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

$Aislamiento semiautomático de la fracción vascular estromal del tejido adiposo blanco murino util...$

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

Aislamiento semiautomático de la fracción vascular estromal del tejido adiposo blanco murino utilizando un disociador tisular

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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 describe el aislamiento semiautomático de la fracción vascular estromal (SVF) del tejido adiposo murino para obtener preadipocitos y lograr la diferenciación de los adipocitos in vitro. El uso de un disociador de tejidos para la digestión de la colagenasa reduce la variación experimental y aumenta la reproducibilidad.

Estamos interesados en los subtipos marrones y beige de adipocito que disipan la energía en forma de calor, mientras que el adipocito blanco generalmente almacena energía como Nuestro objetivo es desarrollar un nuevo tratamiento para la obesidad y la diabetes basado en la promoción del gasto energético, en lugar de la supresión de la ingesta de energía. Hemos identificado un factor de transcripción, NFI, como un regulador crítico de la diferenciación de adipocitos marrones mediante un análisis de cromatina abierta de todo el genoma. NFI y el regulador transcripcional maestro de la adipogénesis, PPAR-gamma, se co-localizan como los potenciadores específicos de la grasa marrón.

En el proceso de aislamiento de la fracción vascular estromal del tejido adiposo del ratón, la picada manual y la digestión de la colagenasa del tejido adiposo pueden conducir a la variabilidad experimental y la contaminación. Nuestro método utiliza un disociador de tejidos para la digestión de la colagenasa, que facilita el aislamiento de SVF, reduce la variabilidad experimental y la contaminación, y mejora la reproducibilidad. Los experimentos con adipocito individualmente diferenciados son indispensables para la investigación en nuestro campo.

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