Methods of tissue dissociation and cell separation have been modified to obtain highly enriched fractions of mouse gastric parietal cells. Suspensions of gastric mucosal cells are prepared by pronase digestion of the glandular portion of the stomach from adult mice. By utilizing the velocity sedimentation technique to separate cells of different sizes, it is possible to recovery parietal cells, which are larger than the other cell types, in fractions with purity of 75-95%. The homogeneity of cell fractions has been assessed by light and electron microscopy. The ability of the isolated cells to exclude the dye trypan blue, to incorporate labeled substrate, to consume oxygen, and to retain their structural integrity indicates that they are viable and still capable of functional activity.Numerous attempts have been made to assign specific functions to the individual cell types in the gastric mucosa. The classical approach to this problem has been that of Holter and LinderstromLang (4) correlating quantitative variations in biochemical activity with the numbers of a particular cell type in the mixed population in frozen sections cut at different levels of the gastric glands. Analysis of the functions of the respective cell types could be accomplished with greater validity and precision if it were possible to isolate relatively pure fractions of the different kinds of cells in the gastric mucosa. Recent advances in gastric cell isolation methods and the unit gravity sedimentation method seemed to us to hold considerable promise of achieving this objective. We are reporting here the results of a modification of the isolation technique of Blum et al. (I) used in conjunction with the velocity sedimentation method of Miller and Phillips (10). This procedure provides a means of recovering highly enriched fractions of gastric parietal cells which are still viable as indicated by biochemical, physiological, and ultrastructural parameters. MATERIALS AND METHODSSuspensions of mouse gastric mucosal cells were prepared by adapting the method described by Blum et al. (I) for separation of cells from amphibian stomach. Male CD-1 mice, 60 90 days old, were killed by cervical dislocation. Stomachs were excised and all but the area containing the gastric glands proper was discarded. This glandular portion of the stomach, including its muscularis and serosa, was washed free of adhering lumenal content by grasping the organ with forceps and vigorously shaking in a Krebs-Ringer buffer solution.All of the in vitro steps used in this study employed a Krebs-Ringer bicarbonate buffer solution (122 mM NaC1, 4.8 mM KCI, 25.2 mM NaHCOs, 1.2 mM 428
A structural analysis of the intact digestive gland epithelium of Busycon canaliculatum was undertaken at the light and ultrastructual levels. Procedures were developed for the isolation and separation of component epithelial cells by modifying existing pronase digestion techniques. Three distinct cell types were identified following separation by velocity sedimentation. Cells identified after isolation, and separation can be epithelium.The physiology of digestion and the morphology of the digestive gland in gastropods are highly variable. Controversy about the number of cell types (from two to four cells) present in the digestive epithelium is a result of the structural complexity of the tissue, and this in turn adds to the difficulty in assigning specific function to the cell types (Walker, '70; Merdsoy and Farley, '73; Boghen and Farley, '74; Gupta, '77). Work has been done to characterize the digestive epithelium of pulmonates
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