Protocol for isolation of lipid-laden hepatocytes from high-fat diet-fed and metabolically challenged mice

We present a protocol to isolate hepatocytes from mice fed a high-fat diet (HFD), modeling features of non-alcoholic fatty liver disease (NAFLD) and metabolically associated fatty liver disease (MAFLD). We perfuse and enzymatically digest the liver and then purify hepatocytes using a modified Percoll gradient (30%, 25%, and 10%) to retain lipid-laden cells. This protocol enables the isolation of both normal and lipid-filled hepatocytes for culture and downstream studies of drug metabolism, hepatotoxicity, and glucose or lipid metabolism in vitro.