Bao Kai, Akguel Baki, Bostanci Nagihan
Oral Translational Research Unit, Institute of Oral Biology, Center of Dental Medicine, University of Zurich, Zurich, Switzerland.
Cells Tissues Organs. 2014;199(4):228-37. doi: 10.1159/000363694. Epub 2014 Nov 29.
In vitro studies using 3D co-cultures of gingival cells can resemble their in vivo counterparts much better than 2D models that typically only utilize monolayer cultures with short-living primary cells. However, the use of 3D gingival models is still limited through lack of appropriate cell lines. We aimed to establish immortalized cell line models of primary human gingival epithelium keratinocytes (HGEK) and gingival fibroblasts (GFB). Immortalized cell lines (HGEK-16 and GFB-16) were induced by E6 and E7 oncoproteins of human papillomavirus. In addition, 3D multilayered organotypic cultures were formed by embedding GFB-16 cells within a collagen (Col) matrix and seeding of HGEK-16 cells on the upper surfaces. Cell growth was analyzed in both immortalized cell lines and their parental primary cells. The expression levels of cell type-specific markers, i.e. cytokeratin (CK) 10, CK13, CK16, CK18, CK19 for HGEK-16 and Col I and Col II for GFB-16, were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Expansion of the primary cultures was impeded at early passages, while the transformed immortalized cell lines could be expanded for more than 30 passages. In 3D cultures, immortalized HGEK formed a multilayer of epithelial cells. qRT-PCR showed that cell-specific marker expression in the 3D cultures was qualitatively and quantitatively closer to that in human gingival tissue than to monolayer cultures. These results indicate that immortalized gingival fibroblastic and epithelial cell lines can successfully form organotypic multilayered cultures and, therefore, may be useful tools for studying gingival tissue in vitro.
使用牙龈细胞的三维共培养进行的体外研究，比通常仅利用原代短寿命细胞单层培养的二维模型，能更好地模拟其体内对应物。然而，由于缺乏合适的细胞系，三维牙龈模型的应用仍然有限。我们旨在建立原代人牙龈上皮角质形成细胞（HGEK）和牙龈成纤维细胞（GFB）的永生化细胞系模型。人乳头瘤病毒的E6和E7癌蛋白诱导产生了永生化细胞系（HGEK - 16和GFB - 16）。此外，通过将GFB - 16细胞包埋在胶原蛋白（Col）基质中，并将HGEK - 16细胞接种在其表面，形成了三维多层器官型培养物。对永生化细胞系及其亲代原代细胞的细胞生长进行了分析。通过定量实时聚合酶链反应（qRT - PCR）评估了细胞类型特异性标志物的表达水平，即HGEK - 16的细胞角蛋白（CK）10、CK13、CK16、CK18、CK19以及GFB - 16的I型胶原和II型胶原。原代培养物在早期传代时生长受阻，而转化后的永生化细胞系可以传代超过30次。在三维培养中，永生化的HGEK形成了多层上皮细胞。qRT - PCR表明三维培养中细胞特异性标志物的表达在定性和定量上比单层培养更接近人牙龈组织。这些结果表明，永生化的牙龈成纤维细胞和上皮细胞系能够成功形成器官型多层培养物，因此可能是体外研究牙龈组织的有用工具。
