Establishment and characterization of feeder cell-dependent bovine fetal liver cell lines

Abstract

The establishment and initial characterization of bovine fetal liver cell lines are described. Bovine fetal hepatocytes were cultured from the liver of a 34-d bovine fetus by physical disruption of the liver tissue. Released liver cells and clumps of cells were plated on STO (SIMS mouse strain, thioguanine- and ouabain-resistant) feeder layers and were cultured in a medium supplemented with 10% fetal bovine serum. After 2-3 wk, primary colonies of hepatocytes were observed by phase-contrast microscopic observation. Individual hepatocyte colonies were colony-cloned into independent bovine fetal liver (BFL) cell lines. Two cell lines, BFL-6 and BFL-9, grew the best of several isolates, and they were further characterized for growth potential and for hepatocyte morphology and function. The two cell lines were found to grow markedly better in the presence of the transforming growth factor (TGF)-beta inhibitor, SB431542 (1 μM). Their continuous culture also depended on a particular medium height-for T12.5 flasks, 3 ml total medium produced optimum growth. Higher or lower amounts of medium caused less cell growth or cessation of growth. The cell lines were propagated for over a year at split ratios of 1:2 or 1:3 at each passage until reaching senescence at approximately 30 passages. The cells were laterally polarized with well-developed canalicular spaces occurring between adjacent BFL cells. Treatment of the cultures with cyclic adenosine monophosphate (cAMP)-stimulating chemicals or peptides (e.g., forskolin or glucagon) caused physical expansion of the canaliculi between the cells within 15 min. The cells secreted a spectrum of serum proteins, were positive for the expression of several hepatocyte-specific genes, and converted ammonia to urea, although at a relatively low rate. The culture system provides an in vitro model of fetal bovine hepatocytes and is the first demonstration of the continuous culture of normal bovine hepatocytes as cell lines.

Widespread basal cytochrome P450 expression in extrahepatic bovine tissues and isolated cells

“…In general, no in vitro cell type expressed a profile of epoxygenases similar to the profile of any tissue. The low expression of CYP3A4 and CYP2E1 in cultured bovine cells, compared with the organ types from which they were originally derived, has indeed been noted previously (Talbot et al, 2016).…”

Evaluation of the hepatocyte-derived cell line BFH12 as an in vitro model for bovine biotransformation

“…For established hepatocyte-derived cell lines from other species, different patterns of gene expression are observed. Cytochromes 3A4 and 2B6 were found in a feeder-cell dependent bovine hepatocyte line, but their expression could not be increased (Talbot et al 2015). A study investigating the CYP system in the coronary arteries and the liver of cattle found CYP 1A, 2B, 2C, 2D, 2E, 2 J, 3A, 4A subfamilies to be present in the liver (Grasso et al 2005).…”

“…The results suggest that, despite the fact that foetal cells have a reduced set of CYPs compared to adult primary hepatocytes, CYP expression can be induced in BFH12. The AhR pathway is likely to be involved in this regulation (Talbot et al 2015).…”