Joan A. McGowan

Stimulation of DNA synthesis in primary cultures of adult rat hepatocytes by rat platelet-associated substance(s)

SummaryExperiments in whole animals have shown that normally quiescent adult rat hepatocytes are induced to proliferate by blood borne substances, which we are now probing in primary monolayer cultures. Under our conditions, freshly isolated adult hepatocytes do not proliferate actively in a defined medium, but are stimulated to synthesize DNA — an essential first step — by either serum or an EGF-hormone combination.Stimulation of [3H]thymidine incorporation into hepatocyte DNA by addition of dialyzed mouse, human, horse, or bovine (fetal, newborn, or calf) serum, whose activities are all similar, is regularly surpassed by an EGF-insulin mixture without serum. This, in turn, is exceeded by dialyzed normal rat serum, which is several times more potent than the other sera tested.Removal of blood platelets reduces the activity of normal rat serum by over 50%. Heat inactivation (56° C) causes a similar loss, but heat treatment of platelet-poor serum fails to cause further reduction. The activity of mouse and human serum is not reduced by platelet removal.Serum from partially hepatectomized rats is not significantly more stimulatory than normal rat serum, and its activity is depressed in the same way by platelet deprivation and heat inactivation. Lack of enhancement by partial hepatectomy is not consonant with whole animal studies and requires further investigation.The heat-labile portion of the DNA synthesis-stimulating activity of rat serum appears to derive from platelets. This activity differs from the well-characterized heat-stable human PDGF. Its relation to other reported platelet-associated growth factors is still undetermined.

Pyruvate promotion of DNA synthesis in serum-free primary cultures of adult rat hepatocytes

SummaryPyruvate (2 to 60 mM), acting alone and in conjunction with insulin and epidermal growth factor (EGF), enhances DNA synthesis in primary monolayer cultures of adult rat liver parenchymal cells. Lactate can replace pyruvate in stimulating DNA synthesis. Several other intermediary metabolites (oxaloacetate, α-ketoglutarate, α-ketobutyrate, succinate, fumarate, and malate), though less potent than pyruvate and lactate, also elevate DNA synthesis, whereas alanine at similar concentrations is inhibitory.

The isolation of adult rat liver parenchymal cells

This report describes procedures for the isolation and maintenance of monolayer culture of adult rat liver hepatic parenchymal cells. Isolation of the cells is accomplished using perfusion in situ with a calcium-free buffer followed by buffered collagenase. Gravity sedimentation and selective media are used to limit the contribution of nonparenchymal cells in the cultures.