Extraction and subculture of neural stem cells from mouse embryonic spinal cord: comparison and analysis on advantages and disadvantages of three commonly used digestive enzymes

doi:10.12307/2025.540

Abstract

Abstract: BACKGROUND: In the research and application of neural stem cells, cell culture and passage are key links, which directly affect the quality of cells and experimental results. It is of great significance to find the most suitable digestive enzymes that can maintain the biological characteristics of embryonic mouse spinal cord neural stem cells and enhance their passage efficiency.
OBJECTIVE: To explore the most suitable digestive enzyme for passage of neural stem cells from the spinal cord of embryonic mice.
METHODS: Microscopic dissection was used to isolate and extract spinal cord tissue from E14 d embryonic mice, which was cultured in DMEM/F12 serum-free medium containing epidermal growth factor, basic fibroblast growth factor, and B27. After spherulation, Nestin and Sox2 immunofluorescence identification was performed. During neural stem cell passage and culture, single-cell suspensions were prepared using trypsin, papain, and TrypLETM Express enzyme digestion combined with blow molding. The cell dispersion and spheroidization were observed, and passage 3 cells were stained with propidium iodide to detect cell death. Cell proliferation was detected by counting the total number of cells. Immunofluorescence staining, western blot assay and RT-PCR were used to detect the expression of Olig2, Tuj1, GFAP, and NeuN at the protein and mRNA levels and to identify cell differentiation.
RESULTS AND CONCLUSION: After 72 hours of culture, E14 d embryonic mouse spinal cord tissue cells could form suspended neurospheres, which could be passaged after 5-7 days. Compared with trypsin and papain, TrypLETM Express enzyme combined with blow beating method was used for passage. The cell dispersion rate was high, the activity was good, and more NeuN- and Tuj1-positive neurons differentiated. This study optimized the culture and passaging process of neural stem cells, laying a foundation for further research on stem cell transplantation therapy for spinal cord diseases.

Key words: neural stem cell, C57BL/6 mouse, embryo, spinal cord, trypsin, papain, TrypLETM Express enzyme, engineered stem cell

CLC Number:

Luo Dan, Ge Zhilin, Hou Yonghui, Wang Wanshun, Zhan Jiheng, Hou Yu, Lin Dingkun, Chen Shudong. Extraction and subculture of neural stem cells from mouse embryonic spinal cord: comparison and analysis on advantages and disadvantages of three commonly used digestive enzymes[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(31): 6609-6615.

Figures/Tables 3

2.1 小鼠胚胎脊髓神经干细胞的形态提取分离出的原代细胞为圆形的单个悬浮细胞，其边界清楚、折光性强，见图1；24 h后，部分细胞贴壁，单个或成对存在；48 h后，可见哑铃型的细胞分裂相；72 h后，数个到数十个细胞形成细胞集落，悬浮生长；随着时间的推移，细胞迅速增加，细胞集落不断增大呈球形的神经球；7-10 d后神经球长大至200 μm左右即可传代，见图2。 2.2 神经干细胞的鉴定结果 Nestin是一种中间丝类型的蛋白，是公认的一种神经干细胞的特征性标志物[16-17]，Sox2 也是神经干细胞的关键标记蛋白之一，对于维持神经干细胞的自我更新和多能性起着至关重要的作用。将Nestin与Sox2的荧光双标作为神经干细胞的鉴定依据。荧光显微镜下观察488标记Nestin发绿光，555标记的Sox2为红光，表明Nestin与Sox2免疫荧光染色为阳性，确定培养的细胞为神经干细胞，见图3。 2.3 神经干细胞的分化情况将神经球分离为单细胞后接种于预先用多聚赖氨酸包被的24孔板中，用神经干细胞分化培养基诱导分化培养；细胞在培养24 h左右开始贴壁，2 d后细胞开始分化，有些细胞长出突起；随着时间的增加，突起逐渐增长，互相联系，分化出具有不同形态的神经细胞，其生长变化见图4。 2.4 不同酶传代对神经干细胞的影响 TrypLE TM Express酶消化后单细胞状态适宜，传代后球体大小适中，形态较好，背景干净；木瓜蛋白酶消化后仍有部分细胞小球无法消化成单个细胞，成球后易贴壁分化；胰蛋白酶虽能将神经球完全消化成单个细胞，但消化后的细胞活性差，成球速度慢，未成球的单个细胞较多，成球后部分贴壁分化，见图5。碘化丙啶不会染色质膜完整的活细胞，仅对死细胞染色。用545 nm激发，可观察到死细胞，TrypLETM Express酶组的死细胞比例明显低于其他两种酶组，差异有显著性意义，证明TrypLETM Express酶消化传代能减少神经干细胞的死亡率，保持细胞活性。见图6。 "

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