Hierarchical Micro-/Nanotopographies Patterned by Tandem Nanosphere Lens Lithography and UV–LED Photolithography for Modulating PC12 Neuronal Differentiation

Modulating neuronal behavior via topographical cues is integral to studying neuronal development, building in vitro neural networks, and designing better neural interfaces and nerve regeneration devices. While numerous topographies with micro- and nanoscale features have been investigated, studies involving hierarchically patterned substrates containing both micro- and nanoscale structures have been limited, which may be due, in part, to the technical challenges involved in their fabrication. In this work, we utilize for the first time a facile, low-cost fabrication method using nanosphere lens lithography and UV–LED photolithography for creating ordered, hierarchical structures consisting of nanodots or nanopillars on microgrooves using the photosensitive polymer SU-8. Pheochromocytoma 12 (PC12) cells are differentiated on the micro-/nanopatterned films in the presence of nerve growth factor, and their neurite outgrowth and orientation are characterized by semi-automatic analysis of fluorescence images. The results show that nanopillars with micron-scale gaps significantly inhibited neurite outgrowth and branching. When combined with microgrooves, the nanopillar array resulted in more neurites and cell somas being localized on the ridges in the hierarchical substrates, which was associated with significantly improved neurite alignment on the ridges compared with the plain microgroove substrate. Visualization of filopodia-like projections and adhesion formation reveal the entrapment of thin projections and the inhibition of cell–matrix interactions on the nanopillar array, which could be responsible for the modulation of neurite development. The hierarchically patterned photosensitive substrates, fabricated using our proposed simple and cost-effective method, can thus be utilized in controlling the neurite outgrowth and alignment of neuronal cells for applications ranging from basic neuroscience to nerve regeneration.