Abstract
A method has been established for the routine culture for normal oral epithelial (NOE) cells based on simple methodology and commercially available materials. Normal mucosa was obtained from surgical resections of cancer patients, oropharyngeal mucosa, or pediatric tonsillectomies, anterior tonsillar pillar. NOE cells were allowed to outgrow using explant outgrowth techniques on whole mucosa or the dispased epithelial component. Outgrowth in AmnioMax-C100 medium (Gibco) followed by cell passaging in KGM (Clonetics) using Primaria (Falcon) tissue culture dishes produced reproducible growth, a reflection of a high rate of cell proliferation. Using this technique, cells maintain log phase growth for 2 cell passages, a minimum of 10–20 population doublings, and over 108 cells can be easily obtained from most specimens, if desired. The growth potential of NOE cells from individual clinical specimens can be predicted by cell size on a particle counter. If the mean population diameter is approximately 15 μm or less, cells exhibit good log phase growth whereas if the diameter is greater than 16 um, growth is poor. NOE cells cultured by this method show typical epithelial morphology, have desmosomal junctions, rest on an extracellular matrix, are positive for cytokeratin expression and are growth inhibited by TGF-β, a normal inhibitory growth regulator. In summary, using this technique, epithelial cells from human oral mucosa can be easily generated in sufficient quantity and quality for cell biological, biochemical and molecular studies.
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Abbreviations
- NOE:
-
normal oral epithelia
- CE:
-
cornified envelopes
- IHC:
-
immunohistochemistry
- TGF-β:
-
transforming growth factor beta
References
Southgate J, Williams HK, Trejdosiewicz LK, Hodges GM (1987). Primary culture of human oral epithelial cells. Growth requirements and expression of differentiated characteristics. Lab Invest 56: 211–223.
Rheinwald JG, Green H (1975). Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizinig colonies from single cells. Cell 6: 331–343.
Lindberg K, Rheinwald JG (1990). Three distinct keratinocyte subtypes identified in human oral epithelium by their patterns of keratin expression in culture and in xenografts. Diff 45: 230–241.
De Luca M, D'Anna F, Bondanza S, Franzi AT, Cancedda R (1988). Human epithelial cells induce human melanocyte growth in vitro but only skin keratinocytes regulate its proper differentiation in the absence of dermis. J Cell Bio 107: 1919–1926.
Boyce ST, Ham RG (1985). Cultivation, frozen storage, and clonal growth of normal human epidermal keratinocytes in serum-free media. J Tissue Cult Meth 9: 83–93.
Sundqvist K, Liu Y, Arvidson K, Ormstad K, Nilsson L, Toftgård R, Grafström RC (1991). Growth regulation of serum-free cultures of epithelial cells from normal human buccal mucosa. In Vitro Cell Dev Biol 27A: 562–568.
Oda D, Watson E (1990). Human oral epithelial cell culture I. Improved conditions for reproducible culture in serum-free medium. In Vitro Cell Dev Biol 26: 589–595.
Kasperbauer JL, Neel HB, Scott RE (1990). Proliferation and differentiation characteristics of normal human squamous mucosal cells of the upper aerodigestive tract. Ann Otol Rhinol Laryngol 99: 29–37.
Rollins BJ, O'Connell TM, Bennett G, Burton LE, Stiles CD, Rheinwald JG (1989). Environment-dependent growth inhibition of human epidermal keratinocytes by recombinant human transforming growth factor-beta. J Cell Physiol 139: 455–462.
Shillitoe EJ, Schantz SP, Spitz MR, Hecht SS (1993). Environmental carcinogenesis and its prevention: the head and neck cancer model. Cancer Res 53: 2189–2191.
Sacks PG, Parnes SM, Gallick GE, Mansouri Z, Lichtner R, Satya-Prakash KL, Pathak S, Parsons DF (1988). Establishment and characterization of two new squamous cell carcinoma cell lines derived from tumors of the head and neck. Cancer Res 48: 2858–2866.
Rice RH, Green H (1979). Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions. Cell 18: 681–694.
Schmidt R, Cathelineau C, Cavey MT, Dionisus V, Michel S, Shroot B, Reichert U (1989). Sodium butyrate selectively antagonizes the inhibitory effect of retinoids on cornified envelope formation in cultured human keratinocytes. J Cell Physiol 140: 218–287.
Xu L, Chen Y-T, Huvos AG, Zlotolow IM, Rettig WJ, Old LJ, Garin-Chesa P (1994). Overexpression of p53 protein in squamous cell carcinomas of head and neck without apparent gene mutations. Diag Mol Pathol 3: 83–92.
Sundqvist K, Kulkarni P, Hybbinette SS, Bertolero F, Liu Y, Grafström RC (1991). Serum-free growth and karyotype analyses of cultured normal and tumorous (SqCC/Y1) human buccal epithelial cells. Cancer Comm 3: 331–340.
Basset-Seguin N, Culard JF, Kerai C, Bernard F, Watrin A, Demaille J, Guilhou JJ (1990). Reconstituted skin in culture: a simple method with optimal differentiation. Diff 44: 232–238.
Prunieras M (1983). Keratinocytes synthesize basallamina proteins in culture. J Invest Dermatol 81: 74s-81s.
Barrandon Y, Green H (1985). Cell size as a determinant of the clone-forming ability of human keratinocytes. Proc Nat Acad Sci USA 82: 5390–5394.
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Xu, L., Schantz, S.P., Edelstein, D. et al. A simplified method for the routine culture of normal oral epithelial (NOE) cells from upper aerodigestive tract mucosa. Methods Cell Sci 18, 31–39 (1996). https://doi.org/10.1007/BF00123521
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DOI: https://doi.org/10.1007/BF00123521