A New Approach to Primary Culture of Human Gastric Epithelium

  • Pierre Chailler
  • Daniel Ménard
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 107)

Abstract

The gastric mucosa assumes several digestive and protective functions including enzyme and mucus secretion, as well as sterilization of the luminal content through the release of hydrochloric acid. Distinct epithelial populations located in specific compartments of the gastric pit-gland units mediate these secretory functions. In addition, previous studies from our laboratory (1-5) and others suggest that local growth factors and extracellular matrix (ECM) proteins may cooperatively regulate epithelial morphogenesis, differentiation, and home-ostasis at the level of the stomach. Owing to the absence of a normal or cancer cell line that could serve as an in vitro model to delineate the molecular mechanisms involved, gastric epithelial biology mostly relied on primary culture of epithelial cells freshly isolated from the stomach mucosae of animal species. The protocols used were based on two different approaches, i.e., microsurgi-cal/scraping techniques and enzymatic treatments. The first requires laboratory expertise and, unless gradient fractionation or elutriation is used to purify glandular cells, these are rapidly overgrown during the first 2 d of culture by dividing mucous cells originating from the pits (foveolae) of gastric units (6-8). The second approach, which commonly uses a combination of collagenase and hyaluronidase, generates cultures that are initially composed of mucus-secreting cells mainly, with a low proportion of acid-secreting parietal cells (9,10). Such cultures are also devoid of glandular zymogenic cells, named chief or pepsino-gen cells. These represent the major cell type found at the base of gastric glands and they are highly specialized for the secretion of pepsinogens. Their absence in epithelial cultures is particularly deleterious for the advancement of gastric physiology in humans. Indeed, human chief cells not only contribute to the digestion of dietary proteins, but also to triglyceride lipolysis through the secretion of a bile salt-independent and acid-tolerent gastric lipase, which is absent in rodents.

Keywords

Formaldehyde Glycerol Chloroform Glycine Aldehyde 

References

  1. 1.
    Tremblay, E. and Ménard, D. (1996) Differential expression of extracellular matrix components during the morphogenesis of human gastric mucosa. Anat. Rec. 245, 668–676.PubMedCrossRefGoogle Scholar
  2. 2.
    Tremblay, E., Monfils, S., and Ménard, D. (1997) Epidermal growth factor influences cell proliferation, glycoproteins and lipase activity in human fetal stomach. Gastroenterology 112, 1188–1196.PubMedCrossRefGoogle Scholar
  3. 3.
    Chénard, M., Basque, J.-R., Chailler, P., Beaulieu, J.-F., and Ménard, D. (2000) Expression of integrin subunits correlates with differentiation of epithelial cell lineages in developing human gastric mucosa. Anat. Embryol. 202, 223–233.PubMedCrossRefGoogle Scholar
  4. 4.
    Chailler, P., Basque, J.-R., Corriveau, L., and Ménard, D. (2000) Functional characterization of the keratinocyte growth factor system in human gastrointestinal tract. Pediatr. Res. 48, 504–510.PubMedCrossRefGoogle Scholar
  5. 5.
    Tremblay, E., Chailler, P., and Ménard, D. (2001) Coordinated control of fetal gastric epithelial functions by insulin-like growth factors and their binding proteins. Endocrinology 142, 1795–1803.PubMedCrossRefGoogle Scholar
  6. 6.
    Sanders, M.J., Amirian, D.A., Ayalon, A., and Soll, A.H. (1983) Regulation of pepsinogen release from canine chief cells in primary monolayer culture. Am. J. Physiol. 245, G641–G646.PubMedGoogle Scholar
  7. 7.
    Rattner, D. W., Ito, S., Rutten, M. J., and Silen, W. (1985) A rapid method for culturing guinea pig gastric mucous cell monolayers. In Vitro (Cell. Dev. Biol.) 21, 453–462.CrossRefGoogle Scholar
  8. 8.
    Chew, C. (1994) Parietal cell culture: new models and directions. Annu. Rev. Physiol. 56, 445–461.PubMedCrossRefGoogle Scholar
  9. 9.
    Terano, A., Ivey, K. J., Stachura, J., et al. (1982) Cell culture of rat gastric fundic mucosa. Gastroenterology 83, 1280–1291.PubMedGoogle Scholar
  10. 10.
    Terano, A., Mach, T., Stachura, J., Sekhon, S., Tarnawski, A., and Ivey, K. J. (1983) A monolayer culture of human gastric epithelial cells. Dig. Dis. Set 28, 595–603.CrossRefGoogle Scholar
  11. 11.
    Fukamachi, H., Ichinose, M., Ishihama, S., et al. (1994) Fetal rat glandular stomach epithelial cells differentiate into surface moucous cells which express cathepsin E in the absence of mesenchymal cells in primary culture. Differentiation 56, 83–89.PubMedGoogle Scholar
  12. 12.
    Fujiwara, Y., Arakawa, T., Fukuda, T., Higuchi, K., Kobayashi, K., and Tarnawski, A. (1995) Role of extracellular matrix in attachment, migration, and repair of wounded rabbit cultured gastric cells. J. Clin. Gastroenterol. 21(snSuppl.), S125–S130.PubMedGoogle Scholar
  13. 13.
    Nakagawa, S., Yoshida, S., Hirao, Y., Kasuga, S., and Fuwa, T. (1985) Biological effects of biosynthetic human EGF on the growth of mammalian cells in vitro. Differentiation 29, 284–288.PubMedCrossRefGoogle Scholar
  14. 14.
    Rutten, M. J., Dempsey, P. J., Solomon, T. E., and Coffey, R. J., Jr. (1993) TGF-alpha is a potent mitogen for primary cultures of guinea pig gastric mucous epithelial cells. Am. J. Physiol. 265, G361–G369.PubMedGoogle Scholar
  15. 15.
    Takahashi, M., Ota, S., Shimada, T., et al. (1995) Hepatocyte growth factor is the most potent endogenous stimuklant of rabbit gastric epithelial cell proliferation and migration in primary culture. J. Clin. Invest. 95, 1994–2003.PubMedCrossRefGoogle Scholar
  16. 16.
    Yoshida, S., Kasuga, S., Hirao, Y., Fuwa, T., and Nakagawa, S. (1987) Effect of biosynthetic human epidermal growth factor on the synthesis and secretion of mucin glycoprotein from primary culture of rabbit fundal mucosal cells. In Vitro (Cell. Dev. Biol.) 23, 460–464.CrossRefGoogle Scholar
  17. 17.
    Boland, C. A., Kraus, E. R., Scheiman, J. M., Black, C., Deshmukh, G. D., and Dobbins, W. O. (1990) Characterization of mucous cell synthetic functions in a new primary canine gastric mucous cell culture system. Am. J. Physiol. 258, G774–G787.PubMedGoogle Scholar
  18. 18.
    Wagner, S., Beil, W., Mai, U.E., Bokemeyer, C., Meyer, H. J., and Manns, M. P. (1994) Interaction between Helicobacter pylori and human gastric epithelial cells in culture: effect of antiulcer drugs. Pharmacology 49, 226–237.PubMedCrossRefGoogle Scholar
  19. 19.
    Quaroni, A. and May, R. J. (1980) Establishment and characterization of intestinal epithelial cell cultures. Methods Cell Biol. 218, 403–427.CrossRefGoogle Scholar
  20. 20.
    Kedinger, M., Haffen, K., and Simon-Assman, P. (1987) Intestinal tissue and cell cultures. Differentiation 36, 71–85.PubMedCrossRefGoogle Scholar
  21. 21.
    Evans, G. S., Flint, N., and Potten, C. S. (1994) Primary cultures for studies of cell regulation and physiology in intestinal epithelium. Annu. Rev. Physiol. 56, 399–417.PubMedCrossRefGoogle Scholar
  22. 22.
    Perreault, N. and Beaulieu, J.-F. (1998) Primary cultures of fully differentiated and pure human intestinal epithelial cells. Exp. Cell Res. 245, 34–42.PubMedCrossRefGoogle Scholar
  23. 23.
    Basque, J.-R., Chailler, P., Perreault, N., Beaulieu, J.-F., and Ménard, D. (1999) A new primary culture system representative of the human gastric epithelium. Exp. Cell Res. 253, 493–502.PubMedCrossRefGoogle Scholar
  24. 24.
    Basque, J.-R. and Ménard, D. (2000) Establishment of culture systems of human gastric epithelium for the study of pepsinogen and gastric lipase synthesis and secretion. Microsc. Res. Tech. 48, 293–302.PubMedCrossRefGoogle Scholar
  25. 25.
    Streeter, G. L. (1920) Weight, sitting head, head size, foot length and menstrual age of the human embryo. Carnegie Inst. Contrib. Embryol. 11, 143–179.Google Scholar
  26. 26.
    Freshney, R. I. (1987) Culture of Animal Cells: a Manual of Basic Technique. 2nd ed., Wiley-Liss, New York, pp. 246–247.Google Scholar
  27. 27.
    Bowie, D. J. (1936) A method for staining the pepsinogen granules in gastric glands. Anat. Rec. 64, 357–365.CrossRefGoogle Scholar
  28. 28.
    Ménard, D., Monfils, S., and Tremblay, E. (1995) Ontogeny of human gastric lipase and pepsin activities. Gastroenterology 108, 1650–1656.PubMedCrossRefGoogle Scholar
  29. 29.
    Lévy, E., Goldstein, R., Freier, S., and Shafrir, E. (1981) Characterization of gastric lipolytic activity. Biochim. Biophys. Acta 664, 316–326.PubMedGoogle Scholar
  30. 30.
    Anson, M. L. and Mirsky, A. E. (1932) The estimation of pepsin with hemoglobin. J. Gen. Physiol. 10, 342–344.Google Scholar
  31. 31.
    Lowry, O. H., Rosebrough, N. E, Farr, A. L., and Randall, R. J. (1951) Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265–275.PubMedGoogle Scholar
  32. 32.
    Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162,156–159.PubMedCrossRefGoogle Scholar
  33. 33.
    Tremblay, E., Basque, J.-R., Rivard, N., and Ménard, D. (1999) Epidermal growth factor and transforming growth factor-α downregulate human gastric lipase gene expression. Gastroenterology 116, 831–841.PubMedCrossRefGoogle Scholar
  34. 34.
    Beaulieu, J.-F. (1997) Extracellular matrix components and integrins in relationship to human intestinal cell differentiation. Prog. Histochem. Cytochem. 31, 1–78.PubMedGoogle Scholar
  35. 35.
    Montgomery, R. K., Mulberg, A. E., and Grand, R. I. (1999) Development of the human gastrointestinal tract: twenty years of progress. Gastroenterology 116, 702–731.PubMedCrossRefGoogle Scholar
  36. 36.
    Podolsky, D. K. and Babyatsky, M. W. (1995) Growth and development of the gastrointestinal tract, in Textbook of Gastroenterology, 2nd ed. (Yamada, T., ed.), Lippincott, Philadelphia, PA, pp. 546–577.Google Scholar
  37. 37.
    Basque, J.-R., Chailler, P., and Ménard, D. (2002) Laminins and TGF-β maintain cell polarity and functionality of human gastric glandular epithelium. Am. J. Physiol. Cell Physiol. 282, C873–C884.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Pierre Chailler
    • 1
  • Daniel Ménard
  1. 1.CIHR Group on the Functional Development and Physiopathology of the Digestive Tract, Department of Anatomy and Cell BiologyUniversité de SherbrookeQuébecCanada

Personalised recommendations