Population Expansion Of Rat Hippocampal Neural Stem Cells And Their Integration With Three-dimensional Microwell Patterns

Neural stem cells ( NSCs) are multipotential stem cells that exist in the central nervous system, and play an important role in neural development and neural injury repair. A major problem in the researches of NSCs is to effectively obtain and easily amplify the cells. Amplification of neural stem cells includes expansion in normal two-dimensional (2-D) culture condition and on three-dimensional (3-D) scaffold materials. It was reported that the morphology and ion channel function of NSCs in two-dimensional culture condition were quite different from those under three-dimensional culture condition, and the latter may be more close to the in vivo situation. Therefore, we studied the means of effective population expansion of hippocampal stem cells as well as their integration with 3-D PLLA microwell patterns.Objective To compare the growth behaviors of rat hippocampal NSCs under two medium conditions, to optimize the proliferation condition, to achieve integration of hippocampal NSCs with our fabricated three-dimensional (3-D) poly-L-lactide (PLLA) microwell patterns and, finally, to find pattern configurations that favor intergration by NSCs or maintaining of the multipotentiality of NSCs.Methods Rat hippocampal cells, obtained with mechanical separation, were expanded in either Neurobasal- or DMEM∕F12-based culture medium. Proliferation behaviors of the isolated hippocampal cells were evaluated with neural sphere counting and the methylthiazolyl tetrazolium (MTT) assay. PLLA microwell patterns were fabricated with UV lithography, silicon etching and soft lithography and then integrated with the expanded NSCs. The NSCs behavior was detected by Optical microscope, and its differentiation under different 3-D microwell patterns was evalued by immunofluorescence.Results Immunocytochemistry and immunofluorescence showed that the isolated hippocampal cells were positive for neural stem cell marker nestin and were capable of differentiated toward the neuronal and glial lineages. In a period of 30 days of expanding, hippocampal NSCs proliferated slowly forming neural spheres in Neurobasal-based culture medium, with few cells attaching to substrates and showing differentiated morphologically. In contrast, cells in DMEM∕F12-based medium proliferated rapidly, with significant cell attachment and morphological differentiation. On day 25 of expanding, neural sphere number in DMEM∕F12-based medium was 4.7 times as much as that in Neurobasal-based medium. Sequential use of DMEM∕F12- and Neurobasal-based medium resulted in successful population expansion of hippocampal NSCs with high purity. The microwell patterns were well defined,of high aspect ratio (≥1) and stable under culture conditions. Population expanded hippocampal NSCs were successfully integrated with the 3-D PLLA microwell patterns, and the number of them could be affected by micro-pattern configuration (with or without micro-channel and the width of micro-channel): with the microwell diameter increasing, the number of NSCs increased; as the width of micro-channel increasing, the number of NSCs increased too. NSCs integrated with no micro-channel microwell patterns had no neurites; NSCs integrated with micro-channel microwell patterns had much neuritis and the neuritis was found to elongate along the micro-channels.Conclusion DMEM∕F12-based medium was favorable to population expansion of rat hippocampal cells while Neurobasal-based medium was conducive to purification of hippocampal NSCs. Sequential use of DMEM∕F12- and Neurobasal-based medium could thus result in expansion of hippocampal NSCs and successful integration of the NSCs with 3-D PLLA microwell patterns.The 3-D microwell patterns with no channel structures were favorable to maintaining stem cell multipotentiality, and the 3-D microwell patterns with microchannels were conducive to differentiation of the NSCs.