海马神经干细胞不同传代方法的比较

背景：体外培养神经干细胞，在悬浮培养时由于自身增殖特性会形成球，传代时将会面临如何将细胞球分离成单细胞的问题。 目的：寻求理想的大鼠海马神经干细胞传代方法，以获得大量可增生的神经干细胞以供研究。 方法：分离新生1 d SD大鼠海马神经干细胞，原代培养至五六天时，分别用机械吹打法、胰蛋白酶、TrypLE和Accutase消化法分离神经干细胞球。之后每7 d传代1次，连续传代3次。分别于每次传代后第1天和传代后第4天计数活细胞比例和细胞球数目，实验重复3次。 结果与结论：神经干细胞球经3种酶消化后获得的均是单细胞；经机械吹打后既有单个细胞，也有小细胞球分布于培养液中。在酶消化法中，Accutase消化法传代后神经干细胞的活细胞比例明显高于胰蛋白酶消化(P < 0.01)和TrypLE消化法 (P < 0.05)。同时，Accutase消化法传代后新形成的细胞球数目也较其余各组多(P < 0.01)。提示在实验条件下，Accutase消化法能够较好地将神经干细胞球分离成存活率较高、能快速形成新的克隆球的单个细胞，是较为理想的神经干细胞分离传代方法。

Comparison of different passage methods for hippocampal neural stem cells

BACKGROUND: Neural stem cells (NSCs) cultured in vitro can form sphere due to its proliferation characteristics during suspension culture. How to isolate cell spheres into single cells is a problem facing us during subculture. OBJECTIVE: To establish an ideal subculture method for harvesting NSCs from the rat hippocampus to obtain a large number of NSCs for investigation. METHODS: NSCs were isolated from neonatal 1-day rat hippocampus. After a growth of typically 5-6 days, primary neurospheres were passaged by mechanical dissociation, trypsin, TrypLE or Accutase digestion. Each method was used to dissociate neurospheres for three passages every 7 days. The ratio of cell viability and neurosphere numbers were estimated on day 1 and day 4 respectively after passage. This experiment was repeated three times. RESULTS AND CONCLUSION: Three enzymatic dissociation each obtained single-cell suspension; while mechanical dissociation resulted in mixtures of single cells and small neurospheres. The cell viability after Accutase treatment was significantly higher than trypsin (P < 0.01) and TrypLE digestion (P < 0.05). Simultaneously, more newly formed neurospheres were generated by Accutase treatment as compared with other groups (P < 0.01). These indicated that in this experimental condition, enzymatic treatment using Accutase can be regarded as an ideal protocol for dissociating neurospheres into single cells with high cell survival which yield secondary neurospheres rapidly.