Isolating and Maintaining Highly Polarized Primary Epithelial Cells from Normal Human Duodenum for Growth as Spheroid-Like Vesicles

A method is described for the three-dimensional (3-D) in vitro culture of nontransformed gastrointestinal epithelial cells from the human duodenal mucosa. Biopsies obtained from human duodenum were finely minced. The tissue fragments were suspended in culture medium supplemented with 5% fetal calf s...

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Bibliographic Details
Published in:In vitro cellular & developmental biology. Animal 1997-07, Vol.33 (7), p.536-545
Main Authors: Hans-Jürgen Boxberger, Meyer, Thomas F., Matthias C. Grausam, Kristian Reich, Horst-Dieter Becker, Michael J. Sessler
Format: Article
Language:English
Subjects:
Antibodies, Monoclonal
Basement membrane
Cell culture techniques
Cell Division
Cell lines
Cell membranes
Cell Polarity
Cell Separation
Cells
Cells, Cultured
Cellular Models
Collagen - analysis
Cultured cells
Duodenum - cytology
Epithelial Cells
Flow Cytometry
Glycocalyx
Humans
Immunohistochemistry
Intestinal Mucosa - cytology
Intestines
Laminin - analysis
Liposomes
Microscopy, Electron
Microscopy, Electron, Scanning
Mucosa
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Summary:A method is described for the three-dimensional (3-D) in vitro culture of nontransformed gastrointestinal epithelial cells from the human duodenal mucosa. Biopsies obtained from human duodenum were finely minced. The tissue fragments were suspended in culture medium supplemented with 5% fetal calf serum and the appropriate antibiotics. The suspended mucosal fragments generated spheroid-like multicellular vesicles consisting of highly prismatic absorptive and goblet cells retaining most of the histological features of the tissue in vivo. We performed immunocytochemical studies to determine the origin of the vesicles using monoclonal antibodies against EP4. The histochemistry of the vesicles showed alkaline phosphatase activity. Ultrastructural studies revealed that these cells exhibit characteristics of normal duodenal cells in vivo: apical microvilli, glycocalyx, tight junctions and desmosomes, lateral membrane interdigitations, mucous droplets, and a well-developed Golgi apparatus. An overgrowth of the vesicles by fibroblasts was not seen during cultivation. In contrast with the two-dimensional cell cultures grown on artificial supports, the vesicle cells show organization similar to that of natural epithelia. The polarization and cytoarchitecture of normal gastrointestinal epithelial cells cultured as 3-D vesicles are comparable to those known for the native tissue. This study was undertaken to provide a morphological baseline for subsequent infection experiments.
ISSN:1071-2690
1543-706X
DOI:10.1007/s11626-997-0096-0