An Improved Two‐Step Collagenase Perfusion Protocol for Isolation of Mouse Primary Hepatocytes

ABSTRACT In metabolic dysfunction‐associated steatotic liver disease (MASLD) or diabetes mechanistic studies, cellular experiments are frequently required. However, liver cancer cell lines are not as representative of the actual state of cellular metabolism as primary hepatocytes. In addition, primary hepatocytes are an essential resource in numerous scientific research fields, functioning as an ex vivo model for liver physiology. It is technically difficult to obtain significant yields of viable primary mouse hepatocytes, which restricts their application. Previous methods often required extended periods of time, sometimes exceeding 30 min, which was detrimental to the viability and yield of hepatocytes. Here, we offer an enhanced procedure that is based on the traditional two‐step collagenase perfusion method. Perfusion I cleanses the liver, and Perfusion II, containing collagenase, dissociates the hepatocytes, which are then isolated from other cells and cultivated. This protocol was refined to enhance hepatocyte viability (over 80%) and yield (over 20,000,000 hepatocytes) while drastically reducing the process time (15 min).