Study Of Metabolic Parameters 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. Therefore, the aim of this study is to analyze the possible reasons which lead to the necrotic core inside the neurosphere.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 diameters' neurospheres, various metabolic parameters of both single NSC and neurosphere were determined. Meanwhile, during the neurosphere culture, the diameters of neurospheres and the growth curve were also determined.The results showed that the cultured cells have the characteristics of NSCs and satisfy its definition. The metabolic rate of single NSC declined with the decrease of glucose concentration and showed significant differences when glucose concentration was lower than 6.444 mmol/L. During the 108-hour culture of neurosphere, cell growth of the neurospheres was inhibited when the concentrations of glucose and glutamine decreased to 36.11 mmol/L and 1.33 mmol/L, respectively, and the average radii of neurospheres reached about 50 μrn. Inside the neurospheres, the death rates of cells would exceed the proliferation rates when the average radius of neurosphere was in the range of 50-75 μrn, and the concentrations of glucose and glutamine in the medium were 31.11 mmol/L and 1.15mmol/L. Moreover, the calculations of nutrient and metabolite concentration distributions were carried out for neurospheres with different radii (50, 55, 60, 65, 75 μm). And the results demonstrated that, when the neurosphere radius was above 55 μrn, the deficiency of dissolved oxygen could result in cell death in the center of neurospheres. Furthermore, the formation of "necrotic core" could affect healthy cells via signal communications and therefore cause rapid death of them. It is concluded that the deficiency of dissolved oxygen is the key factor for the growth inhibition of neurospheres.