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In Vitro Cellular & Developmental Biology

, Volume 23, Issue 7, pp 465–473 | Cite as

Growth of exocrine acinar cells on a reconstituted basement membrane gel

  • Constance Oliver
  • Judith F. Waters
  • Carolyn L. Tolbert
  • Hynda K. Kleinman
Article

Summary

Methods have been developed for culturing a dividing population of morphologically differentiated rat parotid, lacrimal, and pancreatic acinar cells in vitro. Isolated acinar cells were plated onto tissue culture dishes coated with a three-dimensional, reconstituted basement membrane gel. After attachment in Ham’s nutrient mixture F12, the cells were cultured at 35°C in F12 supplemented with 10% heat inactivated rat serum, epidermal growth factor, dexamethasone, insulin, transferrin, selenium, putrescine, reduced glutathione, ascorbate, penicillin, streptomycin, and the appropriate secretagogue. Under these conditions, the cells attached rapidly and DNA synthesis was initiated within 2 to 3 d. Although the cells flattened on the substratum, they continued to maintain their differentiated morphology. The cells contained secretory granules, and the secretory enzymes peroxidase and amylase could be detected. The use of a reconstituted basement membrane gel proved critical for the attachment and growth of exocrine acinar cells.

Key words

pancreas parotid gland exorbital lacrimal gland basement membrane 

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References

  1. 1.
    Amsterdam, A.; Jamieson, J. D. Studies on dispersed pancreatic exocrine cells. I. Dissociation technique and morphologic characteristics of separated cells. J. Cell Biol. 63:1037–1056; 1974.PubMedCrossRefGoogle Scholar
  2. 2.
    Barka, T.; Van Dor Noen, H. Dissociation of rat parotid gland. Lab. Invest. 32:373–381; 1975.PubMedGoogle Scholar
  3. 3.
    Baron-VanEvercooren, A.; Kleinman, H. K.; Ohno, S., et al. Nerve growth factor, laminin, and fibronectin promote neurite growth in human fetal sensory ganglia cultures. J. Neurosci. Res. 8:179–193; 1982.CrossRefGoogle Scholar
  4. 4.
    Barnes, D.; Sato, G. Serum-free cell culture: a unifying approach. Cell 22:649–655; 1980.PubMedCrossRefGoogle Scholar
  5. 5.
    Bissell, M. J.; Hall, H. G.; Parry, G. How does the extracellular matrix direct gene expression. J. Theor. Biol. 99:31–68; 1982.PubMedCrossRefGoogle Scholar
  6. 6.
    Bottenstein, J.; Hayashi, I.; Hutchings, S., et al. The growth of cells in serum-free hormone-supplemented medium. In: Jakoby, W. B.; Pastan, I. H., eds. Methods in enzymology, New York: Academic Press; 1979:94–109.Google Scholar
  7. 7.
    Brannon, P. M.; Orrison, B. M.; Kretchmer, N. Primary cultures of rat pancreatic acinar cells in serum-free medium. In Vitro 21:6–14; 1985.Google Scholar
  8. 8.
    Burton, K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62:315–322; 1956.PubMedGoogle Scholar
  9. 9.
    Carpenter, G.; Cohen, S. Epidermal growth factor. Ann Rev. Biochem. 48:193–216; 1979.PubMedCrossRefGoogle Scholar
  10. 10.
    Cherington, P. V. Regulation of fibroblast growth by multiple growth factors in serum-free medium. In: Mather, J. P., ed. Mammalian cell culture. New York: Plenum Press; 1984:16–52.Google Scholar
  11. 11.
    Di Pasquale, A.; White, D.; McGuire, J. Epidermal growth factor stimulates putrascine transport and ornithine decarboxylase activity in cultivated human fibroblasts. Exp. Cell Res. 116:317–323; 1978.CrossRefGoogle Scholar
  12. 12.
    Ehrmann, R. L.; Gey, G. O. The growth of cells on a transparent gel of reconstituted rat-tail collagen. JCNI 16:1375–1404; 1956.Google Scholar
  13. 13.
    Emerman, J. T.; Burwen, S. J.; Pitelka, D. R. Substrate properties influencing ultrastructural differentiation of mammary epithelial cells in culture. Tissue Cell 11:109–119; 1979.PubMedCrossRefGoogle Scholar
  14. 14.
    Emerman, J. T.; Pitelka, D. R. Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes. In Vitro 13:316–328; 1977.PubMedCrossRefGoogle Scholar
  15. 15.
    Fahimi, H. D. Cytochemical localization of peroxidatic activity of catalase in rat hepatic microbodies (peroxisomes). J. Cell Biol. 43:275–288; 1969.PubMedCrossRefGoogle Scholar
  16. 16.
    Fleckman, P.; Langdon, R.; McGuire, J. Epidermal growth factor stimulates ornithine decarboxylase activity in cultured mammalian keratinocytes. J. Invest. Dermatol. 82:85–89; 1984.PubMedCrossRefGoogle Scholar
  17. 17.
    Germinario, R. J.; McQuillan, A.; Oliveira, M., et al. Enhanced insulin stimulation of sugar transport and DNA synthesis by glucocorticoids in cultured human skin fibroblasts. Arch. Biochem. Biophys. 226:498–505; 1983.PubMedCrossRefGoogle Scholar
  18. 18.
    Gospodarowicz, D. Growth factors and their actionin vivo andin vitro. J. Pathol. 141:201–233; 1983.PubMedCrossRefGoogle Scholar
  19. 19.
    Gospodarowicz, D.; Tauber, J. P. Growth factors and the extracellular matrix. Endocrinol. Rev. 1:201–227; 1980.Google Scholar
  20. 20.
    Hadley, M. A.; Byers, S. W.; Suares-Quian, C. A., et al. Sertoli cell differentiation, testicular cord formation, and germ cell developmentin vitro. J. Cell Biol. 101:1511–1522; 1985.PubMedCrossRefGoogle Scholar
  21. 21.
    Hay, E. D. Cell-matrix interaction in the embryo: cell shape, cell surface, cell skeletons, and their role in differentiation. In: Trelstad, R. L., ed. New York: Alan R. Liss; 1984:1–31.Google Scholar
  22. 22.
    Ham, R. G.; McKeehan, W. L. Development of improved media and culture conditions for clonal growth of normal diploid cells. In Vitro 14:11–22; 1978.PubMedCrossRefGoogle Scholar
  23. 23.
    Heby, O. Role of polyamines in the control of cell proliferation and differentiation. Differentiation 19:1–20; 1981.PubMedCrossRefGoogle Scholar
  24. 24.
    Herzog, V.; Sies, H.; Miller, F. Exocytosis in secretory cells of rat lacrimal gland. Peroxidase release from lobules and isolated cells upon cholinergic stimulation. J. Cell Biol. 70:692–706; 1976.PubMedCrossRefGoogle Scholar
  25. 25.
    Kanamura, S.; Barka, T. Short term culture of dissociated rat submandibular gland cells. Lab. Invest. 32:366–372; 1975.PubMedGoogle Scholar
  26. 26.
    Karnovsky, M. J. Use of ferrocyanide-reduced osmium tetroxide in electron microscopy. J. Cell Biol. 51:146a; 1971.Google Scholar
  27. 27.
    Kleinman, H. K.; Klebe, R. J.; Martin, G. R. Role of collagenous matrices in the adhesion and growth of cells. J. Cell Biol. 88:473–485; 1981.PubMedCrossRefGoogle Scholar
  28. 28.
    Kleinman, H. K.; McGarvey, M. L.; Hassell, J. R., et al. Basement membrane complexes with biological activity. Biochemistry 25:312–318; 1986.PubMedCrossRefGoogle Scholar
  29. 29.
    Korc, M.; Iwamoto, Y.; Sankaran, H., et al. Insulin action in pancreatic acini from streptozotocin-treated rats. I. Stimulation of protein synthesis. Am. J. Physiol. 240:G56-G62; 1981.PubMedGoogle Scholar
  30. 30.
    Korc, M.; Owerbach, D.; Quinto, C., et al. Pancreatic islet-acinar cell interaction: Amylase messenger RNA levels are determined by insulin. Science 213:351–353; 1981.PubMedCrossRefGoogle Scholar
  31. 31.
    Lee, E. Y.-H.; Parry, G.; Bissell, M. J. Modulation of secreted proteins of mouse mammary epithelial cells by the collagenous substrata. J. Cell Biol. 98:146–155; 1984.PubMedCrossRefGoogle Scholar
  32. 32.
    Logsdon, C. D.; Williams, J. A. Pancreatic acinar cells in monolayer culture: direct trophic effects of caerulein in vitro. Am. J. Physiol. 250;G440-G447; 1986.PubMedGoogle Scholar
  33. 33.
    Logsdon, C. D.; Moessner, A.; Williams, J. A. Glucocorticoids increase amylase mRNA levels, secretory organelles, and secretion in pancreatic AR42J cells. J. Cell Biol. 100:1200–1208; 1985.PubMedCrossRefGoogle Scholar
  34. 34.
    Mangos, J. A.; McSherry, N. R.; Butcher, F., et al. Dispersed rat parotid acinar cells. I. Morphological and functional characterization. Am. J. Physiol. 229:553–559; 1975.PubMedGoogle Scholar
  35. 35.
    Manjusri, D. Epidermal growth factor: mechanisms of action. Int. Rev. Cytol. 78:233–256; 1982.CrossRefGoogle Scholar
  36. 36.
    Moriarity, D. M.; DiSorbo, D. M.; Litwack, G., et al. Epidermal growth factor stimulation of ornithine decarboxylase activity in a human hepatoma cell line. Proc. Natl. Acad. Sci. USA 78:2752–2756; 1981.PubMedCrossRefGoogle Scholar
  37. 37.
    Morris, J. G.; Cripe, W. S.; Chapman, H. L., et al. Selenium deficiency in cattle associated with Heinz bodies and anemia. Science 223:491–492; 1984.PubMedCrossRefGoogle Scholar
  38. 38.
    Nicholas, K. R.; Sankaran, L.; Topper, Y. J. A unique and essential role for insulin in the phenotypic expression of rat mammary epithelial cells unrelated to its function in cell maintenance. Biochim. Biophys. Acta 763:309–314; 1983.PubMedCrossRefGoogle Scholar
  39. 39.
    Oliver, C. Isolation and maintenance of differentiated exocrine gland acinar cells in vitro. In Vitro 16:297–305; 1980.PubMedGoogle Scholar
  40. 40.
    Osterman, J.; Hammond, J. M. Effects of epidermal growth factor, fibroblast growth factor and bovine serum albumin on ornithine decarboxylase activity of porcine granulosa cells. Horm. Metab. Res. 11:485–488; 1979.PubMedCrossRefGoogle Scholar
  41. 41.
    Paranjpe, M. S.; DeLarco, J. E.; Todaro, G. J. Retinoids block ornithine decarboxylase induction in cells treated with the tumor promotor TPA or the peptide growth hormones, EGF and SGF. Biochem. Biophys. Res. Commun. 94:586–591; 1980.PubMedCrossRefGoogle Scholar
  42. 42.
    Parry, G.; Lee, E. Y.-H.; Farson, D., et al. Collagenous substrata regulate the nature and distribution of glycosaminoglycans produced by differentiated cultures of mouse mammary epithelial cells. Exp. Cell Res. 156:487–499; 1985.PubMedCrossRefGoogle Scholar
  43. 43.
    Perez Infante, V.; Mather, J. P. The role of transferrin in the growth of testicular cell lines in serum free medium. Exp. Cell Res. 142:325–332; 1982.PubMedCrossRefGoogle Scholar
  44. 44.
    Petersen, O. H.; Iwatsuki, N.; Philpott, H. G., et al. Membrane potential and conductance changes evoked by hormones and neurotransmitters in mammalian exocrine gland cells. In: Hand, A. R.; Oliver, C., eds. Basic mechanisms of cellular secretion. Methods in cell biology, vol. 23. New York: Academic Press; 1981:513–531.Google Scholar
  45. 45.
    Pierre, K. J.; Tung, K. K.; Nadj, H. A new enzymatic kinetic method for determination of α-amylase. Clin. Chem. 22:1219; 1976.Google Scholar
  46. 46.
    Pledger, W. J.; Estes, J. E.; Howe, R. H., et al. Serum factor requirements for the initiation of cellular proliferation. In: Mather, J. P., ed. Mammalian cell culture. New York: Plenum Press; 1984:1–16.Google Scholar
  47. 47.
    Reynolds, E. S. The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J. Cell Biol. 17:208–212; 1963.PubMedCrossRefGoogle Scholar
  48. 48.
    Richman, R. A.; Claus, T. H.; Pilkis, S. J., et al. Hormonal stimulation of DNA synthesis in primary cultures of adult rat hepatocytes. Proc. Natl. Acad. Sci. USA 73:3589–3593; 1976.PubMedCrossRefGoogle Scholar
  49. 49.
    Rillema, J. A.; Linebaugh, B. E. Characteristics of the insulin stimulation of DNA, RNA and protein metabolism in cultured human mammary carcinoma cells. Biochim. Biophys. Acta 475:74–80; 1977.PubMedGoogle Scholar
  50. 50.
    Sunkara, P. S.; Rao, P. N. Differential cell cycle response of normal and transformed cells to polyamine limitation. Adv. Polyamine Res. 3:347–364; 1981.Google Scholar
  51. 51.
    Spurr, A. R. A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26:31–43; 1969.PubMedCrossRefGoogle Scholar
  52. 52.
    Theoharides, T. C.; Canellakis, Z. N. Spermidine inhibits induction of ornithine decarboxylase by cyclic AMP but not by dexamethasone in rat hepatoma cells. Nature 255:733–734; 1975.PubMedCrossRefGoogle Scholar
  53. 53.
    Van Nest, G.; Raman, R. K.; Rutter, W. J. Effects of dexamethasone and 5-bromodeoxyuridine on protein synthesis and secretion duringin vitro pancreatic development. Dev. Biol. 98:295–303; 1983.PubMedCrossRefGoogle Scholar
  54. 54.
    Wakimoto, H.; Oka, T. Involvement of collagen formation in the hormonally induced functional differentiation of mouse mammary gland in organ culture. J. Biol. Chem. 258:3375–3379; 1983.Google Scholar

Copyright information

© Tissue Culture Association, Inc 1987

Authors and Affiliations

  • Constance Oliver
    • 1
  • Judith F. Waters
    • 1
  • Carolyn L. Tolbert
    • 1
  • Hynda K. Kleinman
    • 2
  1. 1.Laboratory of Oral Biology and PhysiologyNational Institute of Dental Research, National Institutes of HealthBethesda
  2. 2.Laboratory of Developmental Biology and Anomalies (H. K. K.)National Institute of Dental Research, National Institutes of HealthBethesda

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