Study Of 3-D Culture In Vitro And Modeling Of NSCs

More and more attention has been paid on neural stem cells (NSCs) since their magic potential in neurologic repair was found. A substantial amount of NSCs will be required for cell transplantation. And high expansion rates of NSCs in culture are thus crucial for the clinic application. However, this demand has not been satisfied because of the NSCs' special growth mode-neurosphere inside which a necrotic core may appear when the size of it reaches a certain critical value. Considering the application of collagen(COL) in vitro culture,if we inoculate cells into a cell-COL system and culture them in a 3-D scaffold environment, the result may become more effective.Combined experimental data with mathematical model, metabolite concentration distribution of NSCs in COL could be obtained, the most suitable thickness and inoculum density can also be determined.In the experiment, NSCs were separated from the hippocampus of E14 mice, and cultured in serum-free medium. The differentiation potential and the ability of proliferation of NSCs were identified by fluorescent dye and immunofluorescence. In order to establish the mass transfer and metabolism model to calculate the different concentration distributions in different thickness' COL, various metabolic parameters of both single NSC and cell-COL were determined. The most suitable preparation parameters of COL were determined by calculating the different concentration distributions in different thickness' COL.The results showed that the cultured cells have the characteristics of NSCs and satisfy its definition. The oxygen metabolism rate of NSC was 2.9×10¹⁴ mmol/cell/s. The ultimate concentration of COL in a cell-COL system should be between 1mg/ml and 2mg/ml.With the initial concentration of glucose 5.08×10^-2 mmol/ml, the thickness of liquid layer 5mm, the inoculum density of NSCs 1×10⁶cells/ml,2mm cell-COL met the growth requirement of NSCs culture in vitro well. Furthermore, combined experimental data with mathematical mode, the most suitable conditions of NSCs 3-D culture in vitro in cell-COL system were determined: NSCs inoculum density 2×10⁵cells/ml, the thickness of COL 2mm, and the thickness of liquid layer 5mm.