During the process of isolating and culturing neural stem cells, trypsin is used to digest tissues and cells, but it is difficult to control the digestion time.OBJECTIVE:
To isolate and cultivate neural stem cells from fetal brain of Kunming mice by trypsinization combined with mechanical isolation and to identify by the immunohistochemical method.DESIGN, TIME AND SETTING:
In vitro observation of cytology. The experiment was performed at Basic Medical Laboratory of Guangxi Medical University from October 2006 to September 2007.MATERIALS:
Kunming mice of pregnant 14 to 16 days were provided by the Laboratory Animal Center of Guangxi Medical University.METHODS:
Brain tissues were isolated from the fetal brain of Kunming mice, pipetted mechanically after trypsinization, and incubated in serum-free DMEM/F12 medium containing B27, basic fibroblast growth factor and epidermal growth factor.MAIN OUTCOME MEASURES:
Immunohistochemistry was performed to identify the cultured neural stem cells.RESULTS:
After incubation in serum-free DMEM/F12 medium for 24 hours, the cells were suspensions with the manner of little neurospheres. After incubation for 48 hours, the cultured cells grew much larger, formed typical neurospheres, with no marked process, and survived well after passaged. In addition, immunocytochemical method showed nestin-positive.CONCLUSION:
Serum-free B27 medium supplemented with basic fibroblast growth factor and epidermal growth factor can benefit culture and promote the proliferation of neural stem cells in vitro.