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Cross-section of the human liver.
- Protein synthesis
- Protein storage
- Transformation of carbohydrates
- Synthesis of cholesterol, bile salts and phospholipids
- Detoxification, modification, and excretion of exogenous and endogenous substances
The hepatocyte also initiates formation and secretion of bile.
The typical hepatocyte forms a cubical cell of 20-30 µm sides (in comparison, a human hair has a diameter of 17 to 180 µm). The typical volume of a hepatocyte is 3.4 x 10−9 cm3.
Hepatocytes display an eosinophilic cytoplasm, reflecting numerous mitochondria, and basophilic stippling due to large amounts of rough endoplasmic reticulum and free ribosomes. Brown lipofuscin granules are also observed (with increasing age) together with irregular unstained areas of cytoplasm; these correspond to cytoplasmic glycogen and lipid stores removed during histological preparation. The average life span of the hepatocyte is 5 months; they are able to regenerate.
Hepatocyte nuclei are round with dispersed chromatin and prominent nucleoli. Anisokaryosis (or variation in the size of the nuclei) is common and often reflects tetraploidy and other degrees of polyploidy, a normal feature of 30-40% of hepatocytes in adult human liver. Binucleate cells are also common.
Hepatocytes are organised into plates separated by vascular channels (sinusoids), an arrangement supported by a reticulin (collagen type III) network. The hepatocyte plates are one cell thick in mammals and two cells thick in the chicken. Sinusoids display a discontinuous, fenestrated endothelial cell lining. The endothelial cells have no basement membrane and are separated from the hepatocytes by the space of Disse, which drains lymph into the portal tract lymphatics.
Kupffer cells are scattered between endothelial cells; they are part of the reticuloendothelial system and phagocytose spent erythrocytes. Stellate (Ito) cells store vitamin A and produce extracellular matrix and collagen; they are also distributed amongst endothelial cells but are difficult to visualise by light microscopy.
The liver forms fatty acids from carbohydrates and synthesizes triglycerides from fatty acids and glycerol. Hepatocytes also synthesize apoproteins with which they then assemble and export lipoproteins (VLDL, HDL).
The liver receives many lipids from the systemic circulation and metabolizes chylomicron remnants. It also synthesizes cholesterol from acetate and further synthesizes bile salts. The liver is the sole site of bile salts formation.
One of the detoxifying functions of hepatocytes is to modify ammonia into urea for excretion.
The most abundant organelle in liver cell is the smooth endoplasmic reticulum.
Hepatocyte isolation and culture
Primary hepatocytes are commonly used in cell biological and 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 in suspension or culture to explore mechanisms of drug metabolism and even predict in vivo drug metabolism. For these purposes, hepatocytes are usually isolated from animal or human whole liver or liver tissue by collagenase digestion, which is 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. Next, a solution containing 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 should first be coated with an extracellular matrix (e.g. collagen, Matrigel) to promote hepatocyte attachment (typically within 1-3 hr after seeding) and maintenance of the hepatic phenotype. In addition, and overlay with an additional layer of extracellular matrix is often performed to establish a sandwich culture of hepatocytes. The application of a sandwich configuration supports prolonged maintenance of hepatocytes in culture. Freshly-isolated hepatocytes that are not used immediately can be cryopreserved and stored. They do not proliferate in culture. Hepatocytes are intensely sensitive to damage during the cycles of cryopreservation including freezing and thawing. Even after the addition of classical cryoprotectants there is still damage done while being cryopreserved. Nevertheless, recent cryopreservation and resuscitation protocols support application of cryopreserved hepatocytes for most biopharmaceutical applications.
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