Investigation of neural cell fate processes on amorphous carbon structures and effect of vertical electric field using porous carbon electrodes

Investigation of neural cell fate processes on amorphous carbon structures and effect of vertical electric field using porous carbon electrodes

Dr Shilpee Jain

Department of Chemical Engineering, Indian Institute of Technology Kanpur, Kanpur, UP-208016

Among various diseases, neural disorder or neural injury is widely recognized as one of the serious issues. In order to address such issues, a number of biomaterials are being developed and the biocompatibility property is being assessed. In this perspective, the amorphous carbon structures have been developed to assess the efficacy for peripheral nerve regeneration. In particular, carbon nanofibers (Polyacrylonitrile and Resorcinol-Formaldehyde (RF) gel derived), carbon films (Polyacrylonitrile and RF gel derived) and SU-8 (epoxy based negative photoresist) derived carbon patterns were fabricated using electrospinning, spin coating and photolithography techniques, respectively. XRD patterns confirms the amorphous nature of carbon, while extensive XPS analysis of carbon fibers and films confirms the presence of different functional groups on different textured carbon surface, synthesized from same polymer precursor. Other functional groups were also introduced on carbon substrates using UV and oxy-plasma surface treatments and analyzed by Raman and FT-IR spectroscopy.

The in vitro cytocompatibility of carbon structures was tested with neuroblastoma (N2a) cells and Schwann cells. It was found that the carbon nanofibrous substrates (~200 nm diameter) provide essential cues to Schwann cells to grow along the fiber direction, whereas N2a cells spread and make contacts with multiple fibers. These changes in Schwann cell morphology can alter the cell fate processes. In this regard, Schwann cell fate processes i.e. cell proliferation, cell metabolic activity and cell apoptosis were analyzed quantitatively using flow cytometry at various time points in culture. The experimental results of Schwann cell fate processes confirmed that the electrospun carbon nanofibrous substrate can physically guide the cultured Schwann cells and do not significantly induce apoptosis, but in fact even facilitate their proliferation and growth as compared to control (gelatin coated glass cover slips).

Further, the effect of varying micro and nanoscale geometries of amorphous carbon substrates on Neuroblastoma (N2a) and Schwann cell proliferation was also investigated. RF gel derived carbon films and electrospun nanofiber (~200 nm diameter) mats, and SU-8 derived carbon micro-patterns. Additionally, the efficacy of amorphous carbon substrates as electrodes to support neuronal cell fate processes in electric field mediated culture conditions was also studied. The porous carbon electrodes provide higher surface area and specific charge storage capacity (0.2mC/cm2) and low impedance (3.3 kΩ) at 1 kHz frequency. 

When uniform or gradient electric field was applied perpendicular to the amorphous carbon substrate, we found that, the N2a cell viability and neurite length were higher at lower electric field strength (≤ 2.5 V/cm), as compared to control (without field). Interestingly, the length of neurite was higher in gradient electric field as compared to uniform and without electric field conditions.