Three-dimensional cell-based platform (microwell-neural stem cell) for screening compounds against connectivity-related targets
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A three-dimensional (3D) cell-based platform for high-throughput screening (HTS) was developed. This platform was comprised of neural stem cell (NSC)-derived neural networks in interconnected microwell structures. To apply SU-8 microwell structures for human neural stem cell (hNSC) culture, SU-8 microstructures were modified to be biocompatible for hNSC culture. Then hNSCs locomotion within SU-8 microwells was studied by time-lapse recording. Combining fluorescent dyes and confocal microscopy, cell morphology, cytoskeleton organization and resting membrane potential (Vm) were measured for cells on 2D flat surface and within SU-8 microstructures. Microwell architecture affected hNSC morphology, cytoskeleton organization as well as Vm establishment in a quasi-3D manner, which is consistent with other 2D/3D comparison studies. This study moves one step forward in combining hNSCs and microwell structures to form a quasi-3D neural network for drug screening. To apply microwell-hNSC platform for HTS, micropatterns were fabricated in nano-fibrous poly-L-lactic acid (PLLA) films by laser micromachining and the resulting scaffolds were characterized with respect to architecture, thermal, mechanical and mass transport properties. Also, hNSCs were successfully cultured in these micropatterned nano-fibrous scaffolds (MNFSs). The scaffolds were incorporated in high-density well plates (e.g., 96-well plates), creating a platform for HTS of drugs with physiologically more relevant neural networks. To establish a protocol for hNSCs synaptic development in early differentiation, ginsenosides Rg1 and Rb1, major pharmacologically active ingredients from Ginseng, were applied in the differentiation media for hNSCs, together with brain-derived neurotrophic factor (BDNF). Cell locomotion and neurite extension were observed by time-lapse microscopy and analyzed by ImageJ software. The expression of synaptic formation was confirmed by immunostaining of synaptophysin (SYN) or/and the co-localization of synapsin I and microtubule associated protein-2 (MAP-2). Results have shown that administration of BDNF/ginsenosides (Rg1 and Rb1) combination in differentiation medium promoted cell survival, enhanced neurite outgrowth and synaptic marker expression during differentiation. High cell density promotes synaptic marker expression in BDNF/ginsenosides combination medium.