The Study Of Culture And Differentiation Of Neural Stem Cells Derived From The Cerebral Cortex Of Fetal Rats And Transplantation Into The Fluid Percussion Injury Rats

Objectives To isolate neural stem cells from the cerebral cortex of fetal rats by floating culture. Study their self-renewal and multipotential capacity by passaging.Methods1. Get materials and culture: We isolated the cerebral cortex from embryonic day 14-16 under bacteria-free condition. After the tissue mass was digested with digest solution(0.25% trypsin+0.02% EDTA), it was dispersed with pipettes until the cells were single. The cells were inoculated under the density of 1×10⁶/ml into Corning culture bottles with culture solution including EGF and bFGF. The culture condition is 37°C, saturated humidity, 5% CO₂ open floating culture without substrate. The 6-7 days old neurospheres were triturated with pipette, in this way the neurospheres could be dissolved into single cells or smaller cell masses. The progeny neurospheres were also dissolved in the same way every 3 days.2. NSCs self-renewal capacity: Added BrdU into the primary and lower neurospheres culture condition. After 24-hour culture, the anti-BrdU antibody was applied in immunocytochemical stain.3. Differentiation: Pasted the 24-well culture plate with I-type collagen, inoculated the primary and lower neurospheres in it. They were cultured by the differential culture solution including BDNF. The migration, proliferation and differentiation of these cells was recorded.