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

Isolamento Semi-Automatizado da Fração Vascular Estromal do Tecido Adiposo Branco Murino Usando u...

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

Isolamento Semi-Automatizado da Fração Vascular Estromal do Tecido Adiposo Branco Murino Usando um Dissociador de Tecido

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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.

Este protocolo descreve o isolamento semi-automatizado da fração vascular estromal (FVS) do tecido adiposo murino para obtenção de pré-adipócitos e diferenciação de adipócitos in vitro. O uso de um dissociador tecidual para digestão da colagenase reduz a variação experimental e aumenta a reprodutibilidade.

Estamos interessados nos subtipos marrom e bege de adipócitos que dissipam energia na forma de calor, enquanto os adipócitos brancos geralmente armazenam energia, pois Nosso objetivo é desenvolver um novo tratamento para obesidade e diabetes baseado na promoção do gasto energético, em vez da supressão da ingestão de energia. Identificamos um fator de transcrição, NFI, como um regulador crítico da diferenciação de adipócitos marrons por uma análise de cromatina aberta em todo o genoma. NFI e o regulador transcricional mestre da adipogênese, PPAR-gama, co-localizam como os potenciadores específicos de gordura marrom.

No processo de isolamento da fração vascular estromal do tecido adiposo de camundongos, a picagem manual e a digestão da colagenase do tecido adiposo podem levar à variabilidade experimental e contaminação. Nosso método utiliza um dissociador tecidual para digestão da colagenase, o que facilita o isolamento da FVS, reduz a variabilidade experimental e a contaminação e melhora a reprodutibilidade. Experimentos com adipócitos diferenciados individualmente são indispensáveis para pesquisas em nosso meio.

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