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

$Isolement semi-automatisé de la fraction vasculaire stromale à partir de tissu adipeux blanc muri...$

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

Isolement semi-automatisé de la fraction vasculaire stromale à partir de tissu adipeux blanc murin à l’aide d’un dissociateur tissulaire

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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.

Ce protocole décrit l’isolement semi-automatisé de la fraction vasculaire stromale (SVF) du tissu adipeux murin pour obtenir des préadipocytes et obtenir une différenciation adipocytaire in vitro. L’utilisation d’un dissociateur tissulaire pour la digestion de la collagénase réduit la variation expérimentale et augmente la reproductibilité.

Nous nous intéressons aux sous-types d’adipocytes bruns et beiges qui dissipent l’énergie sous forme de chaleur, tandis que les adipocytes blancs stockent généralement de l’énergie car Notre objectif est de développer un nouveau traitement pour l’obésité et le diabète basé sur la promotion de la dépense énergétique, plutôt que sur la suppression de l’apport énergétique. Nous avons identifié un facteur de transcription, NFI, comme régulateur critique de la différenciation des adipocytes bruns par une analyse de la chromatine ouverte à l’échelle du génome. NFI et le régulateur transcriptionnel principal de l’adipogenèse, PPAR-gamma, co-localisent en tant qu’activateurs spécifiques de la graisse brune.

Dans le processus d’isolement de la fraction vasculaire stromale du tissu adipeux de souris, le hachage manuel et la digestion de la collagénase du tissu adipeux peuvent entraîner une variabilité expérimentale et une contamination. Notre méthode utilise un dissociateur tissulaire pour la digestion de la collagénase, ce qui facilite l’isolement de la SVF, réduit la variabilité expérimentale et la contamination, et améliore la reproductibilité. L’expérimentation utilisant des adipocytes différenciés individuellement est indispensable pour la recherche dans notre domaine.

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