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Neuroscience
神経系発達中の軸の成長と挙動を解析する3Dコラーゲンベースのヒドロゲルの生成
神経系発達中の軸の成長と挙動を解析する3Dコラーゲンベースのヒドロゲルの生成
JoVE Journal
Neuroscience
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JoVE Journal Neuroscience
Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

神経系発達中の軸の成長と挙動を解析する3Dコラーゲンベースのヒドロゲルの生成

Full Text
6,241 Views
09:10 min
June 25, 2019

DOI: 10.3791/59481-v

Vanessa Gil1,2,3,4, José Antonio Del Río1,2,3,4

1Molecular and Cellular Neurobiotechnology, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST),Parc Científic de Barcelona, 2Department of Cell Biology, Physiology, and Immunology,Universitat de Barcelona, 3Center for Networked Biomedical Research on Neurodegenerative Diseases (CIBERNED), 4Institute of Neuroscience,University of Barcelona

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Overview

This study provides a method for analyzing the behavior of growing axons in three-dimensional matrices that simulate their natural development. The protocol focuses on understanding axonal guidance and neuronal migration during neurodevelopment, enabling researchers to observe the influence of specific molecules.

Key Study Components

Area of Science

  • Neuroscience
  • Neurodevelopment
  • Axon guidance

Background

  • The study emphasizes the importance of axons and their growth in a controlled environment.
  • It highlights the complexity of neuronal migration during brain development.
  • The analysis seeks to disentangle the molecular influences on axonal behavior.

Purpose of Study

  • To analyze the role of specific molecules in axonal guidance.
  • To understand neuronal migration processes in a 3D environment.
  • To provide a quick and accessible method for researchers to utilize.

Methods Used

  • The study employs transfected COS-1 cells embedded in collagen matrices to model axonal growth.
  • Embryonic rat brain tissue is utilized for additional neuronal components.
  • The method emphasizes practicality with minimal software requirements.
  • Molecular interactions are observed through time-sensitive incubation in controlled conditions.

Main Results

  • The method allows for the straightforward observation of axonal behavior influenced by specific molecular cues.
  • It provides insights into the plasticity of developing neurons in response to guided growth.
  • Validation of the method demonstrates its efficacy in monitoring neuronal responses.

Conclusions

  • This study enables researchers to better understand the mechanisms of axonal development and guidance.
  • It simplifies the experimental approach to studying neuronal migration.
  • The implications of this work extend to improving our understanding of neurodevelopmental disorders.

Frequently Asked Questions

What are the advantages of using a 3D matrix for axonal studies?
3D matrices mimic the natural environment for axons, allowing for more realistic growth patterns and interactions compared to traditional 2D cultures.
How is the COS-1 cell line utilized in this research?
COS-1 cells are transfected with candidate molecules to study their effects on axonal guidance in a collagen matrix.
What types of data can be obtained from this method?
Researchers can observe axonal growth patterns, molecular influences, and neuronal migration behaviors in a controlled setup.
Can this method be adapted for other neuronal models?
Yes, the method can be tailored to incorporate different neuronal tissue types or additional experimental variables.
What limitations should be considered when using this technique?
Potential limitations include the need for precise cell culture techniques and the requirement for appropriate biological materials.

ここでは、3Dマトリックスで軸を成長させる行動を分析し、その自然な発達を模倣する方法を提供する。

この方法は、神経系の発達中に軸索誘導および神経移動におけるいくつかの分子の役割を分析するために有用である。この手法の主な利点は、結果の分析が迅速かつ簡単であり、研究者のための多くのトレーニングを意味する複雑なソフトウェアを必要としないということです。この手順のデモンストレーションは、私たちの研究室と私の技術者であるミリアン・セグラ=フェリウです。

プレート200、000 COS-1細胞を35ミリメートルのペトリ皿に開始し、一晩70〜80%のコンフルエンスを読み取るために細胞培養インキュベーターで完全な培養培地でインキュベートする。翌日、リポソームベーストランスフェクション法を用いて候補分子をコードするDNAを用いてCOS-1細胞をトランスフェクトする。まず、250マイクロリットルの無血清培地と1~2マイクログラムのDNAを1.5ミリリットル遠心管に混合し、室温で5分間インキュベートします。

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