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一种手动解离、免疫磁珠分选加TIC培养液改良的小鼠原代海马小胶质细胞培养法

许雅南, 周利君, 揭英桃, 麦春林, 张珺, 林震嘉, 谈智^*

中山大学中山医学院生理教研室及中山大学疼痛研究中心，广州 510080

摘要

本文旨在研究手动解离、免疫磁珠分选加TIC培养液改良原代小胶质细胞的培养方法，获得高活性、高纯度且与在体状态更接近的小鼠海马原代小胶质细胞，以用于小胶质细胞的相关研究。选取2~4周龄SPF级C57BL/6J小鼠，PBS灌注后取海马组织剪碎，使用成年鼠脑组织解离试剂盒手动解离海马组织，获得单细胞悬液，再经去碎片试剂去除髓鞘等组织碎片。在4 °C 孵育CD11b免疫磁珠15 min后，细胞悬液经免疫磁珠细胞分选(magnetic activated cell sorting, MACS)两次过柱提高小胶质细胞纯度。将分选后细胞离心重悬，种在多聚赖氨酸包被后的24孔培养板中，用完全培养基或TIC培养液(以TGF-β、IL-34和胆固醇为主要营养成分的无血清培养基)培养4 d后进行其他检测。结果显示：(1)使用成年鼠脑组织解离试剂盒手动解离可得到含有(56.03 ± 2.10)%活细胞的单细胞悬液；(2)与免疫抗体包被筛选法(immunopanning)相比，经过MACS两步分选后，可得到更多且纯度高达(86.20 ± 0.68)%的小胶质细胞；(3)使用TIC培养液培养4 d后，可获得分枝状、形态类似静息状态的原代小胶质细胞；(4) qRT-PCR检测显示，与完全培养基培养相比，TIC培养液培养的小胶质细胞在4 d和7 d后TNF-α、IL-1β及CCL2 mRNA表达水平更接近急性分离后的小胶质细胞。上述结果提示，采用手动解离海马组织，经MACS两步分选及TIC培养后，可以获得高纯度、高活性且接近在体静息状态的小胶质细胞。

关键词： 小胶质细胞; 海马; 磁珠分选; 原代培养; 细胞因子

分类号：R392.33;R33;Q954.6-33+2;Q813.1+1

A modified protocol of mouse hippocampal primary microglia culture by using manual dissociation, magnetic activated cell sorting and TIC medium

XU Ya-Nan, ZHOU Li-Jun, JIE Ying-Tao, MAI Chun-Lin, ZHANG Jun, LIN Zhen-Jia, TAN Zhi^*

Department of Physiology, Zhongshan School of Medicine and Pain Research Center, Sun Yat-sen University, Guangzhou 510080, China

Abstract

In this study, we improved the culture method of mouse hippocampal primary microglia to obtain hippocampal ramified microglia with high activity and purity, which were resemble to the resting status of normal microglia in healthy brain in vivo. Hippocampal tissue was excised from 2–4-week-old SPF C57BL/6J mice and cut into pieces after PBS perfusion, and then manually dissociated into the single-cell suspension by using Miltenyi Biotec’s Adult Brain Dissociation Kit. The tissue fragments such as myelin in the supernatant were removed by debris removal solution in the kit. The cell suspension was incubated with CD11b immunomagnetic beads for 15 min at 4 °C. To obtain high-purity microglia, we used two consecutive cell-sorting steps by magnetic activated cell sorting (MACS). After centrifugation, the cells were resuspended and seeded in a 24-well culture plate. The primary microglia were cultured with complete medium (CM) or TIC medium (a serum-free medium with TGF-β, IL-34 and cholesterol as the main nutritional components) for 4 days, and then were used for further experiments. The results showed that: (1) The cell viability was (56.03 ± 2.10)% by manual dissociation of hippocampus; (2) Compared with immunopanning, two-step MACS sorting allowed for efficient enrichment of 

microglia with higher purity of (86.20 ± 0.68)%; (3) After being incubated in TIC medium for 4 d, microglia exhibited branching, quiescent morphology; (4) The results from qRT-PCR assay showed that the levels of TNF-α, IL-1β and CCL2 mRNA in TIC cultured-microglia were similar to freshly isolated microglia, while those were much higher in CM cultured-microglia after incubation for 4 d and 7 d (P < 0.05). Taken together, compared to the conventional approaches, this modified protocol of mouse hippocampal primary microglia culture by using MACS and TIC medium enables the increased yield and purity of microglia in the quiescent state, which is similar to normal ramified microglia in healthy brain in vivo. 

Key words: microglia; hippocampus; magnetic activated cell sorting; primary culture; cytokine

收稿日期：2019-03-08　　录用日期：2019-05-13

通讯作者：谈智　　E-mail: tanzhi@mail.sysu.edu.cn

DOI: 10.13294/j.aps.2019.0061

引用本文：

许雅南, 周利君, 揭英桃, 麦春林, 张珺, 林震嘉, 谈智. 一种手动解离、免疫磁珠分选加TIC培养液改良的小鼠原代海马小胶质细胞培养法[J]. 生理学报 2019; 71 (6): 883-893.

XU Ya-Nan, ZHOU Li-Jun, JIE Ying-Tao, MAI Chun-Lin, ZHANG Jun, LIN Zhen-Jia, TAN Zhi. A modified protocol of mouse hippocampal primary microglia culture by using manual dissociation, magnetic activated cell sorting and TIC medium. Acta Physiol Sin 2019; 71 (6): 883-893 (in Chinese with English abstract).