Protocol to isolate and co-culture primary mouse hepatocytes, hepatic stellate cells, and Kupffer cells under physiological conditions

With the rising incidence of metabolic diseases, the ability to isolate and co-culture primary hepatic cells has become increasingly critical for investigating liver pathophysiology. We present a protocol to isolate and co-culture primary mouse hepatocytes, hepatic stellate cells, and Kupffer cells. We describe steps for perfusing the liver without a pump, digesting it with collagenase, and separating cells by density centrifugation. We then detail procedures for culturing hepatocytes in a defined, serum-free, plasma-like medium to mimic in vivo conditions.