改良无血清法培养新生SD乳鼠原代海马神经元细胞

An improved serum-free method for culturing primary hippocampal neurons from SD neonatal rats

目的探讨改良无血清法培养新生SD乳鼠原代海马神经元细胞的效果及注意事项。方法2018年2-12月共解剖分离72只出生24 h内SD乳鼠的海马,采取Neurobasal+B27改良无血清法培养原代海马神经元细胞。铺板前使用Invitroge自动细胞计数仪计数存活及死亡细胞。细胞贴壁生长0 d、3 d、6 d、9 d、12 d时使用激光共聚焦显微镜观察神经细胞形态变化。结果平均每只乳鼠的海马可分离出(8.40±2.86)×10^(5)个神经细胞,其中存活细胞数为(5.48±1.73)×10^(5),死亡细胞数为(3.08±1.30)×10^(5),细胞总存活率为69%。细胞贴壁后第3天可观察到轴突长出,第6天树突长出,第9天和第12天可观察到周围神经细胞轴突、树突连成神经网络。贴壁后第3天胞体逐渐变大变饱满,最后呈扁圆形。细胞可存活4周左右。结论改良无血清法可用于培养原代海马神经元细胞,快速精准解剖分离及轻柔有效吹打海马组织是培养出高存活率和高活性原代海马神经元细胞的关键。

Objective To explore the cell survival of the modified serum-free method for culturing primary hippocampal neurons from SD neonatal rats.Methods From February to December in 2018,the hippocampal tissues of 72 SD neonatal rats within 24 hours of birth were dissected and isolated,and the neuron cells were cultured with Neurobasal+B27 modified serum-free medium.Invitroge automatic cell counter was used to count the number of viable and dead cells.The morphological changes of nerve cells were observed using a laser confocal microscope.Results In average,(8.40±2.86)×10^(5)nerve cells were isolated from two hippocampi of each neonatal rat,among which the number of viable cells was(5.48±1.73)×10^(5)and the number of dead cells was(3.08±1.30)×10^(5).The cell survival rate was 69%.Axons was observed to grow on the third day after the cells adhered to the wall,dendrites grew on the sixth day,and axons and dendrites of peripheral nerve cells could be observed to form a neural network on the ninth and twelfth days.On the third day after adherence,the nerve cell body gradually grew larger and fuller,and finally became oblate.All cells could survive for up to 4 weeks.Conclusion Our improved serum-free method is suitable for culturing primary hippocampal neurons.Accurate and rapid dissection and gentle and effective pipetting of hippocampal tissue are the key points of culturing neuronal cells with higher survival and viability.