Hepatocytes isolated from neonatal (NN) and adult (AD) rats were seeded on fibronectin coated substratum and cultured in arginine-free medium supplemented with various combinations of insulin, dexamethasone, triiodothyronine (T3), albumin, and transferrin, in presence or absence of fibronectin depleted serum (FDS). The main finding is that in response to certain hormone mixtures, both NN and AD hepatocytes can be stimulated to proliferate, as revealed by an increase in cell number, a [3H]thymidine incorporation into nuclei, and extractable DNA as well as the appearance of mitotic figures. Moreover, this proliferative activity is associated with changes in hepatocyte ploidy. However, the proliferative response of NN hepatocytes to hormone action is much different from that of AD hepatocytes, and the addition of FDS amplifies this activity in NN but inhibits it in AD hepatocyte cultures. Measurements of tyrosine aminotransferase and lactate dehydrogenase activities indicate a good preservation of NN and AD hepatocyte functional integrity under certain culture conditions. A good maintenance of albumin production in NN and AD hepatocyte cultures requires the presence of dexamethasone, whereas the alpha-fetoprotein production in NN hepatocyte cultures is reduced quite rapidly under most conditions. No alpha-fetoprotein is detectable in AD hepatocyte cultures.
The two-step collagenase perfusion method originally developed for the high yield isolation of parenchymal cells from adult rat livers has been adapted to rats of 1 day, 1 week, and 2 weeks of age. The use of this method to isolate hepatocytes from five or six rats of the respective ages demonstrated its reliability in terms of cell yield, percentage of single cells, and cell viability. In all cases, hepatocytes attach with high efficiency to fibronectin precoated dishes using serum-free culture medium. The dynamics of spreading is faster for newborn hepatocytes than adult ones. The functional integrity of these parenchymal liver cells was assessed by their capacity to secrete albumin and alpha-fetoprotein in serum-free medium and to express lactate dehydrogenase activity over a 24-hr period in primary culture.
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