Primary hepatocytes are commonly used in biopharmaceutical research. In vitro model systems based on hepatocytes have been of great help to better understand the role of hepatocytes in (patho)physiological processes of the liver. In addition, pharmaceutical industry has heavily relied on the use of hepatocytes to explore mechanisms of drug metabolism and predict in vivo drug metabolism. For these purposes, hepatocytes are usually isolated from human whole liver or liver tissue by enzymatic (collagenase) digestion, in a two-step process. In the first step, the liver is placed in an isotonic solution, in which calcium is removed to disrupt cell-cell tight junctions by the use of a calcium chelating agent. In the second step, a solution containing the collagenase is added to separate the hepatocytes from the liver stroma. This process creates a suspension of hepatocytes, which can be seeded in multi-well plates and cultured for many days or even weeks. For optimal results, culture plates are coated with an extracellular matrix to promote hepatocyte attachment and maintenance of the hepatic phenotype. Freshly-isolated hepatocytes can be cryoconserved and stored. Hepatocytes do not proliferate in culture.
Hepatocytes have the ability to metabolize, detoxify, and inactivate endogenous and exogenous compounds. The drainage of the intestinal venous blood into the liver allows for efficient detoxification of absorbed substances and maintains homeostasis.
The typical hepatocyte is cubical with sides of 20-30 µm. The average life span of the hepatocyte is 5 months. In vivo, hepatocytes are able to regenerate. Hepatocytes cytoplasm contains a high number of mitochondria and endoplasmic reticulum. With increasing age, cytoplasmic glycogen and lipid stores increase.
Hepatocytes synthesize, assemble and export lipoproteins and glycoproteins. The endoplasmic reticulum is involved in the conjugation of proteins to lipid and carbohydrate moieties synthesized by, or modified within, the hepatocytes. Hepatocytes form fatty acids from carbohydrates and synthesizes triglycerides from fatty acids and glycerol. The liver is also the main site in the body for gluconeogenesis, the formation of carbohydrates. The liver plays a crucial role in lipid metabolism and receives many lipids from the systemic circulation.