Skip to main content
SpringerLink
Log in

Isolating and maintaining highly polarized primary epithelial cells from normal human duodenum for growth as spheroid-like vesicles

  • Cellular Models
  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Aggeler, J.; Ward, J.; Blackie, L. M., et al. Cytodifferentiation of mouse mammary epithelial cells cultured on a reconstituted basement membrane reveals striking similarities to development in vivo. J. Cell Sci. 99:407–417; 1991.

    PubMed  Google Scholar 

  • Bayerdörffer, E.; Lehn, N.; Hatz, R., et al. Difference in expression of Helicobacter pylori gastritis in antrum and body. Gastroenterology 102:1575–1582; 1992.

    PubMed  Google Scholar 

  • Boxberger, H.-J.; Meyer, T. F. A new method for the 3-D in vitro growth of human RT112 bladder carcinoma cells using the alginate culture technique. Biol. Cell 82:109–119; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Boxberger, H.-J.; Paweletz, N.; Spiess, E., et al. An in vitro model study of BSp 73 rat tumour cell invasion into endothelial monolayer. Anticancer Res. 9:1777–1786; 1989.

    PubMed  CAS  Google Scholar 

  • Boxberger, H.-J.; Sessler, M. J.; Grund, K.-E., et al. Three-dimensional (3-D) cell culture of epithelial cells from the human colonic mucosa. J. Exp. Clin. Cancer Res. 14 (Suppl. 1):22–23; 1995.

    Google Scholar 

  • Burridge, K.; Fath, K.; Kelly, T., et al. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu. Rev. Cell Biol. 4:487–525; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Buset, M.; Lipkin, M.; Winawer, S., et al. Inhibition of human colonic epithelial cell proliferation in vivo and in vitro by calcium. Cancer Res. 46:5426–5430; 1986.

    PubMed  CAS  Google Scholar 

  • Cairnie, A. B.; Lamerton, L. F.; Steel, G. G. Cell proliferation studies in the intestinal epithelium of the rat. I. Determination of the kinetic parameters. Exp. Cell Res. 39:528–538; 1965.

    Article  PubMed  CAS  Google Scholar 

  • Chevillard, M.; Hinnrasky, J.; Pierrot, D., et al. Differentiation of human surface upper airway epithelial cells in primary culture on a floating collagen gel. Epith. Cell Biol. 2:17–25; 1993.

    CAS  Google Scholar 

  • Ekblom, P.; Vestweber, D.; Kemler, R. Cell-matrix interactions and cell adhesion during development. Annu. Rev. Cell Biol. 2:27–47; 1986.

    Article  PubMed  CAS  Google Scholar 

  • Espanet, H.; Alquier, C.; Mauchamp, J. Polarity reversal of inside-out thyroid follicles cultured on the surface of a reconstituted basement membrane matrix. Exp. Cell Res. 200:473–480; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Freshney, R. I. Culture of animal cells. 2nd rev. ed. New York: Wiley-Liss; 1987.

    Google Scholar 

  • Gibson-D’Ambrosio, R. E.; Samuel, M.; D’Ambrosio, S. M. A method for isolating large numbers of viable disaggregated cells from various human tissues for cell culture establishment. In Vitro Cell. Dev. Biol. 22:529–534; 1986.

    Article  PubMed  CAS  Google Scholar 

  • Harrison, D. D.; Webster, H. L. The preparation of isolated intestinal crypt cells. Exp. Cell Res. 55:257–260; 1969.

    Article  PubMed  CAS  Google Scholar 

  • Herzog, V.; Miller, F. Structural and functional polarity of inside-out follicles prepared from pig thyroid gland. Eur. J. Cell Biol. 24:74–84; 1981.

    PubMed  CAS  Google Scholar 

  • Hohn, H.-P.; Denker, H.-W. The role of cell shape for differentiation of choriocarcinoma cells on extracellular matrix. Exp. Cell Res. 215:40–50; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Jackson, C. D.; Newport, G. A simple method for in vitro studies with rodent urinary bladders. In Vitro 17:107–113; 1981.

    Article  PubMed  CAS  Google Scholar 

  • Kedinger, M.; Simon-Assmann, P. M.; Lacroix, B., et al. Fetal gut mesenchyme induces differentiation of cultured intestinal endodermal and crypt cells. Dev. Biol. 113:474–483; 1986.

    Article  PubMed  CAS  Google Scholar 

  • Knüchel, R.; Sutherland, R. M. Recent developments in research with human tumor spheroids. Cancer J. 3:234–243; 1990.

    Google Scholar 

  • Kondo, Y.; Rose, I.; Young, G. P., et al. Growth and differentiation of fetal rat small intestinal epithelium in tissue culture. Exp. Cell Res. 153:121–134; 1984.

    Article  PubMed  CAS  Google Scholar 

  • Kubat, B. The influence of different substrates on tight junction formation and on ADH-response in renal collecting duct cells in primary cultures. Eur. J. Cell Biol. (Suppl. 40, abstract #114); 1994.

  • Latza, U.; Niedobitek, G.; Schwarting, R., et al. Ber-EP4: new monoclonal antibody which distinguishes epithelia from mesotheia. J. Clin. Pathol. 43:213–219; 1990.

    PubMed  CAS  Google Scholar 

  • Lichtenberger, L.; Miller, L. R.; Erwin, D. N., et al. Effect of pentagastrin on adult rat duodenal cells in culture. Gastroenterology 65:242–251; 1973.

    PubMed  CAS  Google Scholar 

  • Mackenzie, I.; Rittman, G.; Bohnert, A., et al. Influence of connective tissues on the in vitro growth and differentiation of murine epidermis. Epith. Cell Biol. 2:107–119; 1993.

    CAS  Google Scholar 

  • Marshall, B. J.; Royce, H.; Annear, D. I., et al. Original isolation of Campylobacter pyloridis from human gastric mucosa. Microbios Lett. 25:83–88: 1984.

    Google Scholar 

  • McDonald, J. A. Matrix regulation of cell shape and gene expression. Curr. Opin. Cell Biol. 1:995–999; 1989.

    Article  PubMed  CAS  Google Scholar 

  • McNeil, P. L. Cellular and molecular adaptions to injurious mechanical stress. Trends Cell Biol. 3:302–307; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Minuth, W. W.; Stöckl, G.; Kloth, S., et al. Construction of an apparatus for perfusion cell cultures which enables in vitro experiments under organotypic conditions. Eur. J. Cell Biol. 57:132–137; 1992.

    PubMed  CAS  Google Scholar 

  • Moyer, M. P. Culture of human gastrointestinal epithelial cells. Proc. Soc. Exp. Biol. Med. 174:12–15; 1983.

    PubMed  CAS  Google Scholar 

  • Mueller-Klieser, W. Multicellular spheroids. A review on cellular aggregates in cancer research. J. Cancer Res. Clin. Oncol. 113:101–122; 1987.

    Article  PubMed  CAS  Google Scholar 

  • Négrel, R.; Rampal, P.; Nano, J.-L., et al. Establishment and characterization of an epithelial intestinal cell line from rat fetus. Exp. Cell Res. 143:427–437; 1983.

    Article  PubMed  Google Scholar 

  • Nelson, W. J.; Veshnock, P. J. Dynamics of membrane-skeleton (Fodrin) organization during development of polarity in Madin-Darby Canine Kidney epithelial cells. J. Cell Biol. 103:1751–1765; 1986.

    Article  PubMed  CAS  Google Scholar 

  • Nelson, W. J.; Veshnock, P. J. Modulation of Fodrin (membrane skeleton) stability by cell-cell contact in Madin-Darby Canine Kidney epithelial cells. J. Cell Biol. 104:1527–1537; 1987.

    Article  PubMed  CAS  Google Scholar 

  • Prewett, E. Y.; Bickley, J.; Owen, R. J., et al. DNA patterns of Helicobacter pylori isolated from gastric antrum, body, and duodenum. Gastroenterology 102:829–833; 1992.

    PubMed  CAS  Google Scholar 

  • Quaroni, A. Development of fetal rat intestine in organ and monolayer culture. J. Cell Biol. 100:1611–1622; 1985.

    Article  PubMed  CAS  Google Scholar 

  • Raul, F.; Kedinger, M.; Simon, P., et al. Behaviour of isolated rat intestinal cells maintained in suspension or monolayer cultures. Biol. Cell 33:163–168; 1978.

    CAS  Google Scholar 

  • Rodriguez-Boulan, E.; Nelson, W. J. Morphogenesis of the polarized epithelial cell phenotype. Science 245:718–725; 1989.

    Article  PubMed  CAS  Google Scholar 

  • Rugge, M.; DiMario, F.; Cassaro, M., et al. Pathology of the gastric antrum and body associated with Helicobacter pylori infection in non-ulcerous patients: is the bacterium a promoter of intestinal metaplasia? Histopathology 22:9–15; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Schmidt, J. W.; Piepenhagen, P. A.; Nelson, W. J. Modulation of epithelial morphogenesis and cell fate by cell-to-cell signals and regulated cell adhesion. Semin. Cell Biol. 4:161–173; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Starkey, J. R.; Hosick, H. L.; Stanford, D. R., et al. Interaction of metastatic tumor cells with bovine lens capsule basement membrane. Cancer Res. 44:1585–1594; 1984.

    PubMed  CAS  Google Scholar 

  • Streuli, C. H.; Bailey, N.; Bissell, M. J. Control of mammary epithelial differentiation: the separate roles of cell-substratum and cell-cell interaction. J. Cell Biol. 115:1383–1395; 1991.

    Article  PubMed  CAS  Google Scholar 

  • Sutherland, R. M. Cell and environment interactions in tumor microregions: the multicell spheroid model. Science 240:177–184; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Tait, I. S.; Flint, N.; Evans, G. S., et al. Progress with small bowel enterocyte culture and transplantation. Transplant. Proc. 24:1061–1064; 1992.

    PubMed  CAS  Google Scholar 

  • Timpl, R. Structure and biological activity of basement membrane proteins. Eur. J. Biochem. 180:487–502; 1989.

    Article  PubMed  CAS  Google Scholar 

  • Trier, J. S. Organ culture methods in the study of gastrointestinal-mucosal function and development. N. Engl. J. Med. 295:150–155; 1976.

    Article  PubMed  CAS  Google Scholar 

  • Tsuchiya, W.; Okada, Y. Membrane potential changes associated with differentiation of enterocytes in the rat intestinal villi in culture. Dev. Biol. 94:284–290; 1982.

    Article  PubMed  CAS  Google Scholar 

  • Walling, J. M.; Blackmore, M.; Hickman, J. A., et al. Role of the extracellular matrix on the growth and differentiated phenotype of murine colonic adenocarcinoma cells in vitro. Int. J. Cancer 47:776–783; 1991.

    Article  PubMed  CAS  Google Scholar 

  • Watt, F.; Jordan, P. W.; O’Neill, C. H. Cell shape controls terminal differentiation of human epidermal keratinocytes. Proc. Natl. Acad. Sci. USA 85:5576–5580; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Weiser, M. M. Concanavalin A agglutination of intestinal cells from human fetus. Science 177:525–526; 1972.

    Article  PubMed  CAS  Google Scholar 

  • Yamanari, H.; Suganuma, T.; Iwamura, T., et al. Extracellular matrix components regulating glandular differentiation and the formation of basal lamina of a human pancreatic cancer cell line in vitro. Exp. Cell Res. 211:175–182; 1994.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Abteilung Infektionsbiologie, Max-Planck-Institut für Biologie, Spemannstrasse 34, 72076, Tübingen, Germany

    Hans-Jürgen Boxberger & Thomas F. Meyer

  2. Abteilung Allgemeine Chirurgie und Poliklinik, Universität Tübingen, Hoppe-Seyler-Strasse 3, 72076, Tübingen, Germany

    Matthias C. Grausam, Kristian Reich, Horst-Dieter Becker & Michael J. Sessler

Authors
  1. Hans-Jürgen Boxberger
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thomas F. Meyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Matthias C. Grausam
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kristian Reich
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Horst-Dieter Becker
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael J. Sessler
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Boxberger, HJ., Meyer, T.F., Grausam, M.C. et al. Isolating and maintaining highly polarized primary epithelial cells from normal human duodenum for growth as spheroid-like vesicles. In Vitro Cell.Dev.Biol.-Animal 33, 536–545 (1997). https://doi.org/10.1007/s11626-997-0096-0

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11626-997-0096-0

Key words

Search

Navigation