Human Anterior Lens Capsule as Carrier Matrix for Cultivated Human Corneal Endothelial Cells
Human Anterior Lens Capsule as Carrier Matrix for Cultivated Human Corneal Endothelial Cells
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DOI：
10.1097/ico.0b013e31818c2c36
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发表时间：
2009-05-01
期刊：
影响因子：
2.8
通讯作者：
Szurman, Peter
中科院分区：
文献类型：
--
作者：
Yoeruek, Efdal;Saygili, Oguzhan;Szurman, Peter
目的：目的：探讨人透镜膜（HALC）作为角膜内皮细胞（HCECs）培养和移植载体的可行性。从人供体眼睛收获HCEC并接种在HALC上。在孵育6小时后以及第1、3、5和7天，用相差显微镜评估细胞形态。将在每个胶囊处计数的HCEC的数量合并，并且将密度表示为培养7天后的细胞/平方毫米。在融合期间孵育7天后，还进行了活/死活力/细胞毒性测定并通过荧光显微镜进行分析。结果：HALCs接种7 d后，HCECs生长至汇合，形成一个连续的单层，平均细胞密度为3012 +/- 109个细胞/mm 2，与天然角膜内皮细胞相似。免疫组化分析显示AE 5、闭合小带-1、连接蛋白-43和Na+/K+-腺苷三磷酸酶强阳性染色。结论：HALCs上HCECs的细胞密度和形态与健康角膜相似。HALCs上的HCEC的表型性质意味着HCEC片能够在体外维持完整的屏障和离子泵功能。因此，HALCs可能被推荐作为体外扩增HCECs的潜在支架，可能为孤立性角膜内皮疾病的治疗提供自体生物基质。
Purpose: To evaluate the potential of human anterior lens capsule (HALC) as a carrier matrix for cultivating and transplanting human corneal endothelial cells (HCECs).Methods: HALCs obtained from 12 patients during cataract surgery were exposed to enzyme digestion to dissolve the lens epithelium and were plated with the epithelial side up in 6-well plates. HCECs were harvested from human donor eyes and seeded on HALCs. Cell morphology was assessed with a phase-contrast microscope after 6 hours of incubation and at days 1, 3, 5, and 7. The number of HCECs counted at each capsule was pooled, and the density was expressed as cells per square millimeter after 7 days in culture. Live/Dead viability/cytotoxicity assay was also performed and analyzed by fluorescence microscopy after 7 days of incubation during confluence. Expression of zonula occludens-1, connexin-43, Na+/K+-adenosine triphosphatase, and cytokeratin-3 (AE5) were investigated by immunohistochemistry.Results: Seven days after seeding on HALCs, the HCECs were grown to confluence and formed a continuous viable monolayer with a mean cell density of 3012 +/- 109 cells per square millimeter, which mimics native corneal endothelial cells. Immunohistochemistry analysis demonstrated strongly positive staining for AE5, zonula occludens-1, connexin-43, and Na+/K+-adenosine triphosphatase.Conclusions: Cell density and morphology of HCECs on HALCs were similar to those of healthy corneas. Phenotypical properties of HCECs on HALCs imply that the HCEC sheets are capable of maintaining intact barrier and ionic pump functions in vitro. HALCs might, therefore, be recommended as a potential scaffold for ex vivo expansion of HCECs, possibly providing an autologous biologic substrate for therapy of isolated corneal endothelial diseases.