Objective   To compare the growth behaviors of rat hippocampal neural stem cells (NSCs) cultured in 2 medium conditions to optimize the proliferation condition

and to achieve integration of hippocampal NSCs with our fabricated three-dimensional (3-D) poly-L-lactide (PLLA) microwell patterns.    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.    Results   Immunocytochemistry and immunofluorescence showed that the isolated hippocampal cells were positive for neural stem cell marker nestin and were capable of differentiating to the neuronal and glial lineages. In a period of 30 days’  expanding

hippocampal NSCs proliferated slowly forming neural spheres in Neurobasal-based culture medium

with few cells attached to substrates and differentiated morphologically. In contrast

cells in DMEM/F12-based medium proliferated rapidly

with significant cell attachment and morphological differentiation. On the day 25 of expanding

neural sphere number in DMEM/F12-based medium was 4.7 times as much as that in Neurobasal-based medium. 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.    Conclusion   DMEM/F12-based medium is favorable to population expansion of rat hippocampal cells while Neurobasal-based medium is conducive to purification of hippocampal NSCs. Sequential use of DMEM/F12- and Neurobasal-based medium can thus result in expansion of hippocampal NSCs and successful integration of the NSCs with 3-D PLLA microwell patterns.