| Summary: | Significant progress has recently been made in the development of extracellular stimulation technology for the enhancement of nerve axonal extension and network generation and regeneration in three-dimensional (3D) bioreactors for neural tissue engineering. In this study, a 3D cell culture cell culture system was developed to accelerate the regeneration of axons using cyclic stretch stimulation. A modified collagen gel was used as a scaffold to mimic the extracellular matrices of the central nervous system in the human brain. First, a cyclic stretch stimulation cell culture system was designed and fabricated in order to load uniform strain onto a 3D culture. Pheochromocytoma (PC12) cells were then mixed with the collagen gel and poured into the stretch chamber of the cell culture system. The stretch stimulation cell culture system was then used to load the cyclic tensile strain against the PC12 cells embedded in the collagen gel, where an in-situ microscopic observation was performed. Second, cyclic stretch stimulations of the PC12 cells were performed, and the 3D morphologies of the cell bodies, neurites, and axons within the PC12 cells were observed using a multi photon microscope (MPM) system. We evaluated the effectiveness of the cyclic stretch stimulation on the axonal extension of nerves in a 3D cell culture system. Finally, we confirmed the enhancement of the axonal extension and determined the optimum tensile strain and the number of cyclic stimulations required to achieve the maximum axonal extension of the PC12 cells. Using these optimum conditions—2.3% strain, 1 Hz cycles, and 1.7×105 times—the cyclic stretch stimulation was performed on rat cerebral cortex cells, and the effectiveness of the enhancement was also confirmed in these cells.
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