Development of a three-dimensional cell culture system for the enhancement of nerve axonal extension by cyclic stretch stimulation

Abstract

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  10 5 times—the cyclic stretch stimulation was performed on rat cerebral cortex cells, and the effectiveness of the enhancement was also confirmed in these (Visible green). PC12 cells were stained using the Milli-Mark TM FluoroPan neuronal marker (MAB2300X, Merck KGaA, Germany) and DAPI solution (340-07971, Dojindo Laboratories, Japan). MAB2300X stains were used to identify the axons, dendrites, spines, nuclei, and the body of the PC12 cells. DAPI stains were used to investigate the nuclei of the PC12 cells. The PC12 cells were observed in all three selected areas of the collagen gel sample, covering a size of 443  443  500  m. The resolution of MPM observation is 0.43  m in both X 1 and X 2 directions of tomography plane, and 0.85  m in the vertical direction X 3 . The lens is HCX IRAPO L 25x/0.95 WATER and the magnification is 250. We adopted the confocal image because of clear photo using the wave length 405 nm (UV blue) for DAPI and 488 nm (Visible green) for MAB2300X. the stretch chamber was set with the MPM system so that the stretching occurred in the direction of the X 1 -axis. The effectiveness of the stretch stimulation in increasing axonal outgrowth was then