Abstract
The ability to analyze individual epithelial cells in the gastric mucosa would provide important insight into gastric disease, including chronic gastritis and progression to gastric cancer. However, the successful isolation of viable gastric epithelial cells (parietal cells, neck cells, chief cells, and foveolar cells) from gastric glands has been limited due to difficulties in tissue processing. Furthermore, analysis and interpretation of gastric epithelial cell flow cytometry data has been difficult due to the varying sizes and light scatter properties of the different epithelial cells, high levels of autofluorescence, and poor cell viability. These studies were designed to develop a reliable method for isolating viable single cells from the corpus of stomachs and to optimize analyses examining epithelial cells from healthy and diseased stomach tissue by flow cytometry. We performed a two stage enzymatic digestion in which collagenase released individual gastric glands from the stromal tissue of the corpus, followed by a Dispase II digestion that dispersed these glands into greater than 1 × 106 viable single cells per gastric corpus. Single cell suspensions were comprised of all major cell lineages found in the normal gastric glands. A method describing light scatter, size exclusion, doublet discrimination, viability staining, and fluorescently-conjugated antibodies and lectins was used to analyze individual epithelial cells and immune cells. This technique was capable of identifying parietal cells and revealed that gastric epithelial cells in the chronically inflamed mucosa significantly upregulated major histocompatibility complexes (MHC) I and II but not CD80 or CD86, which are costimulatory molecules involved in T cell activation. These studies describe a method for isolating viable single cells and a detailed description of flow cytometric analysis of cells from healthy and diseased stomachs. These studies begin to identify effects of chronic inflammation on individual gastric epithelial cells, a critical consideration for the study of gastric cancer.
Highlights
Chronic atrophic gastritis is a common complication after infection with Helicobacter pylori and in individuals that develop autoimmune gastritis [1]
To assess gastric epithelial cell changes in our model of inflammation-induced gastric atrophy, TxA23, we have developed a reliable method for processing tissue and analyzing via flow cytometry that yields repeatable results
Our method of tissue processing involves a two-step enzymatic digestion: first with collagenase to release the glands from the stromal tissue, followed by a digestion step with Dispase II that further separates glands into single cells
Summary
Chronic atrophic gastritis is a common complication after infection with Helicobacter pylori and in individuals that develop autoimmune gastritis [1]. The pathophysiology of gastric cancer development has been well studied in several mouse models using primarily histopathological microscopy techniques [4]. While these are the standard techniques to analyze progression of pathologic changes in gastric epithelial tissue, there are difficulties in obtaining organ-wide surveys of epithelial cells. Proper statistical analysis would require the counting of numerous cells in many different areas of tissue [5]. With respect to these technical difficulties, flow cytometric analysis is ideal for measuring protein expression on individual gastric epithelial cells
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