Dobrowolski Dariusz, Wylegała Edward, Orzechowska-Wylegała Bogusława, Wowra Bogumił, Bielecka Anna, Obuchowicz Ewa, Małecki Andrzej, Gabryel Bozena
Department of Ophthalmology, District Railway Hospital, Katowice, Poland.
Klin Oczna. 2011;113(7-9):249-53.
Ocular surgery based on cultivated corneal epithelium has become a very promising procedure eligible to restore the ocular surface. Analysis of morphologic features and the phenotype of cultivated epithelial cells determines their quality and eligibility of transplantation.
Corneal epithelial cultures were carried out in 25 patients suffering from limbal deficiency after chemical or thermal burns. Fellow healthy eyes were the source of limbal epithelium for the culture. Limbal cells from a 2 mm2 biopsy were seeded on an amniotic membrane after enzymatic pretreatment. Cultures were carried in standard conditions in a supplemented DMEM HAM/F12 medium in the presence of 3T3 fibroblasts. Light microscopy was used to analyze the regularity of the cultivated epithelial layer, histologic examination was used to establish number of epithelial layers, and immunohistochemistry for epithelial and proliferation markers was applied to confirm cell origin and proliferative potential. Staining for cytokeratin 3, 12, 19, connexin 43, and protein p63 was performed.
In 25 donors, 27 cultures of the epithelium were performed. In 2 cases, plates were contaminated. Both cultures were repeated. In 84% of the cultures, regular stratified growth of the epithelium with complete covering of amniotic membrane was observed. In 16% of cultures, growth was not regular, showing differences in the number of cell layers. Staining for cytokeratin 3/12 confirmed the corneal origin of cultivated epithelia. The number of epithelial layers ranged from 3 to 9; the average was 5.3 +/- 1.9 layers.
Cultures of limbal epithelial cells are a valuable source of tissue for restoration of the corneal epithelium.
基于培养角膜上皮的眼外科手术已成为一种非常有前景的恢复眼表的手术方法。对培养上皮细胞的形态特征和表型进行分析可确定其质量和移植适用性。
对25例化学或热烧伤后角膜缘缺陷患者进行角膜上皮培养。健侧健康眼作为培养角膜缘上皮的来源。酶预处理后，将取自2mm²活检组织的角膜缘细胞接种于羊膜上。在添加了3T3成纤维细胞的补充型DMEM HAM/F12培养基中，于标准条件下进行培养。使用光学显微镜分析培养上皮层的规则性，通过组织学检查确定上皮层数，并应用上皮和增殖标志物的免疫组织化学方法来确认细胞来源和增殖潜能。进行细胞角蛋白3、12、19、连接蛋白43和蛋白p63的染色。
在25例供体中，进行了27次上皮培养。2例培养皿被污染，均重新进行了培养。84%的培养物中观察到上皮呈规则的分层生长，羊膜完全被覆盖。16%的培养物生长不规则，细胞层数存在差异。细胞角蛋白3/12染色证实了培养上皮的角膜来源。上皮层数为3至9层；平均为5.3±1.9层。
角膜缘上皮细胞培养是恢复角膜上皮的宝贵组织来源。
