Zhou Qing-Zhong, Feng Xiao-Lan, Jia Xu-Feng, Mohd Nor Nurul Huda Binti, Harun Mohd Hezery Bin, Feng Da-Xiong, Wan Sulaiman Wan Aliaa
Department of Orthopedics, The Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China.
Department of Neurology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia.
World J Stem Cells. 2023 Jun 26;15(6):607-616. doi: 10.4252/wjsc.v15.i6.607.
Timing of passaging, passage number, passaging approaches and methods for cell identification are critical factors influencing the quality of neural stem cells (NSCs) culture. How to effectively culture and identify NSCs is a continuous interest in NSCs study while these factors are comprehensively considered.
To establish a simplified and efficient method for culture and identification of neonatal rat brain-derived NSCs.
First, curved tip operating scissors were used to dissect brain tissues from new born rats (2 to 3 d) and the brain tissues were cut into approximately 1 mm sections. Filter the single cell suspension through a nylon mesh (200-mesh) and culture the sections in suspensions. Passaging was conducted with TrypL Express combined with mechanical tapping and pipetting techniques. Second, identify the 5th generation of passaged NSCs as well as the revived NSCs from cryopreservation. BrdU incorporation method was used to detect self-renew and proliferation capabilities of cells. Different NSCs specific antibodies (anti-nestin, NF200, NSE and GFAP antibodies) were used to identify NSCs specific surface markers and muti-differentiation capabilities by immunofluorescence staining.
Brain derived cells from newborn rats (2 to 3 d) proliferate and aggregate into spherical-shaped clusters with sustained continuous and stable passaging. When BrdU was incorporated into the 5 generation of passaged cells, positive BrdU cells and nestin cells were observed by immunofluorescence staining. After induction of dissociation using 5% fetal bovine serum, positive NF200, NSE and GFAP cells were observed by immunofluorescence staining.
This is a simplified and efficient method for neonatal rat brain-derived neural stem cell culture and identification.
传代时机、传代数、传代方法以及细胞鉴定方法是影响神经干细胞（NSCs）培养质量的关键因素。在综合考虑这些因素的情况下，如何有效地培养和鉴定NSCs一直是NSCs研究中的一个关注点。
建立一种简化高效的新生大鼠脑源性NSCs培养及鉴定方法。
首先，用弯尖手术剪解剖新生大鼠（2至3日龄）的脑组织，将脑组织切成约1mm的切片。通过尼龙网（200目）过滤单细胞悬液，并将切片培养在悬液中。使用TrypL Express结合机械敲击和吹打技术进行传代。其次，鉴定第5代传代NSCs以及冻存复苏后的NSCs。采用BrdU掺入法检测细胞的自我更新和增殖能力。使用不同的NSCs特异性抗体（抗巢蛋白、NF200、NSE和GFAP抗体）通过免疫荧光染色鉴定NSCs特异性表面标志物和多分化能力。
新生大鼠（2至3日龄）的脑源性细胞增殖并聚集成球形簇，传代持续、稳定。当将BrdU掺入第5代传代细胞时，通过免疫荧光染色观察到BrdU阳性细胞和巢蛋白阳性细胞。使用5%胎牛血清诱导分化后，通过免疫荧光染色观察到NF200、NSE和GFAP阳性细胞。
这是一种简化高效的新生大鼠脑源性神经干细胞培养及鉴定方法。
