Establishment and Characterization of an SV40 Large T Antigen-Transduced Porcine Colonic Epithelial Cell Line.

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.

Cholinergic modulation of epithelial integrity in the proximal colon of pigs.

BACKGROUND: Within the gut, acetylcholine (ACh) is synthesised by enteric neurons, as well as by 'non-neuronal' epithelial cells. In studies of non-intestinal epithelia, ACh was involved in the generation of an intact epithelial barrier. In the present study, primary cultured porcine colonocytes were used to determine whether treatment with exogenous ACh or expression of endogenous epithelium-derived ACh may modulate epithelial tightness in the gastrointestinal tract. METHODS: Piglet colonocytes were cultured on filter membranes for 8 days. The tightness of the growing epithelial cell layer was evaluated by measuring transepithelial electrical resistance (TEER). To determine whether ACh modulates the tightness of the cell layer, cells were treated with cholinergic, muscarinic and/or nicotinic agonists and antagonists. Choline acetyltransferase (ChAT), cholinergic receptors and ACh were determined by immunohistochemistry, RT-PCR and HPLC, respectively. RESULTS: Application of the cholinergic agonist carbachol (10 µm) and the muscarinic agonist oxotremorine (10 µM) resulted in significantly higher TEER values compared to controls. The effect was completely inhibited by the muscarinic antagonist atropine. Application of atropine alone (without any agonist) led to significantly lower TEER values compared to controls. Synthesis of ACh by epithelial cells was proven by detection of muscarinic and nicotinic receptor mRNAs, immunohistochemical detection of ChAT and detection of ACh by HPLC. CONCLUSION: ACh is strongly involved in the regulation of epithelial tightness in the proximal colon of pigs via muscarinic pathways. Non-neuronal ACh seems to be of particular importance for epithelial cells forming a tight barrier.

Establishment and characterization of porcine colonic epithelial cells grown in primary culture.

BACKGROUND: Primary cultures of epithelial cells are suitable models for studying epithelial function and, in particular, the regulation of epithelial tightness in vitro. The aim of our study was to develop a protocol for the isolation and culture of porcine colonic epithelial cells and to establish transepithelial electrical resistance (TEER) as a functional parameter for epithelial tightness. METHODS: Epithelial cells were obtained from the proximal colon of piglets by enzymatic dispase digestion. Cells were cultured on collagen-coated membrane supports for 21 days. The epithelial origin of the cells was shown by immunohistochemical detection of cytokeratin and zonula occludens protein 1 (ZO-1). Scanning electron microscopy, transmission electron microscopy and confocal microscopy were used for further morphological characterization. The integrity and tightness of the artificial epithelium were determined by measuring TEER. RESULTS: The cultured epithelial cells were immunoreactive for cytokeratin and ZO-1. They showed dense microvilli on their apical membranes and expression of Na(+)/K(+)-ATPase on their basolateral membranes. Adjacent cells were connected by tight junctions. We observed TEER to continuously increase up to 870 ± 38 Ω·cm(2) during the culture period. TEER correlated with the amount of epithelial cells expressing ZO-1. CONCLUSIONS: The properties of primary cultured epithelial cells resemble the structural properties of polarized colonic epithelium in vivo. Measurement of TEER seems to be suitable for studying epithelial tightness in vitro. We suggest that these primary epithelial cultures be used to investigate the regulation of the epithelial barrier function.