A Simplified Method For Isolation and Culture of Neural Stem Cells From Adult Mice Brain: in Vitro

Abstract

The present study aimed to develop an uncomplicated method for isolation and culture of mice brain derived-neural stem cells (NSCs) - neurospheres, using a simplified method. In order to obtain single cells suspension, the brain tissue was dissociated using mechanical methods (trituration) instead of enzymatic method. These single cells suspension were in vitro cultured in DMEM)/Ham's F12 (1:1) mixture (high glucose) plus 20 ng/ml Epidermal growth factor (rHuEGF) and 20 ng/ml Fibroblast growth factor-basic (FGF-2). The results showed the combined actions of rHuEGF and bFGF-2 was in inducing proliferation of brain derived-NSCs. So, after 3 days culture numerous number of NSCs were observed floated in culture medium. Then within the time of culture the numbers of NSCs were increased. After 7 days culture some of these cells began to aggregate and form small sized neurosphere of undifferentiated cells (early stage) and within 3 weeks culture, large sized mature neurospheres were formed. When these neurospheres were aspirated and re-cultured on the gelatin pre- coated Petri dish, these neurospheres were attached and several nerve cells began to migrate from these neurospheres and occupy the surrounding area. This result confirms the incidence of NSCs derived neurospheres and it is ability to differentiate to nerve cell.  In conclusion this method is simple and less expensive, less effort, but take longer time to get NSCs derived-neurosphere, as well as it is possible to use mechanical dissociation of cells (trituration) instead of enzymatic method to obtain single cell suspension.