Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator
May 19th, 2023
This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.
We are interested in brown and beige subtypes of adipocyte with disparate energy in the form of heat. While white adipocytes generally store energy as repeat, our goal is to develop a new treatment for obesity and diabetes based on promotion of energy expenditure rather than suppression of energy intake. We have identified a transcription factor in FY as a critical regulator of lung adipocyte differentiation by a canon-wide open chromatic analysis.
In FY and master transcriptional regulator of adipogenesis prepare gamma, co-localize as brown-fat specific enhancers. In the process of isolating stromal vascular fractions from mass adipose tissue, murine method and collagenase digestion of adipose tissue can lead to experimental variability and contamination. Our method uses a tissue dissociator for collagenase digestion, which facilitate XPF isolation, reduce experimental variability and contamination, and improves reproducibility.
Experiment using individual differentiated adipocyte are indispensable for research in our field. Our protocol would offer easy and reproducible SVF isolation for subsequent adipocyte differentiation, and would accelerate research in our community. Currently we are investigating the role of NFIA on formidable metabolism, such as body weight and glucose, using gain and loss of function mass models.
We will also explore other pharmacological activation of NFIA for each downstream pathway in near future. Begin by placing a seven to eight week old euthanized male mouse on clean bench surface. Cut the abdominal skin of the mouse with a pair of scissors towards the lower abdomen.
Excise the adipose tissue from the inner thighs. Place the excised tissue into tube C on ice, containing 2.5 milliliters of an enzyme mixture of enzymes DRA, and 2.35 milliliters of DMEM/F-12 without FBS or antibiotics. Using scissors, cut the adipose tissue into approximately two square millimeter-sized pieces.
Tightly close the cap of tube C and invert the tube. Attach the tube to the sleeve of a tissue dissociator and digest the samples at 37 degrees Celsius for 40 minutes. Next, prewarm DMEM/F-12 media containing FBS and antibiotics to 37 degrees Celsius.
After incubation, remove tube C from the tissue dissociator, and stop digestion by adding five milliliters of DMEM/F-12 with FBS and antibiotics. Gently pipette four times. Centrifuge the suspension at 700 g for 10 minutes at 20 degrees Celsius.
Without disturbing the cell palate, carefully aspirate the supernatant. Re-suspend the palate in 10 milliliters of DMEM/F-12 containing FBS and antibiotics, and gently pipette five times. Filter the cell suspension through a 70 micron diameter cell strainer placed on a 50 milliliter tube.
Centrifuge the filtrate at 250 g for five minutes. Re-suspend the palate in 10 milliliters of PBS. Before plating, centrifuge the cell suspension at 500 g for five minutes.
Discard the supernatant. Re-suspend the pellet in 10 milliliters of DMEM/F-12 containing FBS and antibiotics. Mix by pipetting the suspension gently 10 times.
Plate 10 milliliters of the suspension on a 10 centimeter collagen-coated dish. Place the dish in a cell culture incubator. Aspirate the medium and wash the cells three times with three milliliters of PBS per wash.
Add 10 milliliters of DMEM/F-12 containing FBS and antibiotics, and incubate. Oil Red O staining showed lipid laden adipocytes seven days after inducing adipocyte differentiation. The degree of full differentiation was confirmed by mRNA expression analysis of adipogenesis regulators.
Rosiglitazone induced a dose-dependent effect on the expression levels of brown fat-specific genes, such as Ucp1 one and Ppargc1a. However, for Fabp4, the effect saturated at 0.1 micromole Rosiglitazone concentration. Differentiated adipocytes can be used for various functional and mechanistic analyses, such as oxygen consumption rate analysis, and chromatin immunoprecipitation.
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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
Executive Industry Relevance
Reproducible isolation of the stromal vascular fraction (SVF) from murine adipose tissue is critical for generating primary preadipocytes and differentiated adipocytes, enabling mechanistic studies of adipocyte biology. This semi-automated protocol reduces experimental variability and contamination, supporting robust in vitro models for metabolic disease research. Enhanced reproducibility at this stage strengthens predictive confidence for downstream functional and mechanistic analyses relevant to obesity and diabetes pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of adipocyte differentiation pathways and transcriptional regulators.
- Supports mechanistic de-risking by providing primary cells for functional studies.
- Facilitates target validation for metabolic disease research by enabling controlled manipulation of adipocyte subtypes.
Screening & Assay Development
- Provides standardized primary cell systems for assay development and compound screening.
- Improves reproducibility and reduces contamination risk in cell-based assays.
- Enables quantitative assessment of adipogenic differentiation and gene expression responses.
Translational & Preclinical Research
- Supports translational continuity by modeling disease-relevant adipocyte biology in vitro.
- Allows for functional and mechanistic analyses, such as oxygen consumption and chromatin immunoprecipitation, to inform preclinical decisions.
- Aligns with biomarker discovery efforts by enabling gene expression profiling of differentiated adipocytes.
Pipeline & Workflow Integration
This protocol integrates into the early discovery and assay development continuum, providing a reproducible source of primary adipocytes for downstream functional and mechanistic studies.
- Discovery Biology: Supports hypothesis testing of adipocyte differentiation and transcriptional regulation.
- Screening: Delivers assay-ready primary cells with reduced variability for compound evaluation.
- Analytics: Enables quantitative readouts such as Oil Red O staining and mRNA expression analysis.
- Translational Research: Bridges in vitro findings to preclinical metabolic models by enabling functional assays.
- Enterprise Reuse: Establishes a standardized, semi-automated workflow adaptable across metabolic research programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in adipocyte biology studies.
- Operational Value: Enhances standardization, reproducibility, and scalability of SVF isolation.
- Strategic Value: Improves go/no-go decisions for metabolic disease targets by providing robust in vitro data.
- Portfolio Impact: Enables risk-adjusted prioritization of metabolic disease programs through reliable functional assays.
Implementation Considerations
- Requires expertise in murine tissue handling and primary cell culture.
- Needs access to a tissue dissociator and standard cell culture infrastructure.
- Demands cross-team standardization to ensure reproducibility across studies.
- Adaptable to various adipose tissue depots and potentially other model systems.
- Dependent on careful enzymatic digestion and contamination control as supported by the protocol.
