Growth and functional activities of neonatal and adult rat hepatocytes cultured on fibronectin coated substratum in serum-free medium

Abstract

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.

Differential responsiveness of cultured suckling and adult rat hepatocytes to growth‐promoting factors: Entry into s phase and mitosis

“…However in those studies, hepatocytes represented only 40% of the isolated liver cell preparations and were stimulated after 4 days of culture in the presence of serum; such culture conditions are very different from those used to study the hormone response of adult rat hepatocytes (e.g., Laishes and Williams, 1976a;McGowan et al, 1981;Michalopoulos et al, 1982). In the present work, the collagenase perfusion method yielded suckling rat hepatocytes with a purity of 95 to 97%, a high degree of purity comparable to that reported previously by us (Marceau et al, 1982) and others (McGowan et al, 1981) for the preparation of adult rat hepatocytes. Moreover, as reported before for newborn and adult rat hepatocytes Marceau et al, 1982), suckling rat hepatocytes readily attached with high efficiency to fibronectin-precoated plastic, thus permitting all seeding in the absence of serum.…”

Primary culture, cellular stress and differentiated function

“…Glucocorticoids, which are known to enhance albumin synthesis [81,82], help to maintain its rate when added to hepatocytes soon after their isolation [50,83]. In this respect the full induction of vitellogenin synthesis in primary cultures of amphibian or avian liver parenchymal cells offers many advantages, such as the competence of male liver to respond to estrogen, offering a'zero' background of prior vitellogenin gene expression and being independent of cell division There are many similarities between hormonal induction of gene expression and the induction by drugs of detoxicating enzyme systems [89,90].…”

Chronic hypocalcemia of vitamin D deficiency leads to lower intracellular calcium concentrations in rat hepatocytes.

“…24 h after plating, 1 MCi/ml [methyl-3H]thymidine (ICN Canada, spec act, 70-90 Ci/mmol) was added to fresh medium. DNA was precipitated 24 h later for the determination of [3H]thymidine incorporation according to Marceau et al (13). Protein concentration was measured as described by Bradford (14) using crystalline BSA as standard.…”