Due to lack of in vitro models for bovine hepatocytes apart from primary cells, there is demand for a bovine hepatocyte-derived cell line. Transduction of bovine foetal hepatocytes with SV40 large T-antigen was performed using the vector pRetro-E2 SV40. Phase contrast microscopy was carried out to evaluate morphology. Immunofluorescence staining was conducted to study expression of keratins, tight junction proteins zona occludens-1 and claudin-1, glucose transporter-2 and P-glycoprotein as well as phosphoenolpyruvate carboxykinase. Urea and triglyceride production was quantified photometrically. Histochemical staining of glycogen by Periodic acid-Schiff stain and of lipids with Oil red O was performed after 24 h incubation with 20 mM glucose and 85 μM palmitic acid, respectively. Gene expression analysis of hepatocyte-typical genes was conducted by reverse transcription PCR. We obtained a SV40LTAg-transduced extended passage cell line, referred to as BFH12. Polygonal growth, keratins, tight junction proteins zona occludens-1 and claudin-1 and glucose transporter-2 as well as P-glycoprotein and phosphoenolpyruvate carboxykinase were attested positively. Urea production calculated as cell-specific rate was 14.2 ± 2.0 fmol/h (early passage) and 17.6 ± 3.7 fmol/h (late passage). Cell-specific triglyceride production was 1.6 ± 0.5 fmol/h (early passage) and 2.1 ± 0.3 fmol/h (late passage). Additionally, cells were positive for glycogen and lipid storage and showed a gene expression pattern resembling foetal hepatocytes. With the properties described here, the novel cell line BFH12 is a hepatocyte-derived cell line which can be used as an in vitro whole cell model.

The knowledge of drug metabolising enzymes (DMEs) in cattle is rather limited. The capability of the bovine foetal hepatocyte-derived cell line BFH12 to serve as model for biotransformation was evaluated. Gene expression analysis of DMEs was performed by reverse transcription PCR (RT-PCR). The presence of efflux transporters was visualised by immunocytochemistry, and functional induction of cytochrome P450 (CYP) 1A was assessed by the ethoxyresorufin-O-deethylase (EROD) assay. The production of bile acids was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RT-PCR revealed the expression of cytochromes 1A1, 1A2, 3A4 and phase II enzymes UGT1A1, UGT1A6 and GSTM1. Immunofluorescence demonstrated efflux

Continuous cell lines have become indispensable tools that have enabled investigations into cellular mechanisms by increasing experimental reproducibility and sample availability, and decreasing the use of experimental animals. To facilitate studies of epithelial barrier function of the porcine colon, we aimed to establish an epithelial cell line with an extended replicative capacity. Cells were isolated from the proximal colon of a 3-week-old piglet and transduced using a recombinant retroviral vector construct containing the simian virus 40 large T antigen (SV40 TAg). We established a clonal epithelial cell line, referred to as PoCo83-3, that stably expressed the SV40 TAg, verified at mRNA and protein levels. PoCo83-3 showed epithelial cell-specific features, such as cobblestone-like morphology, dome structure formation, the presence of apical microvilli, and the expression of keratin 18, E-cadherin and the tight junction-associated proteins zonula occludens-1, occludin, and claudin-1. To validate PoCo83-3 as an in vitro model in epithelial barrier research, proinflammatory cytokine-inducible alterations in barrier integrity were demonstrated by incubating the cells with TNF-α and IFN-γ for 48 h. These cytokine treatments promoted a decreased transepithelial electrical resistance. In summary, PoCo83-3 exhibited an extended life span and a differentiated phenotype while maintaining epithelial characteristics. Based on these results, we present this cell line as a valuable in vitro model for investigations of epithelial barrier function in the porcine colon.

High amounts of short‐chain fatty acids (SCFAs) occur in the ovine rumen and constitute the animal's main energy source. However, they lead to an acidification of the ruminal epithelium. Therefore, effective intracellular pH (pHi) regulation by transport proteins like monocarboxylate transporter 1 (MCT1) and Na+/H+ exchangers (NHEs) is pivotal to ruminants to avoid epithelial damage. SCFAs might function not only as nutrients but also as signalling molecules by activating free fatty acid receptors (FFARs) in the ruminal epithelium and thus influence pHi regulation. FFARs work as nutrient sensors, transducing their information by modulating cyclic adenosine monophosphate (cAMP) levels. We hypothesized that (FFAR‐modulated) decreases in cAMP levels stimulate the activity of MCT1 and NHEs in the ruminal epithelium of sheep. We detected two FFARs (GPR109A and FFAR2) immunohistochemically in the ovine ruminal epithelium. Administration of 10 mM butyrate to Ussing chamber‐mounted epithelia provoked a significant reduction in intraepithelial cAMP levels. However, application of the GPR109A agonist niacin did not affect cAMP levels. MCT1 activity was analysed by measuring transepithelial 14C‐acetate fluxes, which were not inhibited by forskolin‐induced increased cAMP levels. The recovery of pHi after acidification was assessed as an indicator of NHE activity in primary cultured ruminal epithelial cells. Recovery was significantly reduced when cells with increased cAMP levels were subjected to the NHE inhibitor 5‐(N‐ethyl‐N‐isopropyl)‐amiloride (10 µM). Nonetheless, with augmented cAMP levels alone, NHE activity tended to decline. We hypothesize that modulation of cAMP levels by butyrate is accomplished by FFAR2 activation, regulating NHE activity for pHi homoeostasis at least in part.

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