title: Proliferation of epidermal growth factor-stimulated hepatocytes in a hormonally defined serum-free medium.
abstract: The present study shows that adult rat hepatocytes in primary culture, which normally exhibit a restricted capacity to proliferate, can proceed through the cell cycle when cultured in a mixture of minimal essential medium (MEM) and Medium 199 (MEM-M199; 3:1, v/v), containing epidermal growth factor (EGF; 50 ng/ml), low glucose (0.75 g/l) and low levels of inorganic salts, amino acids and vitamins. Under these conditions, hepatocytes flatten and cell extensions appear. In contrast, Dulbecco's modified Eagle's medium (DMEM) containing high glucose (4.5g/l) levels enriched with inorganic salts, amino acids and vitamins favours maintenance of differentiated functional hepatocyte capacities (albumin secretion), but does not allow proliferation or cell spreading. Cultivation of hepatocytes in MEM-M199 (3:1, v/v) results in the onset of DNA synthesis at 48 hours of culture and a concomitant induction of cyclin D1 protein. Under these conditions, cells successively progress through the mitogen-dependent restriction point in mid-late G1 phase, G1/S transition and S phase, as evidenced by Western blot analysis of the markers cyclins E and A and cyclin dependent kinase (CDK)2 and CDK1, respectively. Progression through the cell cycle is accompanied by a decrease in albumin secretion, indicating a decline in differentiated capacities. This study demonstrates that hepatocytes cultured in a mixture of MEM-M199 (3:1) provide a useful in vitro model for studying the regulation of hepatocyte proliferation.
authors.affiliation: Department of Toxicology, Vrije Universiteit Brussel, Brussels, Belgium. Peggy.Papeleu@vub.ac.be
authors.forename: Peggy
authors.initials: P
authors.lastname: Papeleu
authors.forename: Pascal
authors.initials: P
authors.lastname: Loyer
authors.forename: Tamara
authors.initials: T
authors.lastname: Vanhaecke
authors.forename: Tom
authors.initials: T
authors.lastname: Henkens
authors.forename: Greetje
authors.initials: G
authors.lastname: Elaut
authors.forename: Christiane
authors.initials: C
authors.lastname: Guguen-Guillouzo
authors.forename: Vera
authors.initials: V
authors.lastname: Rogiers
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004261:DNA Replication; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D015415:Biomarkers; D016895:Culture Media, Serum-Free; D004815:Epidermal Growth Factor