Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

Kaede Saito; Yuta Hiraike; Misato Oguchi; Toshimasa Yamauchi

doi:10.3791/65265

JoVE Journal
Biology

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JoVE Journal Biology

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

組織解離剤を用いたマウス白色脂肪組織からの間質血管画分の半自動単離(英語)

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06:08 min

May 19, 2023

DOI: 10.3791/65265-v

Kaede Saito^1,2, Yuta Hiraike^1,2,3, Misato Oguchi², Toshimasa Yamauchi²

¹Division for Health Service Promotion,The University of Tokyo, ²Department of Diabetes and Metabolic Diseases, Graduate School of Medicine,The University of Tokyo, ³The University of Tokyo Excellent Young Researcher Program,The University of Tokyo

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Overview

This study focuses on the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue, with the aim of obtaining preadipocytes and promoting adipocyte differentiation in vitro. A novel protocol utilizing a tissue dissociator for collagenase digestion is presented, which enhances reproducibility and reduces experimental variation.

Key Study Components

Research Area

Adipocyte differentiation
Energy expenditure in obesity treatments
Murine models for metabolic research

Background

Different functions of brown, beige, and white adipocytes
Need for reproducible methods in adipocyte research
Identification of critical transcription factors regulating differentiation

Methods Used

Semi-automated isolation using collagenase digestion
Murine adipose tissue
Tissue dissociators and enzymatic assays

Main Results

Improved methods for isolating SVF and differentiating adipocytes
Successful differentiation demonstrated via Oil Red O staining and mRNA analysis
Investigation into the effects of Rosiglitazone on brown fat-specific gene expression

Conclusions

This protocol facilitates reliable SVF isolation for adipocyte differentiation.
The findings hold significant implications for obesity and diabetes research.

Frequently Asked Questions

What is the significance of the stromal vascular fraction in adipocyte research?

The SVF is crucial for isolating preadipocytes, which are necessary for studying adipocyte development and differentiation.

How does the tissue dissociator improve the isolation process?

It reduces variability and contamination during collagenase digestion, leading to more reproducible results.

What specific outcomes were measured to confirm adipocyte differentiation?

Outcomes included Oil Red O staining and mRNA expression levels of adipogenesis regulators.

What are the potential applications of the differentiated adipocytes?

They can be used for functional and mechanistic analyses, including oxygen consumption rate analysis.

How might this research contribute to obesity treatments?

By promoting energy expenditure rather than suppressing intake, new obesity treatments can be developed based on these findings.

What role does the transcription factor NFIA play in this study?

NFIA is identified as a critical regulator of adipocyte differentiation and metabolism.

このプロトコルは、マウス脂肪組織から間質血管画分(SVF)を半自動分離して、脂肪前駆細胞を取得し、 in vitroで脂肪細胞分化を達成することを説明しています。コラゲナーゼ消化に組織解離剤を使用すると、実験の変動が減少し、再現性が向上します。

私たちは、熱の形でエネルギーを放散する脂肪細胞の茶色とベージュのサブタイプに興味を持っていますが、白い脂肪細胞は一般的にエネルギーを蓄えます私たちの目標は、エネルギー摂取の抑制ではなく、エネルギー消費の促進に基づく肥満と糖尿病の新しい治療法を開発することです。我々は、ゲノムワイドオープンクロマチン解析により、褐色脂肪細胞分化の重要な制御因子である転写因子NFIを同定しました。NFIと脂肪生成のマスター転写調節因子であるPPAR-γは、褐色脂肪特異的エンハンサーとして共局在します。

マウス脂肪組織から間質血管画分を単離するプロセスにおいて、脂肪組織の手動ミンチおよびコラゲナーゼ消化は、実験の変動性および汚染をもたらし得る。私たちの方法は、コラゲナーゼ消化に組織解離剤を使用しており、SVFの分離を容易にし、実験のばらつきや汚染を減らし、再現性を向上させます。個々に分化した脂肪細胞を用いた実験は、私たちの分野の研究に不可欠です。

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