Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For <em>In Vitro</em> Assays and Transcriptomic Studies

Julie J. Ahn; Robert H. Miller; Yusra Islam

doi:10.3791/65893

Isolamento de Astrócitos Puros e Microglia da Medula Espinhal de Camundongos Adultos para Ensaios...

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JoVE Journal Neuroscience

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Isolamento de Astrócitos Puros e Microglia da Medula Espinhal de Camundongos Adultos para Ensaios In Vitro e Estudos Transcriptômicos

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06:45 min

October 20, 2023

DOI: 10.3791/65893-v

Julie J. Ahn¹, Robert H. Miller¹, Yusra Islam¹

¹Department of Anatomy and Cell Biology,The George Washington University School of Medicine

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Overview

This study characterizes cell populations in the central nervous system, focusing on isolating astrocytes and microglia from the adult mouse spinal cord. Using a refined protocol, the research enables subsequent applications like RNA analysis and cell culture, addressing challenges in studying spinal cord pathologies.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Pathology

Background

Isolation of cells from the spinal cord is challenging due to its complex matrix.
A focus on viable microglia and astrocytes aids in understanding spinal cord diseases.
Existing methods often struggle with yielding high-quality isolated cells.
This protocol specifically tackles the isolation of glial cells without involving the brain.

Purpose of Study

To explore how astrocytes and microglia are affected under pathological conditions.
To facilitate in vitro research for spinal cord-related diseases and fill research gaps.
To develop a method for isolating these cell types from the adult mouse spinal cord.

Methods Used

The primary method is the enzymatic dissociation of spinal cord tissues, followed by cell sorting.
The biological model used is the adult mouse spinal cord, specifically targeting astrocytes and microglia.
No multiomics workflow was mentioned.
Critical steps include careful tissue handling, use of specific enzyme mixes, and density centrifugation.
Cell sorting achieved 92-93% viability of the isolated cultures.

Main Results

Successfully isolated microglia and astrocytes exhibiting viability and cellular responses.
By day four, distinct morphological changes were observed in both cell types.
Astrocytes formed a connected confluent layer, with microglia showing fewer processes.
Confirmation of high cell viability indicates the protocol's effectiveness.

Conclusions

The study demonstrates a reliable method for isolating specific glial cell populations from the spinal cord.
This advancement enables detailed studies of spinal cord pathology at a cellular level.
The findings have implications for understanding disease mechanisms in the spinal cord.

Frequently Asked Questions

What are the advantages of this isolation protocol?

The protocol allows for efficient isolation of viable astrocytes and microglia, minimizing damage to the spinal cord and enhancing research on spinal pathologies.

How is the spinal cord prepared for cell isolation?

The spinal cord is perfused, dissected, and treated with enzyme mixes to facilitate cellular dissociation while maintaining cell viability.

What types of outcomes can be observed from the isolated cells?

Outcomes include cell morphology, viability, and the ability to culture astrocytes and microglia for further analysis.

Can the method be adapted for other types of neural cells?

While this protocol focuses on astrocytes and microglia, adaptations may be possible to isolate other cell types with appropriate adjustments.

What are some limitations of this method?

Potential limitations include the complexity of tissue handling and the need for specific reagents, which may limit generalizability.

How do the results contribute to understanding spinal cord diseases?

The results provide insights into the cellular dynamics of astrocytes and microglia in disease contexts, enhancing the understanding of spinal cord pathologies.

Este protocolo descreve o isolamento de astrócitos purificados e microglia da medula espinhal de camundongos adultos, facilitando aplicações subsequentes, como análise de RNA e cultura celular. Inclui métodos e procedimentos detalhados de dissociação celular projetados para melhorar a qualidade e o rendimento das células isoladas.

Nossa pesquisa visa caracterizar populações celulares específicas no sistema nervoso central, especificamente na medula espinhal, utilizando modelos pré-clínicos de doença. E estamos tentando determinar como essas células são afetadas em condições patológicas e em diferentes pontos de tempo. Pode ser desafiador analisar células, especialmente no sistema nervoso central, porque as células do cérebro e da medula espinhal formam uma matriz altamente complexa e tudo está muito bem conectado, então pode ser difícil isolar essas células sem danificar o sistema.

Nosso protocolo aborda as limitações nos métodos existentes para estudar doenças da medula espinhal, isolando eficientemente astrócitos viáveis da microglia da medula espinhal de camundongos adultos ricos em mielina. Isso permite a pesquisa in vitro sobre doenças relacionadas à medula espinhal na análise a jusante, preenchendo uma lacuna crucial de pesquisa. Este protocolo apresenta várias vantagens, como a análise de astrócitos e microglia em condições patológicas e em momentos específicos.

Ele também se concentra exclusivamente em isolar as células gliais desta medula espinhal adulta sozinho, excluindo o envolvimento cerebral. Isso permite que os pesquisadores estudem patologias da medula espinhal em nível celular.

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