Cell contact but not junctional communication (dye coupling) with biliary epithelial cells is required for hepatocytes to maintain differentiated functions.

Specific differentiated gene expression and the morphology of adult rat hepatocytes can be maintained for as long as 8 weeks in vitro only when they are cultured in the presence of biliary epithelial cells; when primary hepatocytes are cultured alone, they lose these functions within 2 to 3 days. We obtained evidence suggesting that contact between hepatocytes and biliary epithelial cells is necessary for maintaining hepatocyte functions. We examined whether junctional communication between and among hepatocytes and biliary epithelial cells is required for long-term maintenance of hepatocyte functions, using a dye-transfer method, in three co-cultures: (1) hepatocytes and biliary epithelial cells prepared from Sprague-Dawley rats; (2) hepatocytes from Sprague-Dawley rats and epithelial cells of the IAR 20 line, originally established from BDVI rats; and (3) hepatocytes from BDVI rats and IAR 20 epithelial cells. The established epithelial cell line (IAR 20) and early-passage cultures of biliary epithelial cells maintained hepatocyte-specific functions in culture for 40 and 70 days, respectively, but the latter induced more stable maintenance of albumin secretion. Hepatocytes cultured alone lost their characteristic morphology within 5 to 8 days, and almost no dye transfer was observed. In co-cultures, the capacity of biliary epithelial cells to communicate among themselves remained relatively high throughout the culture period, whereas hepatocytes showed almost no junctional communication at an early phase of culture and first began to communicate after 2 weeks, communication capacity increasing for at least the next 10 days of culture. The most notable finding was that there was no dye transfer between hepatocytes and biliary epithelial cells in any co-culture system. These results suggest that the maintenance of hepatocyte-specific functions requires intercellular contact but probably not gap-junctional communication between hepatocytes and biliary epithelial cells. This system is useful for studying heterotypic cell-cell interactions and the control of gene expression.

First evidence of lysogeny in Propionibacterium freudenreichii subsp. shermanii.

Dairy propionic acid bacteria, particularly the species Propionibacterium freudenreichii, play a major role in the ripening of Swiss type cheese. Isometric and filamentous bacteriophages infecting P. freudenreichii have previously been isolated from cheese. In order to determine the origin of these bacteriophages, lysogeny of P. freudenreichii was determined by isometric bacteriophage type analysis. The genomic DNA of 76 strains were hybridized with the DNA of nine bacteriophages isolated from Swiss type cheeses, and the DNA of 25 strains exhibited strong hybridization. Three of these strains released bacteriophage particules following UV irradiation (254 nm) or treatment with low concentrations of mitomycin C. A prophage-cured derivative of P. freudenreichii was readily isolated and subsequently relysogenized. Lysogeny was therefore formally demonstrated in P. freudenreichii.

Isolation and long-term maintenance of differentiated adult chicken hepatocytes in primary culture.

Adult chicken hepatocytes were obtained by an adaptation of the two step in situ collagenase perfusion. Usually 0.5 to 1 x 10(9) cells were obtained, with 75 to 95% viability. Hepatocytes attached within 2 h when plated on plastic cell culture dishes and spread in 4 h, surviving for several months in a specific serum-free medium. These cells retained a typical parenchymal cell morphology and the ability to produce a specific protein (albumin) throughout the culture period. We hereby provide a suitable model for studying hepatic metabolism in birds.

Lipogenic enzyme and apoprotein messenger RNAs in long-term primary culture of chicken hepatocytes.

Hepatocytes isolated from 9-week-old chickens were cultured in a serum-free, hormonally defined medium. Relative amounts of mRNAs coding for lipogenic enzymes (acetyl-CoA carboxylase, fatty acid synthase, delta 9 desaturase, malic enzyme) and apoproteins (apoprotein A1 and apoprotein B) were determined until the 12th day. beta-actin and albumin mRNA, as well as albumin secretion, were also assessed. Cellular metabolic activity appeared to be very low for the first days of culture, but increased after the 7th day. All the mRNAs studied, except for that of malic enzyme, were present from this time throughout the culture lifespan. The biological significance of the observed results and the relevance of this chicken hepatocyte culture system for long-term metabolic and genetic studies are discussed.

Dependence of hepatocyte-specific gene expression on cell-cell interactions in primary culture.

In co-culture with non-parenchymal liver epithelial cells, rat hepatocytes show a marked increase in albumin and total protein synthesis when compared with cells maintained as pure populations in which an early decline in albumin secretion takes place. Analysis of the relative amounts of different mRNA sequences, determined by hybridization, indicated that the increase in protein synthesis resulted essentially from an increased level of the corresponding mRNAs. In addition, when cell-cell contacts were established between the two cell types several days after the seeding of hepatocytes, the stimulation of albumin secretion was similarly observed with a significant increase of the corresponding mRNA on days 10-14 of culture. Transcriptional assays, in which isolated nuclei were used for the study of RNA synthesis, showed that liver-specific gene transcription was significantly increased and maintained for at least 2 weeks. These results demonstrate for the first time long-term stabilization and reversibility of various specific mRNAs at high levels by adult hepatocytes in primary culture. They suggest that establishment of cell-cell contacts between hepatocytes and liver epithelial cells are essential for the maintenance of a high rate of transcription of the liver-specific genes.

Influence of ornithine on albumin synthesis by fetal and neonatal hepatocytes maintained in culture.

Fetal rat as well as fetal and neonatal human hepatocytes were cultured in an arginine-free medium with or without L-ornithine. This amino acid was found greatly to improve cell survival of both rat and human parenchymal cells and to favour their proliferation. In addition, L-ornithine appeared to modulate the expression of various functions and particularly to increase albumin secretion. Since a correlated increase of the corresponding mRNA was observed, it may be postulated that L-ornithine acts at a pre-translational level.