Glimpse into the role of Kupffer cells in a spheroid model of metabolic dysfunction-associated steatohepatitis (MASH).

Metabolic dysfunction-associated steatotic liver disease (MASLD) and its progressive form, metabolic dysfunction-associated steatohepatitis (MASH), are characterized by lipid accumulation, inflammation, and fibrosis. Existing preclinical models often fail to capture multicellular interactions that shape disease progression. In this study, we established a human multicellular 3D liver spheroid model comprising HepaRG hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells, and Kupffer cells. Spheroids were analyzed for lipid accumulation, cytokine secretion, hepatocellular injury, and extracellular matrix remodeling using immunofluorescence, qPCR, ELISA, and LDH release assays. The model exhibited multicellular spatial organization resembling the native liver microarchitecture. Lipid accumulation was most pronounced under MASLD conditions, whereas progression to MASH was associated with stronger inflammatory and fibrosis-related changes. TNF-α showed the clearest Kupffer cell-associated increase, whereas IL-6 changes were less consistent and likely reflected the broader inflammatory response of a multicellular system. LDH release increased with disease severity and was most pronounced in MASH without evidence of a robust Kupffer cell-specific effect. Fibrotic remodeling was reflected by increased expression of fibronectin, collagen III, and procollagen I. Together, these findings establish a human multicellular in vitro platform that recapitulates the key disease-relevant features of MASLD/MASH under controlled conditions.