Investigating the Morphology and Kinetics of Three-Dimensional Neuronal Networks on Electro-Spun Microstructured Scaffolds

Petri dishes and glass slides have been widely used as general substrates for in vitro mammalian cell cultures due to their culture viability, optical transparency, experimental convenience, and relatively low cost. Despite the aforementioned benefit, however, the flat two-dimensional substrates exhibit limited capability in terms of realistically mimicking cellular polarization, intercellular interaction, and differentiation in the non-physiological culture environment. Here, we report a protocol of culturing embryonic rat hippocampal neurons on the electro-spun polymeric network and the results from examination of neuronal cell behavior and network formation on this culture platform. A combinatorial method of laser-scanning confocal fluorescence microscopy and live-cell imaging technique was employed to track axonal outgrowth and synaptic connectivity of the neuronal cells deposited on this model culture environment. The present microfiber-based scaffold supports the prolonged viability of three-dimensionally-formed neuronal networks and their microscopic geometric parameters (i.e., microfiber diameter) strongly influence the axonal outgrowth and synaptic connection pattern. These results implies that electro-spun fiber scaffolds with fine control over surface chemistry and nano/microscopic geometry may be used as an economic and general platform for three-dimensional mammalian culture systems, particularly, neuronal lineage and other network forming cell lines.