A Simple Protocol for the Isolation and Culture of Hepatocytes from MASLD Mice

Metabolic dysfunction-associated steatotic liver disease (MASLD), characterized by pathological lipid accumulation (steatosis) within hepatocytes, represents a significant and growing global health burden. Primary mouse hepatocyte cultures serve as indispensable ex vivo models for elucidating MASLD pathogenesis and therapeutic interventions. However, reliable isolation of high-quality hepatocytes from steatotic livers remains technically challenging. Here, we present a simple protocol for the efficient isolation of primary hepatocytes from MASLD mice using the collagenase perfusion technique. This protocol generates hepatocytes exhibiting inherent steatosis without requiring artificial induction via palmitate and oleic acid supplementation, thereby preserving a more physiologically relevant phenotype. The protocol yields highly viable and high-purity hepatocytes and is universally applicable for isolating high quantities of hepatocytes from MASLD mice. Furthermore, it is suitable for pharmacological evaluation or genetic intervention studies. In summary, we provide a reproducible protocol to isolate high-yield, high-purity, and highly viable hepatocytes for downstream cell biological studies to facilitate the discovery of novel therapeutic targets and drugs for MASLD.