从胚胎小鼠大脑中建立混合神经元和神经胶质细胞培养物以研究感染和先天免疫
Summary
该协议提供了一种从胚胎第17天小鼠大脑生成中枢神经系统细胞培养物的独特方法，用于神经（免疫）学研究。该模型可以使用各种实验技术进行分析，包括RT-qPCR，显微镜，ELISA和流式细胞术。
Abstract
中枢神经系统（CNS）的模型必须概括 体内发现 的相互连接的细胞的复杂网络。中枢神经系统主要由神经元、星形胶质细胞、少突胶质细胞和小胶质细胞组成。由于越来越多的努力替代和减少动物的使用，已经开发了各种 体外 细胞培养系统来探索先天细胞特性，从而可以开发针对CNS感染和病理的治疗方法。虽然某些研究问题可以通过基于人类的细胞培养系统来解决，例如（诱导的）多能干细胞，但与人类细胞一起工作在可用性、成本和伦理方面有其自身的局限性。在这里，我们描述了一种从胚胎小鼠大脑中分离和培养细胞的独特方案。由此产生的混合神经细胞培养物模拟 体内大脑中发现的几种细胞群和相互作用。与目前的等效方法相比，该协议更接近于模拟大脑的特征，并且还获得了更多的细胞，从而允许从一只怀孕的小鼠中研究更多的实验条件。此外，该协议相对容易且高度可重复。这些培养物已经过优化，可用于各种规模，包括基于 96 孔的高通量筛选、24 孔显微镜分析和用于流式细胞术和逆转录定量聚合酶链反应 （RT-qPCR） 分析的 6 孔培养物。这种培养方法是在CNS的一些复杂性背景下研究感染和免疫力的有力工具，并且体 外 方法很方便。
Introduction
提高我们对中枢神经系统（CNS）的理解对于改善许多神经炎症和神经退行性疾病的治疗选择至关重要。中枢神经系统是大脑、脊髓和视神经内相互连接的细胞的复杂网络，包括神经元、少突胶质细胞、星形胶质细胞及其先天免疫细胞小胶质细胞1。 体外 方法通常可以大大减少进行有意义研究所需的小鼠数量;然而，CNS的复杂性使得不可能使用细胞系概括 体内 情况。混合神经细胞培养物提供了一种非常有价值的研究工具，用于研究相关模型中的神经（免疫）学问题，符合替换、减少和改进（3Rs）原则2，3。
Thomson等人描述了一种使用产前脊髓细胞分化为上述所有主要CNS细胞类型4的细胞培养方法。该系统还具有突触形成，髓鞘轴突和Ranvier节点。这种培养方法的主要局限性是，作为脊髓，它不能有效地模拟大脑，并且胚胎第13天（E13）脊髓的细胞产量正在收缩。因此，这限制了可以研究的实验条件的数量。因此，本研究旨在开发一种新的细胞培养系统，通过提高细胞产量来概括大脑的特征，以减少对动物的需求。
以Thomson等人为起点，我们开发了一种纯粹来自产前小鼠大脑的细胞培养模型。这些培养物具有与脊髓培养物相同的细胞群、互连性和治疗选择，只是相比之下髓鞘形成较少。然而，具有大约三倍细胞产量的CNS 体外 模型更有效，需要更少的小鼠和更少的胚胎处理时间。我们针对多种下游应用和规模优化了这种独特的培养系统，包括使用玻璃盖玻片进行显微镜分析，以及使用各种尺寸的塑料孔板，包括用于高通量研究的 96 孔板。
Protocol
所有动物实验均符合当地动物使用法律和准则，并得到格拉斯哥大学当地伦理审查委员会的批准。根据1986年英国动物科学程序法，在英国内政部项目许可证的主持下，动物被饲养在特定的无病原体条件下。在这项研究中，使用了内部繁殖的成年C57BL / 6J小鼠。由于怀孕成功率较高，建议使用年轻女性（8-12周）;雄性可以重复使用进行多轮繁殖。 图1 表示所述生成混合神经元和…
Representative Results
显微术在玻璃盖玻片上生长的培养物非常适合通过显微镜进行分析。为了可视化培养物的发展，将盖玻片固定在从DIV0（一旦细胞附着）到DIV28的多个时间点的4%多聚甲醛（PFA）中。如前 所述5使用三种不同的染色组合对培养物进行免疫荧光成像染色：NG2（未成熟的少突胶质细胞）和巢蛋白（神经元干/祖细胞）作为发育标志物，SMI31（轴突），MBP（髓磷脂）和NeuN（?…
Discussion
中枢神经系统是一个复杂的网络，从大脑到脊髓，由许多细胞类型组成，主要是神经元、少突胶质细胞、星形胶质细胞和小胶质细胞1。由于每个细胞在维持体内平衡和对CNS9，10，11中的挑战产生适当的反应方面都起着重要作用，因此包含所有这些细胞类型的培养系统是研究大脑如何对刺激做出反应的有用且通用的工?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢Edgar和Linington实验室的成员，特别是Chris Linington教授，Diana Arseni博士和Katja Muecklisch博士，感谢他们的建议，有用的评论，以及在我们建立这些文化时为文化提供营养的帮助。特别感谢Muecklisch博士为Cell Profiler管道提供了起点。这项工作得到了MS协会（赠款122）和尤里和洛娜·切尔纳约夫斯基基金会的支持;格拉斯哥大学资助JC和MP;以及惠康信托基金会（217093/Z/19/Z）和医学研究委员会（MRV0109721）给GJG。
Materials
| 10x Trypsin | Sigma | T4549-100ML | To digest tissue |
| 140 mm TC Dish | Fisher | 11339283 | Put 8 35 mm dishes per 1 140 mm dish |
| 15 mL Falcon | Sarstedt | 62554502 | To collect cells into pellet for resuspension in plating media |
| 18 G needle | Henke Sass Wolf | 4710012040 | For trituration of sample |
| 21 G needle | BD | 304432 | For trituration of sample |
| 23 G needle | Henke Sass Wolf | 4710006030 | For trituration of sample |
| 35 mm TC Dish | Corning | 430165 | Plate out 3 PLL coated coverslips per 1 35 mm dish |
| 5 mL syringe | Fisher | 15869152 | For trituration of sample |
| 6 well plate | Corning | 3516 | To plate out cells for RT-qPCR, and flow cytometry |
| 7 mL Bijoux | Fisher | DIS080010R | To put brains intp |
| 96 well plate | Corning | 3596 | To plate out cells for high-throughput testing |
| ACSA-2 Antibody, anti-mouse, PE | Miltenyi | 130-123-284 | For flow cytometry staining of astrocytes |
| Angled forceps | Dumont | 0108-5/45-PO | For dissection |
| Biotin | Sigma | B4501 | For DM+/- |
| Boric Acid | Sigma | B6768-500G | For boric acid buffer |
| Brilliant Violet 421 anti-mouse CD24 Antibody, clone M1-69 | Biolegend | 101825 | For flow cytometry staining of neurons and astrocytes |
| Brilliant Violet 605 anti-mouse CD45 Antibody, clone 30-F11 | Biolegend | 103139 | For flow cytometry staining of microglia |
| Brilliant Violet 785 anti-mouse/human CD11b Antibody, clone M1/70 | Biolegend | 101243 | For flow cytometry staining of microglia |
| BSA Fraction V | Sigma | A3059-10G | For SD Inhibitor |
| CNP | Abcam | AB6319 | Mature oligodendrocytes |
| Coverslip | VWR | 631-0149 | To plate out cells for microscopy |
| Dissection Scissors | Sigma | S3146-1EA | For dissection |
| DMEM High glucose, sodium pyruvate, L-Glutamine | Gibco | 21969-035 | For DM+/-, and for plating media |
| DNase I | Thermofisher | 18047019 | For SD Inhibitor, can use this or the other Dnase from sigma |
| DNase I | Sigma | D4263 | For SD Inhibitor, can use this or the other Dnase from thermofisher |
| eBioscience Fixable Viability Dye eFluor 780 | Thermofisher | 65-0865-14 | Live / Dead stain |
| Fine forceps | Dumont | 0102-SS135-PO | For dissection |
| GFAP | Invitrogen | 13-0300 | Astrocytes |
| HBSS w Ca Mg | Sigma | H9269-500ML | For plating media |
| HBSS w/o Ca Mg | Sigma | H9394-500ML | For brains to be added to |
| Horse Serum | Gibco | 26050-070 | For plating media |
| Hydrocortisone | Sigma | H0396 | For DM+/- |
| Iba1 | Alpha-Laboratories | 019-1971 | Microglia |
| Insulin | Sigma | I1882 | For DM+ |
| Leibovitz L-15 | GIbco | 11415-049 | For SD Inhibitor |
| MBP | Bio-Rad | MCA409S | Myelin |
| Mouse CCL5/RANTES DuoSet ELISA Kit | BioTechne | DY478-05 | ELISA kit for quantifying concentration of CCL5 in supernatants of 96 well plate |
| N1 media supplement | Sigma | N6530-5ML | For DM+/- |
| Nestin | Merck | MAB353 | Neuronal stem/progenitor cells |
| NeuN | Thermofisher | PA578499 | Neuronal cell body |
| NG2 | Sigma | AB5320 | Immature oligodendrocytes |
| O4 Antibody, anti-human/mouse/rat, APC | Miltenyi | 130-119-155 | For flow cytometry staining of oligodendrocytes |
| Pen/Strep | Sigma | P0781-100ML | For DM+/-, and for plating media |
| Poly-L-Lysinehydrobromide | Sigma | P1274 | For Boric acid / poly-L-lysine solution to coat coverslips |
| SMI31 | BioLegend | 801601 | Axons |
| Sodium Tetraborate | Sigma | 221732-100G | For boric acid buffer |
| Trizol | Thermofisher | 15596026 | For lysing cells for RT-qPCR |
| Trypsin inhibitor from soybean | Sigma | T9003-100MG | For SD Inhibitor |
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Cite This Article
Gamble, A., Suessmilch, M., Bonestroo, A., Merits, A., Graham, G. J., Cavanagh, J., Edgar, J. M., Pingen, M. Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity. J. Vis. Exp. (196), e65331, doi:10.3791/65331 (2023).