Establishment and Characterization of Porcine Colonic Epithelial Cells Grown in Primary Culture

Abstract

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·cm² 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.

Cholinergic Modulation of Epithelial Integrity in the Proximal Colon of Pigs

“…Based on these findings, it can be concluded that treatment with exogenous cholinergic agonists, mimicking ACh release by cholinergic enteric neurons, influences TJ assembly rather than cell proliferation. This interpretation is further supported by findings from a recent study showing that the TEER value of a cultured colonocyte layer depends directly on the extent of TJ formation [Petto et al, 2011]. The action of the cholinergic agonist carbachol may be mediated by two classes of AChR [Tapper et al, 1978]: the G-protein-coupled mAChR and the nAChR, which are members of a superfamily of ligand-gated ion channel receptors.…”

“…To elucidate the long-term response of epithelial cells to cholinergic stimulation, a cell culture model free of systemic influences and the neuronal components of the enteric nervous system was employed according to a published protocol [Petto et al, 2011]. The mRNAs for ZO-1 and occludin were detected in the cultured epithelial cells.…”

“…The proximal colon was removed and later opened longitudinally and washed with icecold 0.9% NaCl solution. Epithelial cells were isolated according to the cold isolation method published by Petto et al [2011]. In brief, washed pieces of colon were placed in transport medium (phosphate-buffered saline, PBS; PAA Laboratories GmbH, Coelbe, Germany, supplemented with 500 μg/ml gentamicin, 1,000 U/ml penicillin, 1 mg/ml streptomycin and 25 μg/ml amphotericin B; all from Sigma-Aldrich, Taufkirchen, Germany).…”

“…In this regard, a previously established primary culture of porcine colonic cells [Petto et al, 2011] is a suitable model system for examining the putative effects of ACh on the development and maintenance of a tight gastrointestinal epithelium. In this setup, systemic effects are largely excluded, and the cells can be cultured for up to 3 weeks.…”

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

“…Well-established protocols are available for the isolation and cultivation of intestinal epithelial cells derived from different species that maintain the characteristics of the original cells [Golaz et al, 2007;Kaushik et al, 2008;Petto et al, 2011;Ren et al, 2011]. Nevertheless, the use of primary cultures is limited by small proliferative life spans that precede permanent growth arrest, termed "in vitro cellular senescence" [Hayflick, 1965[Hayflick, , 1985Kuilman et al, 2010;Rodier and Campisi, 2011].…”