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Construction and characterization of a multilayered gingival keratinocyte culture model: the TURK-U model

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Abstract

In construction of epithelial cells as multilayers, the cells are grown submerged to confluence on fibroblast-embedded collagen gels and, then, lifted to air to promote their stratification. We recently demonstrated that gingival epithelial cells form uniform monolayers on semi-permeable nitrocellulose membranes, supported with a semi-solid growth medium, which allows the cells to grow at an air–liquid–solid interface from the beginning of the culturing protocol. In this study, the aim was to further develop our previous model to form a multilayered gingival epithelial culture model. Two different epithelial cell lines (HaCaT from skin and HMK from gingiva) were used in all experiments. Both cell lines were grown first as monolayers for 3 days. After that, keratinocytes were trypsinized, counted and seeded on a sterile semi-permeable nitrocellulose membrane placed on the top of a semi-solid growth medium, forming an air–liquid–solid interface for the cells to grow. At days 1, 4, and 7, epithelial cells were fixed, embedded in paraffin, and sectioned for routine Hematoxylin-Eosin staining and immunohistochemistry for cytokeratin (Ck). At day 1, HMK cells grew as monolayers, while HaCaT cells stratified forming an epithelium with two to three layers. At day 4, a stratified epithelium in the HMK model had four to five layers and its proliferation continued up to day 7. HaCaT cells formed a dense and weakly proliferating epithelium with three to four layers of stratification at day 4 but the proliferation disappeared at day 7. At all days, both models were strongly positive for Ck5, Ck7, and Ck 19, and weakly positive for Ck10. Gingival epithelial cells stratify successfully on semi-permeable nitrocellulose membranes, supported with a semi-solid growth medium. This technique allows researchers to construct uniform gingival epithelial cell multilayers at an air–liquid–solid interface, without using collagen gels, resulting in a more reproducible method.

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Acknowledgments

The authors are grateful for the skillful technical assistance of Katja Sampalahti and Mariia Valkama (Institute of Dentistry, University of Turku, Turku, Finland). This study was supported by the Turku University Foundation and Finnish Dental Society Apollonia. The authors report no conflicts of interest related to this study.

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Authors and Affiliations

  1. Department of Periodontology, Institute of Dentistry, University of Turku, Lemminkäisenkatu 2, 20520, Turku, Finland

    Ulvi K. Gursoy, Mervi Gursoy & Eija Könönen

  2. Oral Health Care, Welfare Division, Turku, Finland

    Eija Könönen

  3. Department of Chemistry and Purdue Institute for Drug Discovery and Purdue Institute of Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, IN, USA

    Herman O. Sintim

  4. Department of Oral and Maxillofacial Diseases, University of Helsinki, Helsinki, Finland

    Veli-Jukka Uitto

  5. Department of Oral Pathology, Institute of Dentistry, University of Turku, Turku, Finland

    Stina Syrjänen

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  1. Ulvi K. Gursoy
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  2. Mervi Gursoy
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  3. Eija Könönen
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  4. Herman O. Sintim
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  5. Veli-Jukka Uitto
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  6. Stina Syrjänen
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Correspondence to Ulvi K. Gursoy.

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Gursoy, U.K., Gursoy, M., Könönen, E. et al. Construction and characterization of a multilayered gingival keratinocyte culture model: the TURK-U model. Cytotechnology 68, 2345–2354 (2016). https://doi.org/10.1007/s10616-016-0029-4

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  • DOI: https://doi.org/10.1007/s10616-016-0029-4

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