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

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 airliquid-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.

Multi-species oral biofilm promotes reconstructed human gingiva epithelial barrier function

“…This finding can be explained by the observation that biofilm stimulated keratinocyte proliferation (number of Ki67 and PCNA positive cells) thus preventing the cell senescence which was observed in unexposed RHG at day 7. This would explain previous observations by us and others where sterile RHG maintained a relatively thinner epithelium with few proliferating basal keratinocytes, which was more comparable to skin epidermis than the relatively thicker gingiva epithelium 15 , 18 , 26 , 27 . It is possible that the moderate degree of inflammation caused by microbes is enough to stimulate an innate immune response which will result in secretion of inflammatory cytokines which also have mitogenic properties.…”

Regulatory effects of PRF and titanium surfaces on cellular adhesion, spread, and cytokine expressions of gingival keratinocytes

“…Finally, present study used monolayer culture model to evaluate the cell spread and cytokine secretion. Non-differentiating cell monolayers do not simulate gingiva, because they lack of stratification, vertical cell contacts, and differentiation (Gursoy et al, 2016).…”

Matrix Metalloproteinase Response of Dendritic Cell, Gingival Epithelial Keratinocyte, and T-Cell Transwell Co-Cultures Treated with Porphyromonas gingivalis Hemagglutinin-B

“…In particular, a more complex multi-dimensional system that can more closely mimic the in vivo inflammatory response is required to generate a comprehensive understanding of the process underlying periodontal disease [16]. There are numerous co-culture models, each with their own intrinsic advantages and disadvantages, which have been extensively described in the current literature [17,18,19,20,21,22,23]. These models generally consist of autologous cells cultured together (e.g., organoids, spheroids, etc.…”