An ex vivo Mouse Model for High-Fat Diet-Like Adipose Tissue Inflammation
July 14th, 2026
Using an ex vivo explant model, small pieces of the rostral portion of visceral white adipose tissue from lean mice are cultured for 7–10 days, enabling the induction and study of adipose tissue inflammation as it occurs during high-fat diet feeding.
We are investigating inflammation in adipose tissue, focusing on the role of adipose tissue macrophages in this process. This model can be applied in a great variety of adipose tissue inflammation research where the focus is on the adipose tissue itself. To begin, prepare the culture medium under sterile conditions.
Warm the culture medium and sterile PBS to 37 degrees Celsius. Clean all equipment and the work area inside the tissue culture hood with 70%ethanol. After placing the sacrificed animal in a glass beaker containing 70%ethanol, wipe the exterior of the glass beaker with ethanol and transfer it into the tissue culture hood.
Using blunt forceps, place the mouse in a supine position, and pin it to a dissection board. Then, add a thin layer of pre-warmed PBS to a Petri dish. Change to a fresh pair of gloves.
Using forceps, lift the lower abdominal skin, and make a small horizontal incision using scissors to open the abdominal cavity. Make a vertical incision extending from the initial cut to expose the internal organs. For male mice, carefully lift the gonadal fat pad, which is attached to the epididymis and testis.
Make a cut above the epididymis. Alternatively, excise the epididymis and testis together with the gonadal fat, and remove the epididymis and testis afterward. For female mice, carefully lift the gonadal fat pad, which is attached to the uterine horn and ovaries, and excise it along the uterine horn and ovaries.
Place the isolated fat pads into a Petri dish filled with PBS. Using a razor blade, remove any residual epididymis, testis, ovaries, or blood vessels from the fat pads. Transfer the cleaned fat pad into a second Petri dish containing pre-warmed PBS.
Using a razor blade, cut the fat pad into strips, and then cut the strips into cubes of approximately 10 cubic millimeters. Add one to 1.5 milliliters of culture medium to each well of a six-well plate. Using a sterile spoon or spatula, transfer five explants into each well.
Arrange the explants evenly in a circular pattern. Using sterile forceps, cover the explants with a cell culture insert. Incubate them at 37 degrees Celsius, 5%carbon dioxide, and 21%oxygen for seven to 10 days.
After a culture period of seven to 10 days, collect explants or culture supernatants for downstream analyses as required for the experimental endpoint. TD tomato expression remains stable throughout the 14-day culture period. Lactate dehydrogenase levels remained low, relative to maximal release following complete tissue lysis, indicating preserved tissue integrity.
Perilipin-positive adipocytes remained intact for up to 10 days of culture. The proportion of DAPI positive cells remained stable throughout the culture period. Expression of PPARG, AdipoQ, LEP, and LPL remained stable throughout the culture period.
A key feature of adipose tissue inflammation is the formation of crown-like structures by adipose tissue macrophages around dying adipocytes, primarily occurring between days five and seven of culture. Expression of inflammatory genes and tumor necrosis factor alpha increased over time. Multinucleated giant cells developed and contributed to dead adipocyte clearance.
Although macrophages accumulated within crown-like structures and fused into multinucleated giant cells, the overall number of adipose tissue macrophages remained relatively stable, as a proliferation peak was observed on day five. The adipose tissue macrophages phenotype shifted from a predominantly M2-like state at day one toward an increased proportion of M1-like macrophages at day seven. Metabolically activated macrophages increased during culture with increased body piece staining and increased lysosomal content.
Multiple macrophage populations were identified, including pro-inflammatory, metabolically activated, proliferating, inflammation-resolving, and anti-inflammatory macrophages. Clodrenate liposomes reduced macrophage numbers by approximately 60%by day seven. And PLX-5622 reduced macrophage numbers by approximately 70%by day seven.
This method can be used to study the intercellular functional processes and alterations that occur during adipose tissue inflammation. The most important challenge when performing this protocol is to handle the tissue as gently as possible, so that the tissue explants remain viable throughout the culture period. Future studies can use this method to dissect adipose tissue inflammation signaling, evaluate cell-specific responses to therapies and reduce animal use.
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Overview
This protocol presents an ex vivo mouse model that mimics key cellular and inflammatory features of visceral adipose tissue (VAT) inflammation observed after high-fat diet (HFD) feeding, without requiring in vivo dietary intervention. By culturing small VAT explants from lean mice for 7–10 days, researchers can study the emergence of obesity-related inflammatory phenotypes in a physiologically relevant tissue context. The model preserves adipose tissue architecture and supports diverse downstream applications for investigating adipocyte death, immune cell dynamics, and therapeutic interventions.
Key Study Components
Area of Science
- Immunology
- Metabolic research
- Adipose tissue biology
Background
- Obesity is associated with chronic inflammation in visceral adipose tissue.
- High-fat diet feeding in mice induces VAT inflammation, characterized by immune cell infiltration and cytokine production.
- In vivo dietary studies are resource-intensive and raise ethical concerns regarding animal use.
- There is a need for physiologically relevant ex vivo models to study adipose tissue inflammation and test interventions.
Purpose of Study
- To establish an ex vivo VAT explant culture model that recapitulates obesity-related inflammatory features without in vivo HFD feeding.
- To provide a platform for investigating the mechanisms of adipocyte death and immune cell dynamics in adipose tissue.
- To enable testing of pharmacological and cytokine-based interventions in a controlled, customizable system.
Methods Used
- Isolation of small VAT explants from lean mice.
- Ex vivo culture of explants for 7–10 days.
- Assessment of inflammatory phenotypes, including crown-like structure (CLS) formation and macrophage accumulation.
- Application of downstream analyses such as live imaging, flow cytometry, qPCR, ELISA, immunostaining, and RNA sequencing.
Main Results
- VAT explants spontaneously develop an inflammatory phenotype similar to that seen in HFD-fed animals.
- Key features include CLS formation, accumulation of metabolically activated macrophages, and increased inflammatory cytokine production.
- The model partially represents metabolic stressors and paracrine signaling relevant to obesity-related inflammation.
- Supports a wide range of experimental applications and interventions.
Conclusions
- This ex vivo VAT explant model is reproducible, cost-effective, and straightforward to implement.
- It preserves tissue architecture and enables physiologically relevant studies of adipose tissue inflammation.
- The model aligns with 3R principles by reducing the need for in vivo animal studies.
Frequently Asked Questions
Executive Industry Relevance
Modeling adipose tissue inflammation ex vivo enables mechanistic de-risking and target validation for metabolic and immunometabolic drug discovery. This system provides predictive confidence by recapitulating key inflammatory features of high-fat diet-induced adipose tissue dysfunction without in vivo dietary intervention. Its reproducibility and physiological relevance support risk-adjusted portfolio decisions in early discovery and translational research.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of inflammatory pathways and cellular interactions in a physiologically relevant adipose tissue context.
- Supports functional target validation by modeling crown-like structure formation and macrophage activation ex vivo.
- Facilitates predictive confidence in target selection for metabolic and inflammatory disease programs.
Screening & Assay Development
- Provides a standardized explant system for quantitative assessment of cytokine production and immune cell dynamics.
- Enables reproducible assay development for screening pharmacological and cytokine-based interventions.
- Supports downstream applications including live imaging, flow cytometry, qPCR, ELISA, and RNA sequencing.
Translational & Preclinical Research
- Aligns with disease-relevant mechanisms observed in obesity-related adipose tissue inflammation.
- Bridges discovery and preclinical validation by preserving tissue architecture and paracrine signaling.
- Reduces reliance on in vivo dietary models, supporting translational continuity and ethical research practices.
Pipeline & Workflow Integration
This ex vivo model fits between early discovery and preclinical validation, enabling mechanistic studies and intervention testing before in vivo studies.
- Discovery Biology: Supports hypothesis testing of inflammatory drivers and immune cell interactions in adipose tissue.
- Screening: Provides quantitative, reproducible outputs for compound and pathway evaluation.
- Analytics: Delivers multiplexed readouts including cytokine levels, immune cell phenotyping, and gene expression.
- Translational Research: Maintains disease-relevant features for biomarker and therapeutic strategy alignment.
- Enterprise Reuse: Offers a customizable, scalable platform for diverse metabolic and immunology R&D programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in adipose tissue inflammation studies.
- Operational Value: Standardizes workflows, enhances reproducibility, and lowers animal use and costs.
- Strategic Value: Improves go/no-go decision-making and capital efficiency in early-stage metabolic and immunometabolic pipelines.
- Portfolio Impact: Enables risk-adjusted prioritization and advancement of inflammation-targeted assets.
Implementation Considerations
- Requires expertise in adipose tissue handling and ex vivo culture techniques.
- Needs access to analytical platforms such as flow cytometry, qPCR, ELISA, and imaging systems.
- Demands cross-team standardization for assay reproducibility and data comparability.
- Adaptable to various pharmacological and cytokine-based intervention studies.
- Partial representation of metabolic stressors and paracrine signaling should be considered in experimental design.