体外培养的神经干细胞球几种离散方法的探讨

Evaluation on several methods for dissociating neurospheres of neural stem cell cultured in vitro

目的探讨一种新的有效离散体外培养的神经干细胞球的方法.方法采用单纯玻璃吸管机械分散,胰蛋白酶消化,不锈钢滤网研磨,单纯滤网吸管分散和滤网吸管结合短时间胰酶消化等五种方法离散神经干细胞球,比较其各自的离散效果和离散后细胞存活情况.结果单纯玻璃吸管吹打不能完全离散神经球;胰蛋白酶消化在30min内不能完全离散神经球,延长酶消化时间虽可离散细胞,但细胞难以存活;不锈钢滤网研磨或单纯滤网吸管吹打离散对细胞损伤大,存活率低(分别为64.1%和71.9%);滤网吸管结合短时间(3～5min)胰酶消化离散细胞效果好,存活率(92.1%)显著高于上述其它方法(P＜0.05).结论滤网吸管结合短时间胰酶消化法离散神经干细胞球,离散效果好,细胞存活率高,是一种新的有效离散体外培养的神经干细胞球的好方法.

Objective To explore an improved method for dissociating neural stem cell neurospheres. Methods Neurospheres were dissociated using the methods of triturating only with glass pipette, digestion of trypsin, grinding with stainless steel mesh and triturating by mesh pipette with or without digestion of trypsin. The five methods were evaluated by comparing their separating ability and cell survivals. Results Neurospheres could not be dissociated completely by triturating only with glass pipette or digestion of trypsin for 30 min. Though trypsin digestion time prolonged led to dissociation of neurospheres, neural stem cells (NSCs) were hardly survival. Grinding with stainless steel mesh or dissociating merely by plastic pipette with metal mesh would damage neurospheres severely (NSC survival rates are 64.1% and 71.9% respectively). In contrast, the mesh pipette combined with trypsin digestion (3-5 min) could separate neurospheres better than the other four methods mentioned above, and have a significantly higher NSC survival rate (92.1%, P<0.05). Conclusion Mesh pipette combined with trypsin digestion for a short time is a novel and improved method for dissociating neurospheres of neural stem cells cultured in vitro with advantages of effective dissociation and high cell survival rate.