Immunochemical, biomolecular and biochemical characterization of bovine epithelial intestinal primocultures

Abstract

BackgroundCultures of enterocytes and colonocytes represent valuable tools to study growth and differentiation of epithelial cells. In vitro models may be used to evaluate passage or toxicity of drugs, interactions of enteropathogenes bacteria strains with intestinal epithelium and other physiologic or pathologic phenomenon involving the digestive tract.ResultsCultures of bovine colonocytes and jejunocytes were obtained from organoid-enriched preparations, using a combination of enzymatic and mechanical disruption of the intestine epithelium, followed by an isopicnic centrifugation discarding most single cells.Confluent cell monolayers arising from plated organoids exhibited epithelium typical features, such as the pavement-like structure, the presence of apical microvilli and tight junctions. Accordingly, cells expressed several markers of enterocyte brush border (i.e. maltase, alkaline phosphatase and fatty acid binding protein) as well as an epithelial cytoskeleton component (cytokeratin 18). However, enterocyte primocultures were also positive for the vimentin immunostaining (mesenchyme marker). Vimentin expression studies showed that this gene is constitutively expressed in bovine enterocytes. Comparison of the vimentin expression profile with the pattern of brush border enzymes activities, suggested that the decrease of cell differentiation level observed during the enterocyte isolation procedure and early passages of the primoculture could result from a post-transcriptional de-repression of vimentin synthesis. The low differentiation level of bovine enterocytes in vitro could partly be counteracted adding butyrate (1–2 mM) or using a glucose-deprived culture medium.ConclusionThe present study describes several complementary approaches to characterize bovine primary cultures of intestinal cells. Cultured cells kept their morphologic and functional characteristics during several generations.

Establishment and Characterization of an Omasal Epithelial Cell Model Derived from Dairy Calves for the Study of Small Peptide Absorption

“…In addition, our cells also identified the omasal epithelium as assessed using electron microscopy, which revealed the presence of intercellular junctions (desmosomes and tight junctions) and tonofilaments, which were also observed in primary epithelial cells from the bovine rumen [23] and colon epithelium [37]. Furthermore, the cells possessed the capacity to continuously proliferate to form a confluent monolayer, and express PEPT1 and cytokeratin 18.…”

Establishment of fetal bovine intestinal epithelial cell cultures susceptible to bovine rotavirus infection

“…The primary objective of the present study was to establish epithelial cell cultures from fetal small intestine. Epithelial cell cultures have been established from the colon of cattle (Birkner et al, 2004;Follmann et al, 2000;Hoey et al, 2003;Rusu et al, 2005) but these studies did not evaluate epithelial cell susceptibility to infection by enteric pathogens. Therefore, a second objective of the present study was to use BRV as a model pathogen to determine if cultured bovine intestinal epithelial cells were competent target cells for viral infection.…”

“…While standardizing the culture conditions we initially used collagenase at 100 U/ml concentrations but to increase the yield of epithelial cells and crypts, we increased the concentrations of collagenase to 800 U/ml in subsequent experiments. Earlier studies also recommended using more than 100 U collagenase/ml (Rusu et al, 2005;Kaeffer, 2002) when establishing primary bovine and murine intestinal epithelial cultures.…”

“…Previous studies used a variety of tissue preparation methods and dissociation agents to isolate epithelial cells from bovine, human and mouse intestine (Birkner et al, 2004;Follmann et al, 2000;Hoey et al, 2003;Kaeffer, 2002;Rusu et al, 2005). Therefore, we investigated the use of different dissociation agents and digestion protocols to identify the best method for isolating and culturing viable epithelial cells from fetal intestine.…”

“…Many studies have described (reviewed in Kaeffer, 2002) the establishment of short-term intestinal epithelial cell cultures from mice (Booth et al, 1995b;Macartney et al, 2000;Whitehead et al, 1999), rat (Booth et al, 1995a;Evans et al, 1992;Kaeffer et al, 1997;Kaeffer and Briollais, 1998), rabbit (Vidrich et al, 1988) and humans (Gibson et al, 1989;Panja, 2000;Perreault and Beaulieu, 1998;Perreault and Jean-Francois, 1996;Whitehead et al, 1999). Presently, only four studies have reported the establishment of cell cultures from bovine intestinal cells (Birkner et al, 2004;Follmann et al, 2000;Hoey et al, 2003;Rusu et al, 2005). These studies described the generation of epithelial cell cultures from the colon of fetal cattle and the jejunum and colon of adult cattle.…”