Zheng Xue-Sheng, Yang Xiao-Feng, Liu Wei-Guo, Shen Gang, Pan De-Sheng, Luo Ming
Department of Neurosurgery, First Affiliated Hospital, School of Medicine, Zhejiang University, No.79 Qingchun Road, Hangzhou 310009, China.
In Vitro Cell Dev Biol Anim. 2007 May-Jun;43(5-6):155-8. doi: 10.1007/s11626-007-9035-3. Epub 2007 Jul 7.
The standard culture method for neural stem cells cannot prevent the attachment of neurospheres, which eventually result in differentiation. This study developed a new method for long-term neural stem cell cultivation. In the antiattachment group, neural stem cells were cultured in flasks coated with 1.5% agarose gel. As a control, cells were cultured in plastic flasks. The 5-bromine-deoxyuridine incorporation assay was used to determine the S-phase labeling index of both groups. The methyl thiazolyl tetrazolium (MTT) colorimetric assay was used to determine the total cell vitality. After a 3-mo culture, the spontaneous differentiation of stem cells was studied using immunocytochemistry for neuroepithelial stem cell protein. We found that neural stem cells grew rapidly in the antiattachment flasks. There was no statistically significant difference between the two groups in terms of the S-phase labeling index or MTT assay. When cultured for 3 mo in vitro, many more cells differentiated in the control than in the antiattachment group (32.05 vs. 0.64%, P < 0.01). Moreover, the neural stem cells in the antiattachment group remained multipotent. Therefore, flasks coated with agarose gel are suitable for long-term neural stem cell culture.
神经干细胞的标准培养方法无法阻止神经球的附着，而这最终会导致分化。本研究开发了一种新的神经干细胞长期培养方法。在抗附着组中，神经干细胞在涂有1.5%琼脂糖凝胶的培养瓶中培养。作为对照，细胞在塑料培养瓶中培养。采用5-溴脱氧尿苷掺入法测定两组的S期标记指数。采用甲基噻唑基四氮唑（MTT）比色法测定细胞总活力。培养3个月后，使用神经上皮干细胞蛋白免疫细胞化学方法研究干细胞的自发分化。我们发现神经干细胞在抗附着培养瓶中生长迅速。两组在S期标记指数或MTT测定方面无统计学显著差异。体外培养3个月时，对照组中分化的细胞比抗附着组多得多（32.05%对0.64%，P<0.01）。此外，抗附着组中的神经干细胞仍具有多能性。因此，涂有琼脂糖凝胶的培养瓶适用于神经干细胞的长期培养。
