Name: Författare i fokus: In vitro co-kulturmodell för att studera mikroglia-neuronala interaktioner vid sjukdomstillstånd
Uploaded: 2024-07-26T15:00:00
Description: Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS ...

JP erkänner finansieringsstöd från Natural Sciences and Engineering Research Council of Canada och University of Saskatchewan College of Medicine. YD erkänner finansieringsstöd från University of Saskatchewan College of Medicine Startup Fund, 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) och Brain Canada Foundation Future Leaders in Canadian Brain Research Grant. Figur 1A, Figur 2A och Figur 3A skapades med BioRender.com.

Alla djur som användes i denna studie inhystes och hanterades med godkännande från University Animal Care Committee (UACC) vid University of Saskatchewan och Canadian Council on Animal Care (CCAC). Efter förlossningen dag 0-2 CD1 han- och honmöss och embryonala dag 16-18 (E16-18) embryon från dräktiga CD1-möss användes för denna studie. Detaljerna för reagenserna och den utrustning som används listas i materialtabellen.
1. Primär odling av mikroglia...

 Primärt protokoll för samodling av mikroglia och neuron för studier av CNS-interaktion

Den här artikeln beskriver ett protokoll för att isolera och odla primära nervceller och primära mikroglia hos möss, som sedan används för att etablera en samkultur mellan mikroglia och neuron som kan användas för att studera hur interaktioner mellan mikroglia och neuroner reglerar deras cellulära hälsa och funktion. Detta relativt enkla och tillgängliga tillvägagångssätt kan ge viktiga insikter om mekanismerna och de funktionella resultaten av mikroglianeuroninteraktioner i CNS.
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Mikroglia är vävnadsresidenta makrofager som underlättar immunövervakning och homeostas i centrala nervsystemet (CNS)1,2,3. De härstammar från gulesäckar erytromyeloida stamceller som koloniserar hjärnan under embryonal utveckling 4,5,6 och bibehålls under hela organismens livscykel genom självförnyelse, vilket innebär proliferation och apoptos7. Vid steady-state har vilande mikroglia förgrenat morfologin och ägnar sig åt vävnadsövervakning 8,9,10.
Mikroglia uttrycker ett stort antal receptorer på cellytan, vilket gör det möjligt för dem att snabbt reagera på förändringar i CNS11,12 och att främja inflammatoriska reaktioner vid infektioner eller vävnadsskada 12,13,14, liksom vid neurodegenerativa sjukdomar 9,15, såsom multipel skleros (MS)16,17. Mikroglia uttrycker också receptorer till olika neurotransmittorer och neuropeptider 18,19,20, vilket tyder på att de också kan reagera på och reglera neuronal aktivitet21,22. Faktum är att mikroglia och neuroner interagerar i olika former av dubbelriktad kommunikation 8,23 såsom direkta interaktioner medierade av membranproteiner eller indirekta interaktioner genom lösliga faktorer eller intermediära celler23,24.
Till exempel kan olika neurotransmittorer som utsöndras av neuroner modulera den neuroprotektiva eller inflammatoriska aktiviteten hos mikroglia 25,26,27. Dessutom bidrar direkta interaktioner mellan neuroner och mikroglia till att upprätthålla mikroglia i ett homeostatiskt tillstånd28. Omvänt kan direkta interaktioner mellan mikroglia och neuroner forma neuronala kretsar29 och påverka neuronal signalering 30,31,32. Eftersom störningar i dessa interaktioner inducerar hyperexcitabilitet hos neuroner30 och mikrogliareaktivitet 33,34, är dysreglerade mikroglia-neuronala interaktioner inblandade som en bidragande faktor till neurologiska sjukdomar33,35. Faktum är att psykotiska23,26 och neurodegenerativa sjukdomar har beskrivits uppvisa dysfunktionella mikroglia-neuronala interaktioner33. Även om dessa observationer belyser vikten av mikroglia-neuronal kommunikation i CNS, är specifika mekanismer för hur dessa interaktioner reglerar mikroglia- och neuronala funktioner i hälsa och sjukdom relativt okända.
I en komplex miljö som CNS kan flera miljöfaktorer påverka interaktioner mellan mikroglia och neurona, vilket begränsar möjligheten att studera transienta cellulära interaktioner in vivo. Här presenterar vi ett in vitro mikroglia-neuronalt co-kultursystem som kan användas för att studera direkta cellulära interaktioner mellan mikroglia och neuroner. Detta protokoll beskriver genereringen av primära mikroglia och neuroner från hjärnbarken hos neonatala möss mellan postnatala dagar 0-2 respektive embryonala möss dag 16-18. Neuroner och mikroglia odlas sedan tillsammans i 96-brunnars plattor för nedströms experiment med hög genomströmning. Vi har tidigare använt detta tillvägagångssätt för att visa att mikroglia fagocytos skyddar nervceller från oxiderad fosfatidylkolinmedierad celldöd37, vilket tyder på att denna metod kan hjälpa till att förstå mikroglias roll i samband med neurodegeneration och MS. På samma sätt kan mikroglia-neuronala samkulturer också vara användbara för att undersöka effekten av mikroglia-neuronal överhörning i andra sammanhang, såsom virusinfektioner38 eller neuronal skada och reparation39. Sammantaget gör in vitro mikroglia-neuronala samkultursystem det möjligt för forskare att studera mikroglia-neuronala interaktioner i en manipulerbar och kontrollerad miljö, vilket kompletterar in vivo-modeller.

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			<td>10 cm Petri dish&#160;</td>
			<td>Fisher&#160;</td>
			<td>07-202-011</td>
			<td>Sterile</td>
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		<tr>
			<td>1x Versene</td>
			<td>Gibco</td>
			<td>15040-066</td>
			<td></td>
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			<td>B-27 Plus Neuronal Culture System&#160;</td>
			<td>Gibco&#160;</td>
			<td>A3653401</td>
			<td></td>
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			<td>Dissection microscope</td>
			<td>VWR</td>
			<td></td>
			<td></td>
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			<td>DNase I</td>
			<td>Roche</td>
			<td>11284932001</td>
			<td></td>
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			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11960-044</td>
			<td></td>
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			<td>Fetal Bovine Serum&#160;</td>
			<td>ThermoFisher Sci</td>
			<td>12483-020</td>
			<td></td>
		</tr>
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			<td>HBSS (10x)</td>
			<td>Gibco</td>
			<td>14065-056</td>
			<td></td>
		</tr>
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			<td>Hemacytometer</td>
			<td>Hausser Scientific</td>
			<td>1475</td>
			<td></td>
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			<td>HEPES&#160;</td>
			<td>ThermoFisher Sci</td>
			<td>15630080</td>
			<td></td>
		</tr>
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			<td>Leibovitz&#8217;s L-15 Medium (1x)</td>
			<td>Fisher Scientific&#160;</td>
			<td>21083027</td>
			<td></td>
		</tr>
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			<td>Macrophage colony stimulating factor&#160;</td>
			<td>Peprotech</td>
			<td>315-02</td>
			<td></td>
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			<td>Micro-Forceps</td>
			<td>RWD</td>
			<td>F11020-11</td>
			<td>Autoclaved/Sterile</td>
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			<td>Non-essential amino acids</td>
			<td>Cytiva</td>
			<td>SH3023801</td>
			<td></td>
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			<td>PBS (10x)</td>
			<td>ThermoFisher Sci</td>
			<td>AM9625</td>
			<td></td>
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			<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>
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			<td>Sodium pyruvate (100 mM)</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
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			<td>Spring scissors</td>
			<td>RWD</td>
			<td>S11008-42</td>
			<td>Autoclaved/Sterile</td>
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			<td>Surgical blade</td>
			<td>Feather</td>
			<td>08-916-5D</td>
			<td>Sterile</td>
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			<td>T-25 flasks</td>
			<td>Fisher</td>
			<td>10-126-9</td>
			<td></td>
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			<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>
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generating and co-culturing murine primary microglia and cortical neurons

Mikroglia är vävnadsresidenta makrofager i centrala nervsystemet (CNS), som utför många funktioner som stöder neuronal hälsa och CNS-homeostas. De är en stor population av immunceller associerade med CNS-sjukdomsaktivitet, och antar reaktiva fenotyper som potentiellt bidrar till neuronal skada under kroniska neurodegenerativa sjukdomar som multipel skleros (MS). De distinkta mekanismerna genom vilka mikroglia reglerar neuronal funktion och överlevnad under hälsa och sjukdom förblir begränsade på grund av utmaningar med att lösa de komplexa in vivo-interaktionerna mellan mikroglia, neuroner och andra CNS-miljöfaktorer. Således är in vitro-metoden att samodla mikroglia och neuroner fortfarande ett värdefullt verktyg för att studera mikroglia-neuronala interaktioner. Här presenterar vi ett protokoll för att generera och samodla primära mikroglia och neuroner från möss. Specifikt isolerades mikroglia efter 9-10 dagar in vitro från en blandad gliakultur etablerad från hjärnhomogenat från neonatala möss mellan postnatal dag 0-2. Neuronala celler isolerades från hjärnbarken hos musembryon mellan embryonal dag 16-18. Efter 4-5 dagar in vitro såddes neuronala celler i 96-brunnars plattor, följt av tillsats av mikroglia för att bilda samkulturen. Noggrann timing är avgörande för detta protokoll eftersom båda celltyperna måste nå experimentell mognad för att etablera samkulturen. Sammantaget kan denna samkultur vara användbar för att studera interaktioner mellan mikroglia och neuron och kan ge flera avläsningar, inklusive immunofluorescensmikroskopi, live-avbildning, samt RNA- och proteinanalyser.

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.

Författare i fokus: In vitro co-kulturmodell för att studera mikroglia-neuronala interaktioner vid sjukdomstillstånd

Generering och samodling av murina primära mikroglia och kortikala neuroner

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.

Ett flödesschema som visar de viktigaste stegen i den blandade gliakulturen för mikroglia visas i figur 1A. Sammantaget förväntas glesa celler och mycket cellulärt skräp på dag 1 (Figur 1B). Vid dag 4 bör ett ökat cellantal observeras, särskilt vid generering av vidhäftande astrocyter, vilket indikeras av deras långsträckta morfologi (Figur 1C). Ett fåtal mikroglia kan observeras ovanpå astrocyterna eller som små run...

Detta protokoll beskriver en mikroglia-neuronal samkultur etablerad från primära neuronala celler isolerade från musembryon vid embryonal dag 15-16 och primära mikroglia genererad från hjärnan hos neonatala möss vid postnatal dag 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

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.

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.

Brain Isolation from Neonatal Mice for Primary Microglia Culture

To begin, add 5 milliliters of 10 micrograms per milliliter Poly-L-ornithine hydrobromide to each T75 flask. Incubate at 37 degrees Celsius in a 5%carbon dioxide incubator for at least one hour. After decapitating the mouse, gently hold the head and create a midline incision in the cranium, starting from the neck to the nose.Peel the skull back with tissue forceps to reveal the brain. To scoop the brain out, insert the tissue forceps under the brain, and lift the brain out from the head. After placing the brain into a Petri dish containing 20 milliliters of Leibovitz's L-15 media under a dissection microscope, using a pair of micro-forceps, carefully remove the brainstem and the meninges.Collect the brains in a 50-milliliter conical tube containing 5 milliliters of Leibovitz's L15 media.

Hjärnisolering från neonatala möss för primär mikrogliaodling

Preparing and Maintaining Mixed Glia Culture Derived from Neonatal Mice Brains

To begin, mince the isolated brains into approximately one-square-millimeter pieces with a sterile disposable scalpel blade. Carefully transfer the minced brains into a new sterile 50 milliliter tube. Add a final concentration of 0.25%trypsin to the minced brains.Incubate the tissue in a 37-degrees-Celsius water bath for 20 minutes. Add five milliliters of complete glia culture media to neutralize the trypsin and gently mix the tissue suspension using a 10 milliliter pipette. Carefully decant the cell suspension through the cell strainer.Then remove the plunger from a sterile one-milliliter syringe and grind the tissue into the mesh using the plunger. Add equal volumes of cell suspension containing one to two brains with glia culture media to each flask resulting in a final volume of 15 milliliters. Sparse cells and excessive cellular debris were observed on day one.By day four, adherent astrocytes were generated. Confluent cells with typical astrocyte morphology were observed by day eight of culture.

Beredning och underhåll av blandad gliakultur härledd från neonatala mösshjärnor

Isolating Brains from E16-E18 Mouse Embryos for Primary Neuron Culture

Begin by coating T25 flasks with five milliliters of 10 micrograms per milliliter poly-L-ornithine, and incubate them. To isolate the brain from mouse embryos, lift the skin of the euthanized pregnant mouse's lower abdomen with tissue forceps. Using dissection scissors, perform a V-shaped incision from this point to the lower rib cage.Grab the uterine horns containing the embryos with tissue forceps, and gently remove the embryos from the abdominal cavity. Using spring scissors, create a midline incision at the top of the skull, starting from the back of the neck to the nose. Lift the skull away from the incision site to reveal the brain.Gently scoop out the brain with tissue forceps and transfer the brain to a 10 centimeter Petri dish containing 15 milliliters of Hanks'balanced salt solution. Under a dissection microscope, carefully remove the brainstem and the meninges on both sides of the brain cortices with a pair of micro forceps.

Isolering av hjärnor från E16-E18 musembryon för primär neuronodling

Brain Dissociation and Seeding of Cortical Neurons in Cell Culture 

To begin, mince the isolated brains into pieces in the 50-milliliter conical tube with a sterile disposable scalpel blade. Add trypsin to the tube at a final concentration of 0.25%Immerse the tube in a 35 to 38 degrees Celsius hot water bath for 15 minutes. Gently pipette the tissue suspension up and down to dissociate cells.Then add 0.5 milliliters of fetal bovine serum to the homogenized suspension to neutralize trypsin activity. Filter the cell suspension through the 70-micrometer nylon mesh cell strainer. Using a one-milliliter syringe plunger, gently grind the tissue into the cell strainer.Then centrifuge the conical tube at 300 g for five minutes at four degrees Celsius. Discard the Poly-L-Ornithine in the T25 flasks and rinse the flasks three times with five milliliters of PBS. Carefully decant the tube to remove the supernatant and resuspend the cell pellet by gently pipetting with two to three milliliters of complete neurobasal media.Plate the cells in the Poly-L-Ornithine-coated T25 flasks with approximately 20 million cells per flask in five milliliters of complete neurobasal media. Incubate the cell culture at 37 degrees Celsius in a 5%carbon dioxide incubator. Neurons in T25 flasks and 96-well plates adhered and formed neurite processes by day three.Suboptimal neuron cultures contained cell clumps and debris.

Hjärndissociation och sådd av kortikala neuroner i cellkultur 

Co-Culture of Primary Neurons and Microglia for Studying Microglia-Neuron Interactions

To begin, take neurons grown in T25 flasks for co-culture. Replace the media with five milliliters of versene solution containing trypsin and Dnase I.Add 0.5 milliliters of fetal bovine serum to neutralize the trypsin and Dnase I.Transfer cells to a 15 milliliter conical tube. Wash the flask with five milliliters of complete neurobasal media once to maximize the collected neurons.Then, centrifuge the cell suspension at 300 G for five minutes at four degrees celsius. After discarding the supernatant, gently resuspend the cell pellet in two to three milliliters of complete neurobasal media. Seed 100 microliters of the cell suspension to each well to obtain 75, 000 neurons per well, between eight to 10 days of mixed glia cultivation.Using a 10 milliliter pipette, gently wash the T75 flasks with glia culture media to collect microglia and transfer the cells and media into a sterile 50 milliliter conical tube. Centrifuge the cell collection at 300 G for five minutes at four degrees celsius. Once the supernatant is removed, gently resuspend the pellet in two milliliters of complete neurobasal media.From the 96-well neuronal culture plate, remove 100 microliters of neurobasal media and add 100 microliters of 250, 000 cells per milliliter cell suspension. In co-culture round and large microglia were observed on top or between neurons and neurite processes.