Histology
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 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.
Hepatocytes are an important physiological example for evalutation of both biological and metabolic effects of xenobiotics. They are separated from the liver 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 cultured and plated on 96 well plates for immediate use, or cryopreserved by freezing. 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.
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