JP 感谢加拿大自然科学与工程研究委员会和萨斯喀彻温大学医学院的资金支持。YD 感谢萨斯喀彻温大学医学院启动基金、加拿大自然科学与工程研究委员会发现补助金 （RGPIN-2023-03659）、MS Canada 催化剂补助金 （1019973）、萨斯喀彻温省健康研究基金会建立补助金 （6368） 和 Brain Canada Foundation 加拿大脑研究未来领袖补助金的资金支持。 图 1A、 图 2A 和 图 3A 是使用 BioRender.com 创建的。

本研究中使用的所有动物均经萨斯喀彻温大学动物护理委员会 （UACC） 和加拿大动物护理委员会 （CCAC） 批准进行饲养和处理。本研究使用怀孕 CD1 小鼠出生后 0-2 天 CD1 雄性和雌性小鼠以及胚胎第 16-18 天 （E16-18） 胚胎。材料 表中列出了试剂和所用设备的详细信息。
1. 原代小胶质细胞培养
注意：对混合神经胶质细胞和神经元培养...

 用于 CNS 相互作用研究的原代小胶质细胞和神经元共培养方案

作者声明没有利益冲突。

本文介绍了一种分离和培养小鼠原代神经元和原代小胶质细胞的方案，随后用于建立小胶质细胞-神经元共培养物，可用于研究小胶质细胞和神经元相互作用如何调节其细胞健康和功能。这种相对简单易行的方法可以为 CNS 中小胶质细胞神经元相互作用的机制和功能结果提供关键见解。
为了实现最佳的共培养，该方案中的几个关键方面需要格外小心。在脑分离期间（步骤 1.2.6 ?...

小胶质细胞是组织驻留的巨噬细胞，可促进中枢神经系统 （CNS） 的免疫监视和稳态1,2,3。它们起源于卵黄囊红髓样祖细胞，在胚胎发育过程中定植于大脑 4,5,6，并通过自我更新（涉及增殖和细胞凋亡）在生物体的整个生命周期中维持7。在稳态时，静息小胶质细胞分化形态并参与组织监测 8,9,10。
小胶质细胞表达许多细胞表面受体，这使它们能够快速响应 CNS 的变化11,12，并在感染或组织损伤的情况下促进炎症反应 12,13,14，以及在神经退行性疾病期间 9,15，如多发性硬化症 （MS）16,17.小胶质细胞还表达各种神经递质和神经肽的受体 18,19,20，这表明它们也可能响应并调节神经元活动 21,22。事实上，小胶质细胞和神经元以各种形式的双向通讯相互作用 8,23，例如由膜蛋白介导的直接相互作用或通过可溶性因子或中间细胞的间接相互作用23,24。
例如，神经元分泌的各种神经递质可以调节小胶质细胞的神经保护或炎症活性 25,26,27。此外，神经元和小胶质细胞之间的直接相互作用有助于将小胶质细胞维持在稳态28。相反，小胶质细胞与神经元的直接相互作用可以塑造神经元回路29 并影响神经元信号转导 30,31,32。由于这些相互作用的破坏会诱导神经元的过度兴奋30 和小胶质细胞反应性33,34，失调的小胶质细胞-神经元相互作用被认为是神经系统疾病的一个促成因素33,35。事实上，精神病23,26 和神经退行性疾病已被描述为表现出功能失调的小胶质细胞-神经元相互作用33。虽然这些观察结果强调了小胶质细胞-神经元通讯在 CNS 中的重要性，但这些相互作用如何调节健康和疾病中小胶质细胞和神经元功能的具体机制相对未知。
在 CNS 等复杂环境中，多种环境因素会影响小胶质细胞-神经元相互作用，从而限制了研究 体内瞬时细胞相互作用的能力。在这里，我们提出了一种 体外 小胶质细胞-神经元共培养系统，可用于研究小胶质细胞和神经元之间的直接细胞相互作用。该方案分别描述了出生后第 0-2 天和胚胎小鼠第 16-18 天之间从新生小鼠皮层产生原代小胶质细胞和神经元。然后将神经元和小胶质细胞在 96 孔板中共培养，用于下游高通量实验。我们之前使用这种方法来证明小胶质细胞吞噬作用保护神经元免受氧化磷脂酰胆碱介导的细胞死亡37，这表明这种方法可以帮助理解小胶质细胞在神经变性和 MS 中的作用。同样，小胶质细胞-神经元共培养也可能有助于研究小胶质细胞-神经元串扰在其他情况下的影响，例如病毒感染38 或神经元损伤和修复39。总体而言， 体外小 胶质细胞-神经元共培养系统使研究人员能够在可操作和受控的环境中研究小胶质细胞-神经元相互作用，这与 体内 模型相辅相成。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cm Petri dish&#160;</td>
			<td>Fisher&#160;</td>
			<td>07-202-011</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>1x Versene</td>
			<td>Gibco</td>
			<td>15040-066</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Plus Neuronal Culture System&#160;</td>
			<td>Gibco&#160;</td>
			<td>A3653401</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Roche</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11960-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>ThermoFisher Sci</td>
			<td>12483-020</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (10x)</td>
			<td>Gibco</td>
			<td>14065-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Hausser Scientific</td>
			<td>1475</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES&#160;</td>
			<td>ThermoFisher Sci</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#8217;s L-15 Medium (1x)</td>
			<td>Fisher Scientific&#160;</td>
			<td>21083027</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrophage colony stimulating factor&#160;</td>
			<td>Peprotech</td>
			<td>315-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Forceps</td>
			<td>RWD</td>
			<td>F11020-11</td>
			<td>Autoclaved/Sterile</td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Cytiva</td>
			<td>SH3023801</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10x)</td>
			<td>ThermoFisher Sci</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin Glutamine (100x)</td>
			<td>Gibco</td>
			<td>103780-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide&#160;</td>
			<td>Sigma</td>
			<td>P3655-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate (100 mM)</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring scissors</td>
			<td>RWD</td>
			<td>S11008-42</td>
			<td>Autoclaved/Sterile</td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Feather</td>
			<td>08-916-5D</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>T-25 flasks</td>
			<td>Fisher</td>
			<td>10-126-9</td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 flasks&#160;</td>
			<td>Fisher</td>
			<td>13-680-65</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue forceps</td>
			<td>Codman</td>
			<td>30-4218</td>
			<td>Autoclaved/Sterile</td>
		</tr>
		<tr>
			<td>Tissue scissors</td>
			<td>RWD</td>
			<td>S12052-10</td>
			<td>Autoclaved/Sterile</td>
		</tr>
		<tr>
			<td>Trypan Blue&#160;</td>
			<td>Thermofisher Sci&#160;</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin (2.5%)</td>
			<td>ThermoFisher Sci</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>Widefield Immunofluorescence Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generating and co-culturing murine primary microglia and cortical neurons

小胶质细胞是中枢神经系统 （CNS） 的组织驻留巨噬细胞，执行支持神经元健康和 CNS 稳态的许多功能。它们是与 CNS 疾病活动相关的免疫细胞的主要群体，采用反应性表型，在慢性神经退行性疾病（如多发性硬化症 （MS））期间可能导致神经元损伤。由于解决小胶质细胞、神经元和其他 CNS 环境因素之间复杂的 体内 相互作用的挑战，小胶质细胞在健康和疾病期间调节神经元功能和存活的不同机制仍然有限。因此，共培养小胶质细胞和神经元的 体外 方法仍然是研究小胶质细胞-神经元相互作用的宝贵工具。在这里，我们提出了一种从小鼠生成和共培养原代小胶质细胞和神经元的方案。具体来说，在出生后 0-2 天之间，从来自新生小鼠脑匀浆的混合胶质细胞培养物 中 分离出小胶质细胞。在胚胎第 16-18 天之间从小鼠胚胎的脑皮层中分离出神经元细胞。 体外 4-5 天后，将神经元细胞接种在 96 孔板中，然后加入小胶质细胞以形成共培养物。仔细的时间安排对于该方案至关重要，因为两种细胞类型都需要达到实验成熟度才能建立共培养。总体而言，这种共培养可用于研究小胶质细胞-神经元相互作用，并且可以提供多种读数，包括免疫荧光显微镜检查、实时成像以及 RNA 和蛋白质检测。

Microglia are tissue-resident macrophages that facilitate immunosurveillance and homeostasis in the central nervous system (CNS)1,2,3. They originate from yolk sac erythromyeloid progenitor cells that colonize the brain during embryonic development4,5,6&#160;and are maintained throughout the organism&#39;s life span through self-renewal, which involves proliferation and apoptosis7. At steady-state, resting microglia have ramified morphology and engage in tissue surveillance8,9,10.
Microglia express numerous cell-surface receptors, which enables them to rapidly respond to changes in the CNS11,12 and to promote inflammatory responses in the event of infections or tissue injury12,13,14, as well as during neurodegenerative diseases9,15, such as&#160;multiple sclerosis (MS)16,17. Microglia also express receptors to various neurotransmitters and neuropeptides18,19,20, which suggests they may also respond to and regulate neuronal activity21,22. Indeed, microglia and neurons interact in various forms of bidirectional communication8,23 such as direct interactions mediated by membrane proteins or indirect interactions through soluble factors or intermediate cells23,24.
For instance, various neurotransmitters secreted by neurons can modulate the neuroprotective or inflammatory activity of microglia25,26,27. Additionally, direct interactions between neurons and microglia help to maintain microglia in a homeostatic state28. Conversely, direct interactions of microglia with neurons can shape neuronal circuitry29 and influence neuronal signaling30,31,32. As disruptions of these interactions induce hyperexcitability of neurons30 and&#160;microglia reactivity33,34, dysregulated microglia-neuronal interactions&#160;are implicated&#160;as a contributing factor to&#160;neurological diseases33,35. Indeed, psychotic23,26 and neurodegenerative diseases have been described to exhibit dysfunctional microglia-neuronal interactions33. While these observations highlight the importance of microglia-neuronal communication in the CNS, specific mechanisms of how these interactions regulate microglial and neuronal functions in health and disease are relatively unknown.
Within a complex milieu such as the CNS, multiple environmental factors can influence microglia-neuronal interactions, which limits the ability to study transient cellular interactions in vivo. Here, we present an in vitro microglia-neuronal co-culture system that can be used to study direct cellular interactions between microglia and neurons. This protocol describes the generation of primary microglia and neurons from the cortices of neonatal mice between post-natal days 0-2 and embryonic mice days 16-18, respectively. Neurons and microglia are then co-cultured in 96-well plates for downstream high-throughput experiments. We previously used this approach to demonstrate that microglia phagocytosis protects neurons from oxidized phosphatidylcholine mediated cell death37, suggesting that this method can help to understand the roles of microglia in the context of neurodegeneration and MS. Similarly, microglia-neuronal co-cultures may also be useful for investigating the impact of microglia-neuronal crosstalk in other contexts such as viral infections38 or neuronal injury and repair39. Overall, in vitro microglia-neuronal co-culture systems&#160;enable researchers to study microglia-neuronal interactions in a manipulatable and controlled environment, which complements in vivo models.

作者聚焦：用于研究疾病条件下小胶质细胞-神经元相互作用的体外共培养模型

生成和共培养小鼠原代小胶质细胞和皮质神经元

Our research aims to investigate the interactions between neurons and microglia during disease conditions such as multiple sclerosis. We aim to recapitulate microglia neuronal interactions in vitro to better understand their functional importance in the CNS. We previously demonstrated that microglia phagocytosis protects neurons from oxidized phosphatidylcholine-mediated cell death with this co-culture.While other co-culture systems are also employed, this mixed culture system enables direct and close contact between microglia and neurons. In addition, it can remain healthy from 12 days to potentially three weeks, allowing for long-term studies of neuronal microglia interactions. Our protocol offers a simple and accessible method to co-culture microglia neurons that are easily manipulatable and can be further used in downstream assays to determine cellular functional changes.

图 1A 显示了小胶质细胞混合胶质细胞培养关键步骤的流程图。总体而言，预计第 1 天会出现稀疏的细胞和过多的细胞碎片（图 1B）。到第 4 天，应观察到细胞数量增加，尤其是贴壁星形胶质细胞的产生，如其细长的形态所示（图 1C）。可以在星形胶质细胞顶部观察到一些小胶质细胞，或者作为漂浮在培养基中的小圆形细胞。到第...

该方案描述了从胚胎第 15-16 天从小鼠胚胎中分离的原代神经元细胞和出生后第 1-2 天从新生小鼠大脑产生的原代小胶质细胞建立的小胶质细胞-神经元共培养物。

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS ...

generating-co-culturing-murine-primary-microglia-cortical

Research

JoVE Journal

Neuroscience

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

523 Views.  University of Saskatchewan. Our research aims to investigate the interactions between neurons and microglia during disease conditions such as multiple sclerosis. We aim to recapitulate microglia neuronal interactions in vitro to better understand their functional importance in the CNS. We previously demonstrated that microglia phagocytosis protects neurons from oxidized phosphatidylcholine-mediated cell death with this co-culture.While other co-culture systems are also employed, this mixed culture system enables ...