Name: Autor destacado: Modelo de cocultivo in vitro para el estudio de las interacciones microglía-neuronas en enfermedades
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 agradece el apoyo financiero del Consejo de Investigación de Ciencias Naturales e Ingeniería de Canadá y la Facultad de Medicina de la Universidad de Saskatchewan. YD agradece el apoyo financiero del Fondo de Inversión de la Facultad de Medicina de la Universidad de Saskatchewan, la Subvención de Descubrimiento del Consejo de Investigación de Ciencias Naturales e Ingeniería de Canadá (RGPIN-2023-03659), la Subvención Catalyst de MS Canada (1019973), la Subvención de Establecimiento de la Fundación de Investigación en Salud de Saskatchewan (6368) y la Subvención de Investigación del Cerebro de Futuros Líderes en el Cerebro Canadiense de la Fundación Brain Canada. Las figuras 1A, 2A y 3A se crearon con BioRender.com.

Todos los animales utilizados en este estudio fueron alojados y manejados con la aprobación del Comité Universitario de Cuidado de Animales (UACC) de la Universidad de Saskatchewan y el Consejo Canadiense de Cuidado de Animales (CCAC). Para este estudio se utilizaron ratones macho y hembra CD1 de los días postnatales 0-2 y embriones de los días 16-18 (E16-18) de ratones CD1 preñados. Los detalles de los reactivos y el equipo utilizado se enumeran en la Tabla de Materiales.
...

 Protocolo de cocultivo primario de microglía y neurona para estudios de interacción con el SNC

Este artículo describe un protocolo para aislar y cultivar neuronas primarias y microglía primaria de ratón, que posteriormente se utilizan para establecer un cocultivo microglía-neurona que se puede utilizar para estudiar cómo las interacciones de la microglía y las neuronas regulan su salud y función celular. Este enfoque relativamente simple y accesible puede proporcionar información crítica sobre los mecanismos y los resultados funcionales de las interacciones de las neuronas de la microglía en el SNC.
...

Las microglías son macrófagos residentes en los tejidos que facilitan la inmunovigilancia y la homeostasis en el sistema nervioso central (SNC)1,2,3. Se originan a partir de células progenitoras eritromieloides del saco vitelino que colonizan el cerebro durante el desarrollo embrionario 4,5,6 y se mantienen a lo largo de la vida del organismo a través de la autorrenovación, que implica proliferación y apoptosis7. En estado estacionario, la microglía en reposo tiene una morfología ramificada y realiza vigilancia tisular 8,9,10.
La microglía expresa numerosos receptores en la superficie celular, lo que les permite responder rápidamente a los cambios en el SNC11,12 y promover respuestas inflamatorias en caso de infecciones o lesiones tisulares 12,13,14, así como durante enfermedades neurodegenerativas 9,15, como la esclerosis múltiple (EM)16,17. La microglía también expresa receptores a varios neurotransmisores y neuropéptidos 18,19,20, lo que sugiere que también pueden responder y regular la actividad neuronal 21,22. De hecho, la microglía y las neuronas interactúan en diversas formas de comunicación bidireccional 8,23 como las interacciones directas mediadas por proteínas de membrana o las interacciones indirectas a través de factores solubles o células intermedias23,24.
Por ejemplo, varios neurotransmisores secretados por las neuronas pueden modular la actividad neuroprotectora o inflamatoria de la microglía 25,26,27. Además, las interacciones directas entre las neuronas y la microglía ayudan a mantener la microglía en un estado homeostático28. Por el contrario, las interacciones directas de la microglía con las neuronas pueden dar forma a los circuitos neuronales29 e influir en la señalización neuronal 30,31,32. Dado que las interrupciones de estas interacciones inducen hiperexcitabilidad de las neuronas30 y reactividad de la microglía33,34, las interacciones microglía-neuronas desreguladas están implicadas como un factor contribuyente a las enfermedades neurológicas33,35. De hecho, se ha descrito que las enfermedades psicóticas23,26 y neurodegenerativas presentan interacciones disfuncionales entre la microglía y las neuronas33. Si bien estas observaciones resaltan la importancia de la comunicación microglía-neurona en el SNC, los mecanismos específicos de cómo estas interacciones regulan las funciones microgliales y neuronales en la salud y la enfermedad son relativamente desconocidos.
Dentro de un entorno complejo como el SNC, múltiples factores ambientales pueden influir en las interacciones microglía-neurona, lo que limita la capacidad de estudiar las interacciones celulares transitorias in vivo. Aquí, presentamos un sistema de cocultivo in vitro de microglía-neurona que se puede utilizar para estudiar las interacciones celulares directas entre la microglía y las neuronas. Este protocolo describe la generación de microglía primaria y neuronas a partir de las cortezas de ratones neonatos entre los días postnatales 0-2 y los días 16-18 de ratones embrionarios, respectivamente. A continuación, las neuronas y la microglía se cultivan conjuntamente en placas de 96 pocillos para realizar experimentos posteriores de alto rendimiento. Anteriormente utilizamos este enfoque para demostrar que la fagocitosis de la microglía protege a las neuronas de la muerte celular mediada por fosfatidilcolina oxidada37, lo que sugiere que este método puede ayudar a comprender las funciones de la microglía en el contexto de la neurodegeneración y la EM. Del mismo modo, los cocultivos microglía-neuronal también pueden ser útiles para investigar el impacto de la diafonía microglía-neurona en otros contextos, como las infecciones virales38 o las lesiones y reparaciones neuronales39. En general, los sistemas de cocultivo in vitro microglía-neurona permiten a los investigadores estudiar las interacciones microglía-neurona en un entorno manipulable y controlado, que complementa los modelos in vivo.

<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

Las microglías son macrófagos residentes en los tejidos del sistema nervioso central (SNC), que realizan numerosas funciones que apoyan la salud neuronal y la homeostasis del SNC. Son una población importante de células inmunitarias asociadas con la actividad de la enfermedad del SNC, que adoptan fenotipos reactivos que potencialmente contribuyen a la lesión neuronal durante enfermedades neurodegenerativas crónicas como la esclerosis múltiple (EM). Los distintos mecanismos por los cuales la microglía regula la función neuronal y la supervivencia durante la salud y la enfermedad siguen siendo limitados debido a los desafíos para resolver las complejas interacciones in vivo entre la microglía, las neuronas y otros factores ambientales del SNC. Por lo tanto, el enfoque in vitro de co-cultivo de microglía y neuronas sigue siendo una herramienta valiosa para estudiar las interacciones microglía-neurona. Aquí, presentamos un protocolo para generar y co-cultivar microglía primaria y neuronas a partir de ratones. Específicamente, se aislaron microglía después de 9-10 días in vitro a partir de un cultivo de glía mixta establecido a partir de homogeneizados cerebrales derivados de ratones neonatos entre los días postnatales 0-2. Las células neuronales se aislaron de las cortezas cerebrales de embriones de ratón entre los días embrionarios 16-18. Después de 4-5 días in vitro, las células neuronales se sembraron en placas de 96 pocillos, seguidas de la adición de microglía para formar el cocultivo. El tiempo cuidadoso es crítico para este protocolo, ya que ambos tipos de células deben alcanzar la madurez experimental para establecer el cocultivo. En general, este cocultivo puede ser útil para estudiar las interacciones entre la microglía y las neuronas y puede proporcionar múltiples lecturas, incluida la microscopía de inmunofluorescencia, imágenes en vivo, así como ensayos de ARN y proteínas.

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.

Autor destacado: Modelo de cocultivo in vitro para el estudio de las interacciones microglía-neuronas en enfermedades

Generación y cocultivo de microglía primaria murina y neuronas corticales

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.

En la Figura 1A se muestra un diagrama de flujo que muestra los pasos clave del cultivo de glía mixta para microglía. En general, se esperan células dispersas y desechos celulares excesivos en el día 1 (Figura 1B). Para el día 4, se debe observar un aumento en el número de células, especialmente con la generación de astrocitos adherentes, como lo indica su morfología alargada (Figura 1C). Se pueden observar algunas microgl?...

Este protocolo describe un cocultivo microglía-neuronal establecido a partir de células neuronales primarias aisladas de embriones de ratón en los días embrionarios 15-16 y microglía primaria generada a partir de los cerebros de ratones neonatos en los días postnatales 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.

Investigación

Neurociencias

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.

Aislamiento cerebral de ratones neonatos para cultivo primario de microglía

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.

Preparación y mantenimiento de un cultivo mixto de glía derivado de cerebros de ratones neonatos

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.

Aislamiento de cerebros de embriones de ratón E16-E18 para el cultivo de neuronas primarias

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.

Disociación cerebral y siembra de neuronas corticales en cultivo celular 

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.