The fabrication sequence is described below. First, a p-type 4-inch wafer was RCA cleaned and then a layer of aluminum or gold was deposited (Fig.1a). This was followed by lithographic patterning (Fig. 1c) and removal of undesired metal (Fig. 1d). Each 4-inch Si wafer has about 13 nanopillar arrays. Next, silicon is dry etched on Lam Rainbow 4400 Reactive Ion Etch System. Silicon etching was carried in two steps. The anisotropic vertical etch (Fig. 1e) was followed by isotropic etch to get the desired undercutting effect. For anisotropic etch, SF6  and RFB were used at low pressure (Fig. 1e), while for isotropic etch, SF6  and high RFB were used at high pressure (Fig. 1f). Next, photoresist and metal were etched (Fig. 1g). Finally, a thin layer of Au was deposited for electrode stimulation studies (Not shown in Fig 1).

Cell culture

The precise placement of cells on the nanoelectrodes is a challenging process. After surface functionalization with silane coupling (uncoated Si) or thiol chemistry (Au nanoelectrodes), cells are cultured on the nanoelectrodes. Three different cells were tested on the silicon nanopillar arrays.  First,  NIH 3T3  cells were cultured in  Dulbecco’s modified Eagle’s medium (DMEM) containing 10% Fetal Bovine  Serum  (FBS)  and  1%  antibiotic/antimycotic. 3T3 cells were seeded at a density of 5,000 cells/cm2. 
Next, PC12 cells were grown in RPMI supplemented with 10%  heat activated horse serum,  5% calf serum and 1% penicillin and streptomycin. After PC12 cells were seeded at a density of 10,000 cells/cm2  on the nanopillars, differentiation was carried out using nerve growth factor (NGF) alone or NGF along with electrical stimulation.
Finally, Adipose-derived stem cells (ADSCs) were cultured in DMEM/Ham’s F-12 medium with 10% FBS and 1% Penstrap. For neurogenic differentiation of ADSC, (a) the stromal media was replaced by pre-induction medium (DMEM, 20% FBS, and 1nM beta-mercaptoethanol) for up to
24 hours followed by induction medium (DMEM/2% DMSO/200uM BHA) for up to two weeks or (2) a regime electrical stimulation protocol (as described below) was used to facilitate conventional induction medium based differentiation.

Electrical stimulation protocol

For cell differentiation, the DC stimuli were optimized. The pulsed DC stimuli were in the range of 100 mV/cm field strength with monophasic 100 ms square pulse and 1 Hz frequency.
  

Immunofluorescence staining

Cells were fixed with 4% paraformaldehyde in PBS for 15 min, washed thrice with PBS solution, and subsequently permeabilized in 0.25% Triton X solution for 15 minutes. Before staining, all the samples were blocked using 1% BSA in PBS for one hour. The samples were then incubated with
Actin green and DAPI to visualize actin and nuclei respectively.  To validate the differentiation process,  gene expression markers and morphological changes were monitored.