Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

Hannah Y. Collins; Christopher J. Bohlen

doi:10.3791/57122

Isolement et Culture des microglies rongeurs pour promouvoir une dynamique ramifié morphologie da...

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Neuroscience

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JoVE Journal Neuroscience

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

Isolement et Culture des microglies rongeurs pour promouvoir une dynamique ramifié morphologie dans un milieu sans sérum

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12:00 min

March 9, 2018

DOI: 10.3791/57122-v

Hannah Y. Collins¹, Christopher J. Bohlen¹

¹Department of Neurobiology,Stanford University

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Overview

This study presents a protocol for isolating and culturing microglia from developing or adult rodents. The method allows for the maintenance of highly pure microglial cultures without serum exposure, which supports investigations into microglial morphology and their interactions with other CNS cell types.

Key Study Components

Area of Science

Neuroscience
Cell Culture
Microglial Function

Background

The investigation of microglial function has been limited by a lack of effective culture models.
Mature in vivo microglia exhibit properties that are challenging to replicate in vitro.
Understanding microglial interactions is essential for insight into CNS functioning.
This protocol aims to overcome limitations by providing a defined-medium culture method.

Purpose of Study

To create a reliable method for isolating microglia.
To maintain the viability and functionality of cultured microglia in a defined medium.
To provide a foundation for studying microglial roles in neuroscience.

Methods Used

Cell culture protocol involving isolation and culture of microglia from rodent brains.
Utilized developing or adult rodents as biological models without serum exposure in culture.
The procedure includes careful brain removal, tissue dissociation, and cell isolation techniques.
Involves specific steps to preserve microglial viability during homogenization and filtration.
Adherence to panning dishes and careful trypsinization for subsequent cell recovery.

Main Results

Successfully isolated and cultured highly pure microglia from rodent tissue.
Maintained cellular morphology consistent with mature microglia.
Established methodology enables further study of microglial interactions and activation states.
Highlighted the protocol's potential to clarify microglial roles in health and disease.

Conclusions

This study demonstrates a novel approach for microglial isolation that enhances the understanding of their function.
The method's emphasis on serum-free culture supports the investigation of microglial biology more effectively.
Provides implications for future research in neuronal mechanisms and plasticity in the CNS.

Frequently Asked Questions

What are the advantages of using this isolation method?

The method allows for the generation of highly pure microglial cultures that can be maintained in defined medium without serum, enhancing the study of microglia.

How are the microglia prepared for culture?

Microglia are isolated from brains through careful dissection, tissue homogenization, and a series of centrifugations to remove debris.

What types of data can be obtained from these cultured microglia?

Data can include insights into microglial morphology, interactions with other CNS cells, and potential functional assays relevant to neuroinflammation.

Can this method be adapted for different ages of rodents?

Yes, the protocol is designed for use with developing or adult rodents, making it versatile for various research applications.

What are key considerations when implementing this protocol?

Attention to detail during tissue dissociation and bacterial contamination prevention are critical to ensure cell viability and culture success.

Efforts pour comprendre en détail le fonctionnement du microgliales ont été entravées par le manque de modèles de culture microgliales qui récapitule les propriétés des matures in vivo de cellules microgliales. Ce protocole décrit une démarche d’isolement et de la culture visant à maintenir la survie robuste des microglies rat mature très ramifié dans des conditions définies et moyen.

L’objectif global de ce protocole de culture cellulaire est de générer des cultures microgliales très pures de rongeurs en développement ou adultes, qui peuvent être maintenues sans exposition au sérum. Cette procédure peut aider à répondre à des questions clés en neurosciences liées à la morphologie de la microglie, à la cétose fibreuse et à l’interaction de la microglie avec d’autres types de cellules du SNC. Le principal avantage de ce protocole est que la microglie peut être rapidement isolée des tissus de rongeurs de tout âge et cultivée sans exposition au sérum.

Pour enlever le cerveau, coupez le condyle occipital de la moelle épinière d’un animal des deux côtés avec des ciseaux de dissection, en faisant attention de ne pas endommager le cerveau. Ensuite, coupez la peau le long du haut du crâne et coupez un côté le long de l’os pariétal et frontal vers l’os nasal. Tirez délicatement le haut du crâne vers l’arrière avec une pince, retirez rapidement le cerveau et placez-le dans du DPBS pré-refroidi sur de la glace.

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