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Neurosciences

从新生小鼠中磁分离小胶质细胞用于原代细胞培养

Published: July 25, 2022
doi:
10.3791/62964
, , , , , , , , , ,
1NeuroDiderot, Inserm UMR-1141, Hôpital Robert Debré 48,Université de Paris, 2Department of anesthesia and critical care,APHP-Sorbonne university

Summary

原代小胶质细胞培养通常用于评估新的抗炎分子。本协议描述了一种可重复且相关的方法，用于从新生儿幼崽中磁性分离小胶质细胞。

Abstract

小胶质细胞作为大脑驻留巨噬细胞，是多种功能的基础，包括对环境压力的反应和大脑稳态。小胶质细胞可以采用大谱活化表型。此外，支持促炎表型的小胶质细胞与神经发育和神经退行性疾病有关。 体外 研究广泛用于评估特定细胞类型的潜在治疗策略的研究。在这种情况下，使用原代小胶质细胞培养物在 体外研究 小胶质细胞活化和神经炎症比小胶质细胞系或干细胞衍生的小胶质细胞更相关。然而，某些原代培养物的使用可能会受到缺乏可重复性的影响。该协议提出了一种从新生儿幼崽中磁性分离小胶质细胞的可重复和相关方法。这里展示了在4小时和24小时后通过mRNA表达定量和Cy3-珠吞噬细胞测定使用几种刺激物的小胶质细胞活化。目前的工作有望提供一种易于复制的技术，用于从青少年发育阶段分离生理相关的小胶质细胞。

Introduction

小胶质细胞是中枢神经系统驻留的巨噬细胞样细胞，来源于卵黄囊的红细胞生成前体，在胚胎早期发育期间迁移到神经上皮1。除了免疫功能外，它们在神经发育过程中也起着重要作用，特别是在突触发生、神经元稳态和髓鞘形成方面2。在成年期，小胶质细胞发展出长细胞过程以连续扫描环境。在脑损伤或脑部疾病等稳态破裂的情况下，小胶质细胞可以改变其形态外观，采用变形虫形状，迁移到受伤区域，增加和释放许多细胞保护或细胞毒性因子。小胶质细胞具有异质性激活状态，具体取决于其发育阶段和所受损伤的类型3，4，5。在这项研究中，这些激活状态大致分为三种不同的表型：促炎/吞噬细胞，抗炎和免疫调节，请记住，实际上，情况可能更复杂6。

由于（1）断奶前动物的脆弱性和（2）小胶质细胞细胞数量少，在大脑发育的早期阶段研究体内小胶质细胞活化和神经保护策略的筛选可能具有挑战性。因此，小胶质细胞的体外研究广泛用于毒性7，8，9，神经保护策略5，10，11，12，13，14和共培养15，16，17，18，19，20，21.体外研究可以使用小胶质细胞系、干细胞来源的小胶质细胞或原代小胶质细胞培养。所有这些方法都有优点和缺点，选择取决于最初的生物学问题。使用原代小胶质细胞培养物的好处是同质的遗传背景、无病原体的历史以及控制动物死亡后小胶质细胞被刺激的时间22。

多年来，开发了不同的方法（流式细胞术，摇动或磁性标记）用于培养啮齿动物的初级小胶质细胞，包括新生儿和成人23，24，25，26，27，28，29。在本工作中，使用先前描述的磁激活细胞分选技术使用微珠包被的抗小鼠CD11b 25，27，29进行小鼠新生幼崽的小胶质细胞分离。CD11b是一种在骨髓细胞（包括小胶质细胞）表面表达的整合素受体。当大脑内没有炎症挑战时，几乎所有的CD11b +细胞都是小胶质细胞30。与先前发表的其他方法23，24，25，26，27，28，29相比，本协议平衡了即时离体小胶质细胞活化分析和常见的体外原代小胶质细胞培养。 因此，小胶质细胞（1）在出生后第（P）8天分离，没有髓磷脂去除，（2）无血清培养，（3）在脑分离后仅48小时暴露于siRNA，miRNA，药理化合物和/或炎症刺激。这三个方面中的每一个都使当前的协议具有相关性和快速性。首先，使用小儿小胶质细胞可以在培养中获得动态和反应性活细胞，而无需额外的脱髓鞘步骤，这可能会在体外改变小胶质细胞的反应性。本协议旨在尽可能接近小胶质细胞的生理环境。事实上，小胶质细胞从未遇到过血清，并且该方案也不需要使用血清。此外，早在培养后48小时暴露小胶质细胞可以防止它们失去生理能力。

Protocol

该协议获得批准，所有动物均根据国家卫生和科学研究研究所（法国Inserm）的机构准则进行处理。介绍了 P8 处 24 只 OF1 小鼠幼崽（雄性和雌性）大脑中小胶质细胞的磁分离，分为 6 孔、12 孔或 96 孔板。实验工作在引擎盖下进行，以保持无菌条件。 1. 制备用于分离和细胞培养的无菌溶液 从市售的 10x 溶液中制备 50 mL 不含 Ca 2+ 和 Mg2+ （HBSS<s…

Representative Results

小胶质细胞是中枢神经系统驻留的巨噬细胞，当暴露于环境挑战（创伤，有毒分子，炎症）时被激活4，5，6，34（图3A）。小胶质细胞的体外研究通常用于评估与这些环境挑战相关的细胞自主机制，并表征药理学或遗传操作后的激活状态。本文提出了一种使用磁耦合珠在幼…

Discussion

目前的工作展示了使用磁分选CD11b +细胞的原代小胶质细胞培养物。除了小胶质细胞功能评估（RT-qPCR和吞噬细胞测定）外，还测定了小胶质细胞培养纯度。

经典的小胶质细胞培养物通常由 P1 或 P2 啮齿动物新生儿脑生成，并与星形胶质细胞共培养至少 10 天。然后使用轨道摇床机械分离小胶质细胞。体 外分离 和培养小胶质细胞的方法在1990年代末首次从新生大鼠的大脑<su…

Divulgations

The authors have nothing to disclose.

Acknowledgements

图形是使用BioRender创建的。该研究由Inserm，巴黎大学，地平线2020（PREMSTEM-874721），法国基金会，ARSEP基金会，切尔沃研究基金会，摩纳哥格雷斯基金会资助，以及未来投资-ANR-11-INBS-0011-NeurATRIS和Investissement d’Avenir -ANR-17-EURE-001-EUR G.E.N.E.的额外资助。

Materials

Anti mouse ACSA-2 PE Vio 615 Miltenyi Biotec 130-116-246
Anti mouse CD11b BV421 Sony Biotechnology 1106255
Anti mouse CD45 BV510 Sony Biotechnology 1115690
Anti mouse CX3CR1 PE Cy7 Sony Biotechnology 1345075
Anti mouse NeuN PE Milli-Mark FCMAB317PE
anti mouse O4 Vio Bright B515 Miltenyi Biotec 130-120-016
BD Cytofix/Cytoperm permeabilization kit BD Biosciences 554655
Bovine Serum Albumin Miltenyi Biotec 130-091-376
CD11b (Microglia) MicroBeads, h, m Miltenyi Biotec 130-093-634
Confocal microscope Leica TCS SP8
D-PBS (10x) Thermo Scientific 14200067
EDTA Sigma-Aldrich E1644
Falcon Cell culture 12-well plate, flat bottom + lid Dutscher 353043
Falcon Cell culture 96-well plate, flat bottom + lid Dutscher 353072
Falcon tubes 50 mL Dutscher 352098
Fc blocking reagent (Mouse CD16/32) BD Biosciences 553142
Fluorescence microscope Nikon ECLIPSE TE300
gentleMACS C Tubes (4 x 25 tubes) Miltenyi Biotec 130-096-334
gentleMACS Octo Dissociator with Heaters Miltenyi Biotec 130-096-427
Hanks' Balanced Salt Solution (HBSS) +CaCl2 +MgCl2 10x Thermo Scientific 14065049
Hanks' Balanced Salt Solution (HBSS) -CaCl2 -MgCl2 10x Thermo Scientific 14185045
iQ SYBR Green Supermix Bio-rad 1725006CUST
Iscript c-DNA synthesis Bio-rad 1708890
Latex beads, amine-modified polystyrene, fluorescent red Sigma-Aldrich L2776-1mL
Lipopolysaccharides (LPS) from Escherichia coli O55:B5 Sigma-Aldrich L2880
Macrophage-SFM serum-free medium Thermo Scientific 12065074
MACS BSA Stock Solution Miltenyi Biotec 130-091-376
MACS SmartStrainers (70 μm), 4 x 25 pcs Miltenyi Biotec 130-110-916
Mouse IgG1 PE Millipore MABC002H
Mouse IgG2a PE Cy7 Sony Biotechnology 2601265
Mouse IL1 beta Miltenyi Biotec 130-101-684
Multi-24 Column Blocks Miltenyi Biotec 130-095-691
MultiMACS Cell24 Separator Miltenyi Biotec
Neural Tissue Dissociation Kit – Papain Miltenyi Biotec 130-092-628
Nucleocounter NC-200 Chemometec
Nucleospin RNA Plus XS Macherey Nagel 740990.5
Nun EZFlip Top Conical Centrifuge Tubes Thermo Scientific 362694
OPTILUX Petri dish – 100 x 20 mm Dutscher 353003
Pénicilline-streptomycine (10 000 U/mL) Thermo Scientific 15140122
Rat IgG2b, k BV421 BD Biosciences 562603
Rat IgG2b, k BV510 Sony Biotechnology 2603230
REA control (S) PE vio 615 Miltenyi Biotec 130-104-616
REA control (S) Vio Bright B515 Miltenyi Biotec 130-113-445
Recombinant Mouse IFN-gamma Protein R&D System 485-MI
Recombinant Mouse IL-10 Protein R&D System 417-ML
Recombinant Mouse IL-4 Protein R&D System 404-ML
RIPA Buffer Sigma-Aldrich R0278
Viability probe (FVS780) BD Biosciences 565388
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Tags

MicrogliaMagnetic Cell SortingNeonate MousePrimary Cell CultureCD11bCell DissociationHBSSSorting BufferCell SeparationCell Count
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Bokobza, C., Jacquens, A., Zinni, M., Faivre, V., Hua, J., Guenoun, D., Userovici, C., Mani, S., Degos, V., Gressens, P., Van Steenwinckel, J. Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures. J. Vis. Exp. (185), e62964, doi:10.3791/62964 (2022).
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