Abstract:

Objective To discuss the method of primary culture and activity evaluation system of the neural stem cells （NSCs） of the fetal rats， and to confirm the optimal subculture conditions of the NSCs. Methods The SD rats with 11－14 d gestation were chosen，and the primary NSCs of the fetal rats were extracted. Nestin immunofluorescence staining was used to identify the NSCs. The NSCs were subcultured at the cell densities of 1.0×10⁴，2.0×10⁴，6.0×10⁴，1.0×10⁵， 1.6×10⁵ and 2.0×10⁵ mL^－1， then the morpholgy of the neurospheres was observed 48 h after passage and the diameter of the neurospheres was calculated. The survival rates of NSCs in the neurospheres with different diameters were detected by live-dead cell staining. Results The expression of nestin in the NSCs was positive， indicating that the cultured cells were the NSCs. The NSCs showed aggregation growth and strong cell-cell adhesion pattern forming neurospheres with an average diameter of （152.72±47.52） μm， and there were few single cells scattered between the neurospheres. Compared with 2.0×10⁴ mL^－1 subculture density，the diameters of the neurospheres at the subculture densities of differences 6.0×10⁴ mL^－1 and 1.0×10⁵ mL^－1 were increased （P<0.05）. There were no significant differences in the diameters of the neurospheres at the subculture densities of 1.0×10⁵ mL^－1， 1.6×10⁵ mL^－1， and 2.0×10⁵ mL^－1 （P>0.05）. Compared with neurospheres with diameters of 0—40 μm， 40—60 μm， 60—80 μm，and 80—100 μm， the survival rate of NSCs in the neurospheres with the diameters of 100—200 μm was decreased （P<0.05）. Conclusion The neurospheres with the diameters of 80—100 μm when subcultured at the density of 1.0×10⁵ mL^－1 can effectively improve the efficiency and activity of subculture of the NSCs.

Key words: Neural stem cell, Fetal rat, Primary culture, Subculture condition, Neurosphere