An ex vivo Mouse Model for High-Fat Diet-Like Adipose Tissue Inflammation.

This protocol describes an ex vivo mouse model that recapitulates selected cellular and inflammatory features observed in visceral adipose tissue (VAT) following high-fat diet (HFD) feeding, without dietary intervention in vivo. Small VAT explants from lean mice are cultured for 7-10 days, during which they spontaneously develop an inflammatory phenotype that closely resembles that of VAT from HFD-fed animals. Importantly, the model preserves adipose tissue (AT) architecture, enabling the investigation of inflammatory processes in a physiologically relevant context. During culture, key hallmarks of obesity-related inflammation emerge, including the formation of crown-like structures (CLS), the accumulation of metabolically activated macrophages (MMes), and increased inflammatory cytokine production. Despite adipocyte death, other drivers of obesity-related inflammation, including metabolic stressors such as saturated fatty acids, altered lipid handling, glucose excess, oxidative stress, hypoxia, adipokine imbalance, and paracrine adipocyte-immune cell signaling, can be partially represented in the model. The system supports a wide range of downstream applications, including live imaging, flow cytometry, quantitative PCR, ELISA, immunostaining, and bulk or single-cell RNA sequencing. In addition, pharmacological and cytokine-based treatments can be applied to investigate signaling pathways and cell type-specific responses. This easily customizable model aligns with the 3R principles by reducing animal use for in vivo dietary studies. The model is reproducible, cost-effective, and straightforward to implement, providing a versatile platform for studying adipocyte death, immune cell dynamics, and therapeutic strategies in AT inflammation.