Comparison Of Different Culture Mode For Long-term Expansion Of Neural Stem Cells

Neural stem cells (NSCs) can be cultured in two modes of suspension and monolayer in vitro. In order to find the appropriate methods for large-scale expansion of NSCs, we compared the NSCs cultured in suspension with those cultured in monolayer systematically. Firstly, the forebrain tissue was removed from the embryonic day 14 (E14) mice, then the tissue was dissociated into single-cell suspension by Accutase? and mechanical trituration. The cells were cultured in both suspension and monolayer. The cells cultured in suspension and monolayer were identified to be NSCs by the assay of differentiation and immunofluorescence. Furthermore, the NSCs cultured in suspension and in monolayer were compared on viability, ability of proliferation and heterogeneity by fluorescent dye, immunofluorescence and FACS on DIV21, DF/56 and DIV113 respectively. The results indicated that the NSCs cultured in both suspension and monolayer represented good viability in long-term culture. But they displayed distinct ability of proliferation in long-term culture. The NSCs cultured in monolayer preceded those cultured in suspension on the ability of proliferation on DFV21 and DIV56, but no obvious difference on DIV113. The NSCs population cultured in suspension displayed more Nestin-positive cells than those in monolayer in the whole processes of culture. The NSCs population cultured in monolayer, however, displayed more βtubulinIII-positive cells than those in suspension in the same period. Additionally, we determined the suitable diameter of neurospheres to be passed was about 200μm. The results of this study further indicate that the cultured cells are different in both the ability of proliferation and the heterogeneity. And the suspended culture mode excels the monolayer culture mode for large-scale expansion of NSCs.