Redifferentiation of proliferated rat hepatocytes cultured in L15 medium supplemented with EGF and DMSO

Abstract

Primary adult rat hepatocytes were cultured in serum-free L15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml epidermal growth factor in a 5% CO2:95% air incubator. The number of cells increased and reached about 180% of the initial value by Day 4, and after 2% dimethyl sulfoxide (DMSO) was added to the culture medium at Day 4, the cells continued to proliferate until Day 6. The number of cells reached about 210% at Day 6 and they were well maintained until Day 18. The cell number gradually decreased with time in culture, but many cells remained for more than 2 mo. On the other hand, without 2% DMSO, the cells proliferated until Day 5, but thereafter they rapidly decreased. After DMSO addition, albumin and transferrin were secreted into the medium and the production of both proteins continued for more than 2 mo. Immunocytochemically both proteins were strongly stained in the cells treated with 2% DMSO. Although the expression of G6Pase in the cells disappeared at Day 6 without DMSO, the cells treated with 2% DMSO recovered G6Pase activity at Day 16. In addition, induction of peroxisomes by 2 mM sodium clofibric acid was clearly shown in the hepatocytes at Day 14 and Day 25 using enzyme-cytochemistry. Ultrastructurally, DMSO-treated hepatocytes had many mitochondria and large peroxisomes with a crystalline nucleoid, and both gap junctions and desmosomes were well developed between the cells even at Day 40. Thus, the number of cells doubled, some differentiated functions of the primary hepatocytes were well restored by the use of 2% DMSO, and these functions were maintained for more than 2 mo.

Propagation and functional characterization of serum-free cultured porcine hepatocytes for downstream applications

“…The agents prevented or even rescued the loss of P450 activity or urea synthetic capacity, when added to the cells. This corroborates previous findings with cultured primary hepatocytes showing the stabilization of distinct CYP450 enzyme activity by all-trans retinoic acid (Bertagnolo et al 2003;Jurima-Romet et al 1997), by xenobiotics such as phenobarbital, rifampicin and β-naphthoflavone (Richert et al 2002;Kern et al 1997), by DMSO (Mitaka et al 1993), by growth factors (Runge et al 2000) or by specific extracellular matrix components such as a distinct growth substratum (Hodgkinson et al 2000) and the induction of CPS by glucocorticoids and cAMP (Kitagawa 1987;Nebes and Morris 1988).…”

Formation of Actin Filament Networks in Cultured Rat Hepatocytes Treated with DMSO and Glucagon.

“…The cell shape and volume may play an important role in the induction and maintenance of differentiated function in hepatocytes (29). In fact, the hepatocytes in this culture system highly maintained differentiated hepatic functions such as albumin, transferrin, tryptophan oxygenase, and cytochrome P-450 production (30,31). Furthermore, hepatocytes cultured with DMSOand glucagon also have extensive gap junctional communication between adjacent cells, which is thought to be one of the highly differentiated functions, and they have high expression of Cx32 mRNAand Cx26 mRNA(9).…”

Biogenesis of peroxisomes in fetal liver

“…This hypothesis was inferred from the facts that (1) hepatocyte-specific features are determined by tissue interactions early during development (e.g. Caron, 1990;Cascio and Zaret, 1991) and (2) the number, size, shape, and catalase activity of peroxisomes in cultured hepatocytes depend on the culture conditions (Furukawa et al, 1988;Mitaka et al, 1993;Roels et al, 1996a). Thus far, studies of early embryonic rat liver have not confirmed a relationship between peroxisome maturation and the presence of extracellular matrix proteins laminin and collagen IV (Depreter et al, 1997).…”