Institute of Biomedical Technology, University of Tampere, Tampere, FinlandBioMediTech, Tampere, FinlandDepartment of Eye, Ear, and Oral Diseases, Tampere University Hospital, Tampere, FinlandSILK, Department of Ophthalmology, University of Tampere, Tampere, FinlandEye Center, Tampere University Hospital, Tampere, Finland.
Acta Ophthalmol. 2013 Dec;91(8):744-50. doi: 10.1111/j.1755-3768.2012.02523.x. Epub 2012 Sep 11.
Ocular surface reconstruction with cultivated oral mucosal epithelial transplantation technique is a viable treatment option for severe ocular surface injuries and diseases with limbal stem cell deficiency. Currently, this technique is based on utilization of xenogenic, allogenic or undefined components such as murine 3T3 feeders, serum and amniotic membrane. In this study, we aimed to find a more defined culture method to generate stratified human oral mucosal epithelium.
In this study, we have examined the formation of stratified cell sheets from human oral mucosal epithelial cells under serum-free culture environment both in the absence and presence of fibroblast-conditioned culture medium and elevated epidermal growth factor (EGF) concentration.
In all examined culture conditions, the cultivated oral epithelial cells formed a stratified tissue, which was positive for keratins K3/12, K4 and K13. The tissue-engineered oral epithelia also expressed proliferation and progenitor markers Ki67 and p63 in the basal layer of the cell sheets, suggesting that the epithelia still had regenerative capacity. The cultures presented expression of tight junction proteins ZO-1 and occludin and high transepithelial electrical resistance values.
In this culture method, we have been able to produce stratified cell sheets successfully without serum, conditioning of the medium or increased EGF concentration. We provide a novel protocol to produce tight multi-layered epithelium with proliferative potential, which can be easily adapted for cultivated oral mucosal epithelial transplantation.
利用培养的口腔黏膜上皮移植技术进行眼表重建，是治疗严重眼表损伤和角膜缘干细胞缺乏症的一种可行方法。目前，该技术基于利用异种、同种或未定义的成分，如鼠 3T3 饲养细胞、血清和羊膜。在这项研究中，我们旨在寻找一种更明确的培养方法来生成分层的人口腔黏膜上皮。
在本研究中，我们在无血清培养环境中，检查了人口腔黏膜上皮细胞在无成纤维细胞条件培养基和高表皮生长因子（EGF）浓度存在下形成分层细胞片的情况。
在所有检查的培养条件下，培养的口腔上皮细胞形成了一种分层组织，该组织对角蛋白 K3/12、K4 和 K13 呈阳性。组织工程化的口腔上皮细胞片的基底层还表达增殖和祖细胞标志物 Ki67 和 p63，表明上皮细胞仍具有再生能力。培养物表达紧密连接蛋白 ZO-1 和闭合蛋白，并具有高的跨上皮电阻值。
在这种培养方法中，我们成功地在无血清、无条件培养基或增加 EGF 浓度的情况下生产出了分层细胞片。我们提供了一种生产具有增殖潜力的紧密多层上皮的新方案，该方案可轻松适应口腔黏膜上皮的培养移植。
