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-automatico della frazione vascolare stromale dal tessuto adiposo bianco murino me...

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

Isolamento semi-automatico della frazione vascolare stromale dal tessuto adiposo bianco murino mediante un dissociatore tissutale

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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.

Questo protocollo descrive l'isolamento semi-automatico della frazione vascolare stromale (SVF) dal tessuto adiposo murino per ottenere preadipociti e ottenere la differenziazione degli adipociti in vitro. L'uso di un dissociatore tissutale per la digestione della collagenasi riduce la variazione sperimentale e aumenta la riproducibilità.

Siamo interessati ai sottotipi di adipociti marroni e beige che dissipano energia sotto forma di calore, mentre gli adipociti bianchi generalmente immagazzinano energia come Il nostro obiettivo è sviluppare un nuovo trattamento per l'obesità e il diabete basato sulla promozione del dispendio energetico, piuttosto che sulla soppressione dell'assunzione di energia. Abbiamo identificato un fattore di trascrizione, NFI, come regolatore critico della differenziazione degli adipociti bruni mediante un'analisi della cromatina aperta a livello di genoma. NFI e il principale regolatore trascrizionale dell'adipogenesi, PPAR-gamma, co-localizzano come potenziatori specifici del grasso bruno.

Nel processo di isolamento della frazione vascolare stromale dal tessuto adiposo del topo, la tritatura manuale e la digestione della collagenasi del tessuto adiposo possono portare a variabilità e contaminazione sperimentali. Il nostro metodo utilizza un dissociatore tissutale per la digestione della collagenasi, che facilita l'isolamento SVF, riduce la variabilità sperimentale e la contaminazione e migliora la riproducibilità. Gli esperimenti con adipociti differenziati individualmente sono indispensabili per la ricerca nel nostro campo.

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