中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (7): 1395-1400.doi: 10.12307/2025.011

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

SD大鼠乳鼠原代皮质神经元和小胶质细胞同时提取并培养的实验方法

何龙才1,宋文学1,明  江1,陈光唐1,王军浩1,廖益东2,崔君拴3,徐卡娅1,3,4   

  1. 1贵州医科大学,贵州省贵阳市  550004;2贵阳市第一人民医院,贵州省贵阳市   550002;贵州医科大学附属医院,3神经外科,4高压氧科,贵州省贵阳市   550004
  • 收稿日期:2023-11-01 接受日期:2024-01-14 出版日期:2025-03-08 发布日期:2024-06-27
  • 通讯作者: 徐卡娅,博士,主任医师,贵州医科大学,贵州省贵阳市 550004;贵州医科大学附属医院,神经外科,高压氧科,贵州省贵阳市 550004
  • 作者简介:何龙才,男,1998年生,四川省巴中市人,汉族,贵州医科大学在读硕士,主要从事内皮祖细胞移植对脑缺血性卒中神经血管再生与神经功能网络重塑的影响及机制研究。
  • 基金资助:
    国家自然科学基金项目(81901173,82060230),项目负责人:徐卡娅;贵州省基础研究计划(自然科学类)项目(2023039),项目负责人:徐卡娅;贵州医科大学附属医院国家自然科学基金面上项目培育计划项目(gyfynsfc-2022-08),项目负责人:徐卡娅;贵州医科大学附属医院博士科研启动基金项目(gyfybsky-2021-6),项目负责人:徐卡娅

An experimental method for simultaneous extraction and culture of primary cortical neurons and microglial cells from SD rats

He Longcai1, Song Wenxue1, Ming Jiang1, Chen Guangtang1, Wang Junhao1, Liao Yidong2, Cui Junshuan3, Xu Kaya1, 3, 4   

  1. 1Guizhou Medical University, Guiyang 550004, Guizhou Province, China; 2The First People’s Hospital of Guiyang, Guiyang 550002, Guizhou Province, China; 3Department of Neurosurgery, 4Department of Hyperbaric Oxygen, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • Received:2023-11-01 Accepted:2024-01-14 Online:2025-03-08 Published:2024-06-27
  • Contact: Xu Kaya, MD, Chief physician, Guizhou Medical University, Guiyang 550004, Guizhou Province, China; Department of Neurosurgery, and Department of Hyperbaric Oxygen, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • About author:He Longcai, Master candidate, Guizhou Medical University, Guiyang 550004, Guizhou Province, China
  • Supported by:
    National Natural Science Foundation of China, Nos. 81901173 and 82060230 (to XKY); Guizhou Basic Research Program (Natural Science category) Project, No. 2023039 (to XKY); National Natural Science Foundation of China, Affiliated Hospital of Guizhou Medical University, No. gyfynsfc-2022-08 (to XKY); Doctoral Research Initiation Fund of Affiliated Hospital of Guizhou Medical University, No. gyfybsky-2021-6 (to XKY)

摘要:

文题释义:

原代皮质神经元:是指从动物大脑的皮质区域中直接分离和培养出的神经元。它们是大脑皮质区域的基本组成单位,承担着信息传递和处理等重要功能。因此,原代皮质神经元常被用作细胞模型,用于探索缺血性脑卒中、阿尔茨海默病、帕金森病等方面的研究。
小胶质细胞:是中枢神经系统内固有的免疫效应细胞,其分泌的激素、神经递质或蛋白质等细胞外信号可与神经元上的特异性受体结合,促进神经元的存活或启动神经元发生程序性细胞死亡,从而对中枢神经系统内神经元的数量进行调控。


背景:原代皮质神经元和小胶质细胞在探索神经系统疾病细胞疗法中起着至关重要的作用,而目前获得2种细胞的方法大多较繁琐,需分别单独提取。因此找到一种便捷、快速可同时提取2种细胞的方法十分关键。
目的:探索一种新型同时提取原代皮质神经元及小胶质细胞的方法。
方法:取24 h内新生SD大鼠乳鼠,将大脑取出放入装有DMEM的培养皿中,去除软脑膜后备用。同一脑组织,先提取原代神经元,再将剩余脑组织用于提取小胶质细胞,整个过程在冰上操作。原代皮质神经元提取培养步骤:镊子夹取2.0-3.0 mm厚度大脑皮质组织置于培养皿,用木瓜蛋白酶消化20 min,中止消化后吹打组织呈互相粘连的组织悬液,吸上清细胞悬液,过滤、分装到15 mL离心管中,离心并重悬后将细胞接种于左旋多聚赖氨酸包被的6孔板爬片上放入细胞培养箱,每隔1 d观察神经元形态,第7天进行MAP2和β-Tubulin免疫荧光染色鉴定。小胶质细胞提取培养步骤:夹取上一步取皮质后剩余脑组织,厚度8-10 mm,置于培养皿,用胰酶消化20 min,中止消化后吹打组织呈匀浆,然后将匀浆转移到培养瓶中培养,第14天将培养瓶封口后进行恒温水平摇床振荡2 h,小胶质细胞脱落于上清中;取纯化后的小胶质细胞继续培养3 d进行Iba1免疫荧光染色鉴定。
结果与结论:①神经元培养24 h后贴壁、胞体变大,部分神经元长出突触;培养到第3,5天时胞体进一步增大,大部分神经元已呈突触形态,部分神经元成团生长;第7天时,神经元突触延长增粗并互相连接成网。经β-Tubulin、MAP2免疫荧光染色鉴定为神经元。②小胶质细胞培养第1天换液后可见细胞贴壁生长;第3,5,7天时细胞密度均较小,细胞形态呈高亮椭圆或圆形,但已基本成团块状生长于其他细胞上层;第10天时小胶质细胞密度明显增大;第14天时小胶质细胞呈致密团块状生长于其他细胞上层,此时可进行分离纯化。取分离纯化后细胞再培养至第3天,经Iba1免疫荧光鉴定为小胶质细胞,且纯度大于95%。③结果表明,经此法提取并培养获得的原代皮质神经元和小胶质细胞纯度高、形态好、成活率高。

https://orcid.org/0009-0009-7291-9730 (何龙才) 


中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程

关键词: 原代皮质神经元, 小胶质细胞, SD大鼠, 细胞提取, 细胞培养

Abstract: BACKGROUND: Primary cortical neurons and microglial cells play a crucial role in exploring cell therapies for neurological disorders, and most of the current methods for obtaining the two types of cells are cumbersome and require separate extraction. It is therefore crucial to find a convenient and rapid method to extract both types of cells simultaneously.
OBJECTIVE: To explore a novel method for simultaneous extraction of primary cortical neurons and microglial cells. 
METHODS: Newborn suckling SD rats were taken within 24 hours. The brain was removed and placed in a dish with DMEM, and the pia mater was removed for later use. Primary neurons were extracted from the same brain tissue, and then the remaining brain tissue was used to extract microglial cells. The whole process was performed on ice. Extraction and culture steps of primary cortical neurons: The cerebral cortex was taken 2.0-3.0 mm with forceps, and the tissue was digested with papain for 20 minutes. After aborting digestion, the blown tissue presented an adherent tissue suspension. The supernatant cell suspension was obtained, filtered, and dispensed into 15 mL centrifuge tubes. After centrifugation and re-suspension, the cells were inoculated onto 6-well plate crawls coated with L-polylysine. Neuronal morphology was observed at 1-day intervals, and staining could be performed for identification using immunofluorescence staining of MAP2 and β-Tubulin by day 7. Microglia extraction and culture steps: The remaining brain tissue at 8-10 mm thick was subjected to microglial cell extraction, digested by trypsin for 20 minutes. After digestion was stopped, the tissue was blown to a homogenate, and then the homogenate was transferred to the culture bottle for culture. On day 14, the culture flasks were sealed and subjected to constant temperature horizontal shaking for 2 hours. Microglial cells were shed in the supernatant. Purified microglial cells were taken and continued to be cultured for 3 days for identification by Iba1 immunofluorescence staining.
RESULTS AND CONCLUSION: (1) After 24 hours of culture, the neurons were adherent to the wall, the cytosol was enlarged, and some neurons developed synapses. After 3 and 5 days of culture, the cytosol was further enlarged, and most of the neurons were in the form of synapses, and some neurons were growing in clusters. On day 7, neuronal synapses were prolonged and thickened, and they were connected with each other to form a network. The neurons were identified by β-Tubulin and MAP2 immunofluorescence staining. (2) The cells grew close to the wall on day 1 of culture. On days 3, 5, and 7, the density of microglial cells was small, and the cell morphology was bright oval or round, but the cells basically grew in clumps on the upper layer of other cells. On day 10, the density of microglial cells increased significantly. On day 14, microglial cells grew in dense clumps on the upper layer of other cells, and then they could be isolated and purified. The isolated and purified cells were taken and re-cultured to day 3 and identified as microglial cells by Iba1 immunofluorescence; their purity was greater than 95%. (3) The results show that primary cortical neurons and microglial cells obtained by this method after extraction and culture are of high purity, good morphology, and high viability.

Key words: primary cortical neuron, microglial cell, SD rat, cell extraction, cell culture

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