JP acknowledges funding support from the Natural Sciences and Engineering Research Council of Canada and the University of Saskatchewan College of Medicine. YD acknowledges funding support from the University of Saskatchewan College of Medicine Startup Fund, the Natural Sciences and Engineering Research Council of Canada Discovery Grant (RGPIN-2023-03659), MS Canada Catalyst Grant (1019973), Saskatchewan Health Research Foundation Establishment Grant (6368), and Brain Canada Foundation Future Leaders in Canadian Brain Research Grant. Figure 1A, Figure 2A, and Figure 3A were created with BioRender.com.

All animals used in this study were housed and handled with approval from the University Animal Care Committee (UACC) of the University of Saskatchewan and the Canadian Council on Animal Care (CCAC). Post-natal days 0-2 CD1 male and female mice and&#160;embryonic days&#160;16-18 (E16-18) embryos from pregnant CD1 mice were used for this study. The details of the reagents and the equipment used are listed in the Table of Materials.
1. Primary microglia culture</p...

 Primary Microglia and Neuron Co-Culture Protocol for CNS Interaction Studies

This article describes a protocol for isolating and culturing mouse primary neurons and primary microglia, which are subsequently used to establish a microglia-neuronal co-culture that can be used to study how microglia and neuron interactions regulate their cellular health and function. This relatively simple and accessible approach can provide critical insights into the mechanisms and functional outcomes of microglia neuron interactions in the CNS.
To achieve an optimal co-culture, several c...

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.

<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

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

Author Spotlight: In Vitro Co-Culture Model for Studying Microglia-Neuronal Interactions in Disease Conditions

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

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.

A flowchart showing the key steps of the mixed glia&#160;culture for microglia is shown in Figure 1A. Overall, sparse cells and excessive cellular debris are expected on day 1 (Figure 1B). By day 4, increased cell number should be observed, especially with the generation of adherent astrocytes, as indicated by their elongated morphology (Figure 1C). A few microglia may be observed on top of the astrocytes or as small round cells flo...

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

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

485 vistas.  University of Saskatchewan. Nuestra investigación tiene como objetivo investigar las interacciones entre las neuronas y la microglía durante enfermedades como la esclerosis múltiple. Nuestro objetivo es recapitular las interacciones neuronales de la microglía in vitro para comprender mejor su importancia funcional en el SNC. Previamente demostramos que la fagocitosis de la microglía protege a las neuronas de la muerte celular mediada por fosfatidilcolina oxidada con este cocultivo....