Summary
Gingival tissue from healthy adult human donors was used as a source of epithelial cells for culture. An overnight incubation of this tissue with dispase facilitated the mechanical separation of the surface epithelium from the underlying fibrous connective tissue. This step minimized culture contamination with fibroblasts. The epithelium was then trypsinized to prepare a single cell suspension. The cell pellets were collected by centrifugation and resuspended in keratinocyte growth medium, incubated at 37° C and 5% CO2 in a humid atmosphere. Primary cultures grew in small islands that coalesced at confluency. Immunohistochemistry demonstrated uniform staining of the cells with antibodies to keratins of stratified squamous epithelium. Ultrastructurally, the cells contained distinct intermediate filaments. When cells were grown in media with low calcium (0.15 mM), cell-to-cell contacts were via interlacing papillary projections with no desmosomes. However, when cells were grown under physiologic calcium (1.2 mM), desmosomes were prominent and well developed. Cells were maintained in culture for over 100 d (7 passages).
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Boyce, S. T.; Ham, R. G. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. J. Invest. Dermatol. 81:335–405; 1983.
Boynton, A. L.; Whitfield, J. F.; Isaacs, R. J., et al. Control of 3T3 cell proliferation by calcium. In Vitro 10:12–17; 1974.
Boynton, A. L.; Whitfield, J. F.; Isaacs, J. R., et al. The control of human wl-38 cell proliferation by extracellular calcium and its elimination by sv-40 virus-induced proliferative transformation. J. Cell Physiol. 92:241–247; 1977.
Chen, T. R. In situ detection of mytoplasma contamination in cell cultures by fluorescent Hoechst 3325 8 stain. Exp. Cell Res. 104:255–762; 1977.
Gilchrest, B. A.; Nemore, R. E.; Maciag, T. Growth of human keratinocytes on fibronectin coated plates. Cell Biol. Int. Rep. 4:1009–1016; 1980.
Gown, A. M.; Vogel, A. M. Monoclonal antibodies to human intermediate filament proteins. Distribution of filament proteins in normal human tissue. Am. J. Pathol. 114:309–321; 1984.
Gusterson, B. A.; Edwards, P. A. W.; Foster, C. S., et al. The selective culture of keratinocytes using a cytotoxic antifibroblast monoclonal antibody. Br. J. Dermatol. 105:273–277; 1981.
Hennings, H.; Michael, D.; Cheng, C., et al. Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell 19:245–254; 1980.
Kaighn, M. E.; Camalier, R. F.; Bertolero, F., et al. Spontaneous establishment and characterization of mouse keratinocyte cell lines in serum-free medium. In Vitro Cell. Dev. Biol. 24:845–854; 1988.
Kleinman, H.; McGravey, M. L.; Hassell, J. R., et al. Basement membrane complexes with biological activity. Biochemistry 25:312–318; 1986.
Lillie, J. H.; MacCallum, D. K.; Jepsen, A. Fine structure of subcultivated stratified squamous epithelium grown on collagen rafts. Exp. Cell. Res. 125:153–165; 1980.
Lillie, J. H.; MacCallum, D. K. The behavior of subcultivated stratified squamous epithelial cells on reconstituted extracellular matrices: initial interaction. Eur. J. Cell Biol. 29:50–60; 1982.
Liu, S. C.; Karasek, M. Isolation and growth of adult human epidermal keratinocytes in cell culture. J. Invest. Dermatol. 71:157–162; 1978.
McKeehan, W. J.; Ham, R. G. Calcium and magnesium ions and the regulation of multiplication in normal and transformed cells. Nature 275:756–758; 1958.
Muller-Glauser, W.; Preisig, E. The effect of cholera toxin and epidermal growth factor on the “in vitro” growth of human oral epithelial cells. Arch. Oral Biol. 28:765–771; 1983.
Rheinwald, J. G.; Green, H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331–344; 1975.
Salonen, J. L.; Kautsky, M. B.; Ode, B. A. Changes in cell phenotype during regeneration of junctional epithelium of human gingiva in vitro. J. Periodont. Res. 24:1–8; 1989.
Southgate, J.; Williams, H. K.; Trejdosiewicz, L. K., et al. Primary culture of human oral epithelial cells. Growth requirement and expression of differentiated characteristics. Lab. Invest. 56:211–223; 1987.
Stenn, K. S.; Link, R.; Moellmann, G., et al. Dispase, a neutral protease from bacillus polymyxa, is a powerful fibronectinase and type IV collagen. J. Invest. Dermatol. 93:287–290; 1989.
Taichman, L.; Reilly, S.; Garant, P. R. In vitro cultivation of human oral keratinocytes. Arch. Oral Biol. 24:335–341; 1979.
Tupper, J. T.; del Rosso, M.; Hazelton, B., et al. Serum-stimulated changes in calcium transport and distribution in mouse 3T3 cells and their modification by dibutyryl cyclic AMP. J. Cell. Physiol. 95:71–84; 1978.
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This work was supported by Biomedical Research grant RR 05346 from the National Institute of Health, Bethesda, MD, and the University of Washington Graduate School Research Fund.
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Oda, D., Watson, E. Human oral epithelial cell culture I. Improved conditions for reproducible culture in serum-free medium. In Vitro Cell Dev Biol 26, 589–595 (1990). https://doi.org/10.1007/BF02624208
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DOI: https://doi.org/10.1007/BF02624208