8A). days after seeding. However, tight junctions were discontinuous, TER was less than 300 cm2, and permeability was high. In contrast, chief cells incubated with hepatocyte growth factor (HGF) were confluent in 3 days and had a TER greater than 2,000 cm2, continuous tight junctions, and low permeability. EGF was intermediate. HGF facilitated monolayer development by increasing cell number, which occurred by the proliferation of chief cells. Chief cell cultures, produced with HGF, consisted of more than 99% gastric intrinsic factor-expressing cells and showed strong pepsinogen secretion. Coexpression studies for neck and chief cell markers suggest that the cultures are a mixture of mature, immature, and transitional zone cells. This model will be useful for investigating mechanisms that regulate chief cell physiology in health and disease. Keywords:gastric, methods, zymogenic cells helicobacter pylori(HP) orfelisinfection in animal models including mice, guinea pigs, and Mongolian gerbils has been instrumental in helping to elucidate mechanisms that are important in the pathogenesis of corpus-predominant HP disease, including inflammation, atrophy, metaplasia, and the progression to gastric cancer (9,11,14,23,32). However, detailed mechanistic studies concerning HP contamination in gastric epithelial cells are often not done because of a lack of appropriate culture models. For instance, it is difficult to study how bacterial virulence factors such as vacA Norepinephrine hydrochloride and cagA alter cell-specific signaling pathways and function in gastric epithelial cells. In addition, it is currently not possible to study the way in which HP contamination alters the gastric mucosal barrier, both at the surface and within gastric glands, Norepinephrine hydrochloride or to determine how HP-associated inflammation influences cell-specific survival and death pathways in gastric epithelial cells, especially in chief cells. For such studies, primary epithelial cells in culture that form a confluent monolayer and have intact barrier properties would be required. Primary cells from the gastric mucosa of most species are difficult to isolate and grow in culture and do not form a tight barrier. Because of this limitation, malignancy cell lines or transformed cells, which may or may not reflect mechanisms that occur in normal gastric epithelial Norepinephrine hydrochloride cells, are currently used. To study the physiology of chief cells at the base of gastric glands, Ayalon et al. (2) developed a technique to produce cultures using chief cells isolated from the canine gastric mucosa. For this, cells isolated by collagenase digestion were purified by centrifugal elutriation to yield cultures with a high transepithelial resistance (TER) and low permeability that maintained differentiated function as shown by agonist-induced pepsinogen secretion (2,26). Since the procedure was developed, short-term cultures have been produced from isolated human and rabbit (10), guinea pig (25), pig (12), and rat (20,30,31) chief cells. Although isolated chief cells and short-term cultured cells from all species secrete pepsinogen (10,12,25,31), it is not currently possible to produce a confluent monolayer of chief cells with a high TER, low permeability, and high rate of agonist-induced pepsinogen secretion from a species other than doggie. Because probes are now available to facilitate studies concerned with gastric physiology and pathophysiology in rodent species, development of cultured chief cells from the rat or mouse stomach would be timely and important. Thus the aim of the present work was to develop a chief cell culture model using the rat stomach. We selected rat because the stomach is relatively large and the isolation and purification procedures Rabbit Polyclonal to CLIP1 yield a considerable number of enriched isolated chief cells. By using hepatocyte growth factor (HGF), isolated chief cells can be seeded at low density in culture to produce a large number of confluent monolayers with one preparation. The resulting cultures produced with HGF have significantly more cells and a high TER, low permeability, and significant agonist-stimulated.
