Integrating fine needle aspiration and single-cell RNA sequencing for studying metabolic dysfunction-associated steatotic liver disease.

Introduction: Metabolic dysfunction-associated steatotic liver disease (MASLD) is currently the leading cause of chronic liver disease and hepatocellular carcinoma. The immune response plays a central role in disease onset and progression and is the focus of many experimental studies. However, traditional models typically rely on terminal sampling procedures that require large tissue quantities, a substantial number of animals per experimental condition, and cross-sectional study designs. Methods: Here, we propose integrating two powerful techniques to longitudinally study a MASLD animal model: image-guided fine-needle aspiration (FNA) and single-cell RNA sequencing (scRNA-seq). Results: This framework enables safe, high-precision longitudinal sampling of a limited number of animals, allowing simultaneous profiling of innate and adaptive immune cells during metabolic dysfunction-associated steatohepatitis (MASH) progression induced by a high-fat, high-cholesterol, and high-fructose diet. We further identified dynamic shifts in the hepatic immune landscape following dietary intervention. Conclusion: Our findings support the feasibility of the FNA-scRNA-seq framework as a novel tool for longitudinal immune profiling in preclinical MASLD and MASH studies and highlight its potential to reduce animal use in chronic liver disease research.