neural network and exhibited excellent cell adhesion (Fig. 2a & 2b). High resolution SEM image shows that the cells spread extensively and formed strong filopodia/GF interaction (insert of Fig. 2b). Furthermore, in cross section fluorescence image of 3D-GF scaffold with NSCs (DAPI staining), a number of cells were observed inside the scaffold as well as on the surface, clearly indicating that the cells grew in a 3D fashion (Supplementary Fig. S4). Additionally, the SEM observation presents the 3D-GFs remained intact during cell culture process over 2 weeks.

Figure 2 NSC adhesion and proliferation on 3D-GF scaffold. Low- (a) and high- (b) magnified SEM images of NSCs cultured on 3D-GFs under the proliferation medium. The inset illustrates the interaction between the cell filopodia and 3D-GF surface. (c) Cell viability assay of NSCs on 3D-GFs after 5 days of culture as determined by live/dead assay, live cells are stained green and dead cells are red, white arrow points to dead cell. The lower right inset depicts the percentage of live cell on 2D graphene films and 3D-GFs. (d) Fluorescence images of NSCs proliferated on 3D-GFs for 5 days, immunostaining markers were nestin (green) for neural stem cells and DAPI (blue) for nuclei. (e) NSCs were double-immunostained with anti-Ki67 (red) and anti-nestin (green) antibodies, Ki67 is a marker for cell proliferation. (f) Western Blot analysis of Ki67 expression on 2D graphene films and 3D-GFs. The Data are presented as mean ± standard error (s. e. m.), *p < 0.05, **p < 0.01. Full size image

Biocompatibility of 3D-GFs

3D-GF cytotoxicity was evaluated by Calcein-AM and EthD-I staining assay with 2D graphene film as control. Fig. 2c shows that almost 90% of the cells cultured on 3D-GFs for 5 days were viable, while the difference in cell viability between 3D-GFs and 2D graphene films is neglectable (lower inset in Fig. 2c & Supplementary Fig. S5). Nor could a TUNEL assay find any abnormal cell apoptosis on 3D-GFs (Supplementary Fig. S6). Those data demonstrated good biocompatibility of 3D-GFs, consistent with previous studies10,11,18. The cells were also stained with antibody against nestin, a protein marker of NSCs. Fig. 2d shows that nearly all of cells on 3D-GFs were immunopositive for nestin (green), with no obvious difference from that on 2D graphene films (Supplementary Fig. S5), indicating that NSCs proliferated well on 3D-GFs while maintained their stemness.

NSC proliferation on 3D-GFs

NSC proliferation on 3D-GFs was examined by measuring the expression of Ki-67 protein, a cellular marker for proliferation19. High specific surface area of 3D-GFs (200–800 m2/g) could provide a large surface area for cell attachment and growth5. Also, macroporous structure of 3D-GFs is believed to ensure efficient mass transport of nutrition for NSC metabolic demands, which should facilitate cell proliferation. As expected, Fig. 2e shows that a majority of (nearly 80%) cells on 3D-GFs were stained positively for Ki-67. Further quantitative western blot analysis illustrates that the expression of Ki-67 was significantly higher in 3D-GF groups than in 2D graphene film groups (Fig. 2f), suggesting that NSCs on 3D-GFs sustained a more active proliferation state (consistent with WST-based cell proliferation assay, Supplementary Fig. S7), which could be of a huge advantage as a scaffold material to increase NSC number after the transplantation3.

NSC differentiation on 3D-GFs

The phenotypic changes of differentiated NSCs on 3D-GFs were further investigated. After 5 days differentiation, the cells exhibited elongated cell shape with healthy neurite outgrowth, leading to a confluent neural network covering almost the whole 3D-GF surface (Supplementary Fig. S8). Immunofluorescence staining shows that all Tuj-1+ (neuron marker), O4+ (oligodendrocyte marker) and GFAP+ (astrocyte marker) cells were observed on 3D-GFs (Fig. 3a & 3b) and 2D graphene films (Supplementary Fig. S9), indicating that NSCs keep the pluripotency to differentiate into all three neural subtypes. For a quantitative analysis, cells were harvested and subjected to western blot assay. As shown in Fig. 3c & 3d, compared with 2D graphene film groups, NSCs cultured on 3D-GFs exhibited tremendously lower nestin expression, while the expression of Tuj-1 and GFAP were enhanced in cells by ~2.5 and ~1.5 folds, respectively. Additionally, there was no significant difference in RIP (also oligodendrocyte marker) expression between the two experimental groups. The results indicated that 3D-GFs can greatly enhance NSC differentiation into neurons and astrocytes, especially neuronal lineage.

Figure 3 The differentiation of NSCs on 3D-GF scaffold. (a, b) Representative fluorescence images of differentiated NSCs under differentiation conditions, the cells were immunostained with Tuj-1 for neuron (green, a), GFAP for astrocyte (red, a&b), O4 for oligodendrocyte (green, b) and DAPI for nuclei (blue, a&b). (c) Western blot analysis of nestin, Tuj-1, GFAP and RIP protein expression of differentiated NSCs on 2D graphene films and 3D-GFs. (d) Relative optical densities of nestin, Tuj1, GFAP and RIP bands shown in (c). The Data are presented as mean ± standard error (s. e. m.), *p < 0.05, **p < 0.01. Full size image

Electrical stimulation

To utilize 3D-GFs as a cell stimulation electrode, the electrochemical property of 3D-GF was measured by cyclic voltammetry using a potentiostat with three-electrode system in phosphate buffered solution (PBS). As shown in Supplementary Fig. S10, basically featureless voltammogram curves of the 3D-GF and 2D graphene film electrode are observed within the operational potential window from −0.3 V to 1.0 V, indicating that the current is delivered primarily through charging and discharging the interfacial double layer instead of through faradic reactions. It's indicted that the capacitive charge injection is ideal for neural stimulation since no chemical change occurs to either the tissue or the electrode14. Meanwhile, under the same potential, the current of 3D-GF electrode is significantly higher than that of 2D graphene film owing to its higher double-layer capacitance originated from the larger specific surface area, which can lead to a stronger charge injection ability.

Right before electrical stimulation, differentiated NSCs on 3D-GFs were stained with Fluo-4 AM dye to monitor the change of intracellular calcium ion concentrations caused by electrical stimuli. Fluo-4 AM is a membrane-permeable, Ca2+ dependent dye and exhibits a large fluorescence intensity increase on binding of free Ca2+. Previous report showed that the voltage pulse stimuli on a neuron could open calcium ion channels and increased the calcium ion concentration of cell, resulting in the enhanced fluorescence intensity of Fluo-4 AM dye in the neuron20. In our study, a series of monophasic cathodic pulses were applied using a function generator and the stimulation threshold was 20–30 μA. Fig. 4a shows the fluorescence level of the differentiated NSCs on 3D-GFs increased during a stimulus. The relative change in fluorescence intensity ΔF/F was plotted versus stimulation time in Fig. 4b, the cells exhibited over 50–60% fluorescence intensity increase by electrical stimuli. The results clearly imply that 3D-GFs can work as a conductive scaffold to electrically stimulate cells.