Isolation and Culture of Mouse Hepatocytes and Kupffer Cells (KCs)

Nonalcoholic steatohepatitis (NASH) is characterized by accumulation of lipids in the hepatocytes (steatosis) and chronic inflammation. Liver resident macrophages (Kupffer cells) play a pivotal role in inducing inflammation. Cross-talk between hepatocytes and Kupffer cells (KCs) regulate both steatosis and inflammation during the pathogenesis of NASH. Isolated hepatocytes and KC serve as important tools to study mechanistic events during NASH in an in vitro setting. Because mice and humans share identical genes, primary mouse hepatocytes and KC are valuable ex vivo models for NASH studies. However, isolation of mouse liver cells is challenging and requires specific technical procedure and skills. Here, we elaborate a method for effective isolation of both primary hepatocytes and KC from adult liver of the same mouse. This protocol can be used for isolation of liver cells from both wild-type (WT) and genetically-engineered mice. The principle of the method is based on a two-step collagenase perfusion technique in which the liver is washed by perfusion, liver cells are segregated by collagenase treatment, and hepatocytes and KC are then purified and cultured. We optimized this protocol in terms of reproducibility, yield of different population of liver cells, and viability.