Gursoy Ulvi K, Gursoy Mervi, Könönen Eija, Sintim Herman O, Uitto Veli-Jukka, Syrjänen Stina
Department of Periodontology, Institute of Dentistry, University of Turku, Lemminkäisenkatu 2, 20520, Turku, Finland.
Oral Health Care, Welfare Division, Turku, Finland.
Cytotechnology. 2016 Dec;68(6):2345-2354. doi: 10.1007/s10616-016-0029-4. Epub 2016 Oct 17.
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.
在构建多层上皮细胞时，将细胞在包埋有成纤维细胞的胶原凝胶上贴壁生长至汇合，然后提升至空气中以促进其分层。我们最近证明，牙龈上皮细胞在半透性硝酸纤维素膜上形成均匀的单层，由半固体生长培养基支持，这使得细胞从培养方案开始时就在气-液-固界面生长。在本研究中，目的是进一步改进我们之前的模型，以形成多层牙龈上皮培养模型。所有实验均使用两种不同的上皮细胞系（来自皮肤的HaCaT和来自牙龈的HMK）。两种细胞系均先作为单层培养3天。之后，将角质形成细胞用胰蛋白酶消化、计数并接种到置于半固体生长培养基顶部的无菌半透性硝酸纤维素膜上，形成气-液-固界面供细胞生长。在第1、4和7天，将上皮细胞固定、石蜡包埋并切片，用于常规苏木精-伊红染色和细胞角蛋白（Ck）免疫组织化学染色。在第1天，HMK细胞单层生长，而HaCaT细胞分层形成两到三层的上皮。在第4天，HMK模型中的分层上皮有四到五层，其增殖持续到第7天。HaCaT细胞在第4天形成了密集且增殖较弱的三到四层分层上皮，但在第7天增殖消失。在所有天数，两种模型的Ck5、Ck7和Ck19均呈强阳性，Ck10呈弱阳性。牙龈上皮细胞在由半固体生长培养基支持的半透性硝酸纤维素膜上成功分层。该技术使研究人员能够在气-液-固界面构建均匀的牙龈上皮细胞多层，无需使用胶原凝胶，从而产生一种更具可重复性的方法。
