A live-cell medium-throughput assay for quantifying lipid accumulation in hepatocytes.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global health burden contributing to the high prevalence of associated metabolic diseases such as cardiovascular disease and thus global mortality rates. MASLD is diagnosed by ≥ 5 % lipid accumulation in the liver, and candidate therapies are often evaluated for lipid lowering effects. Current cell-based lipid imaging approaches rely on fixed-cell staining with dyes such as BODIPY 493/503, limiting the ability to dynamically monitor lipid accumulation in living hepatocytes. To gain a better understanding of the effect of different culture conditions and pharmacological interventions on lipid dynamics, we have developed a live-cell assay of fatty acid stimulated lipid accumulation based on BODIPY 493/503, using two hepatocyte cell lines (HepG2 and AML12) in a simplified workflow. This workflow was validated by its success in helping characterise the lipid lowering activity of known pharmacological agents under different conditions and as a function of time. The agents included impact both lipid biosynthesis (diacylglycerol acyltransferase and acetyl-CoA carboxylase inhibitors) and metabolism (THR-β agonist). Collectively, this assay enables the temporal interrogation of hepatic lipid dynamics and represents a valuable tool for screening and characterising emerging MASLD therapeutics.