Decellularized Bovine Corneal Posterior Lamellae as Carrier Matrix for Cultivated Human Corneal Endothelial Cells
Decellularized Bovine Corneal Posterior Lamellae as Carrier Matrix for Cultivated Human Corneal Endothelial Cells
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DOI：
10.3109/02713683.2011.644382
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发表时间：
2012-03-01
影响因子：
2
通讯作者：
Yoeruek, Efdal
中科院分区：
文献类型：
--
作者：
Bayyoud, Tarek;Thaler, Sebastian;Yoeruek, Efdal
目的：目的：探讨脱细胞牛角膜（DBCs）作为培养和移植人角膜内皮细胞（HCECs）的载体材料的可行性。方法：采用0.1%乙二胺四乙酸（EDTA）、10 KIU/mL抑肽酶和0.3%十二烷基硫酸钠（SDS）对10只牛角膜进行脱细胞处理。进行苏木精-伊红（HE）和4，6-二脒基-2-苯基吲哚（DAPI）染色，以确认不存在牛细胞。使用DNA纯化试剂盒进行定量分析，以确定脱氧核糖核酸（DNA）的水平。从人供体眼睛收获HCEC并接种在DBC的后弹力膜上。在孵育6小时后以及第1、4、7、10和14天评估细胞形态。结果：HE染色和DAPI染色显示，牛角膜基质层和后弹力层中的细胞基本被去除，细胞膜中的细胞数量明显减少。观察到显著的DNA减少（脱细胞前的平均值为365.3 +/-88.6ng/mg，脱细胞后的平均值为23.2 +/-7.9ng/mg，p < 0.001）。HCEC在DBCs上形成连续的、有活力的、主要为多边形的单层，平均细胞密度为2380 +/- 179个细胞/mm（2）。免疫组化分析显示AE 5、VIII型胶原、ZO-1、CX-43、Na+/HCO 3-和Na+/K+-ATPase.Conclusions阳性染色。因此，DBCs可能是体外扩增HCECs的有前途的支架。该异种基质可用于治疗孤立性角膜内皮疾病。
Purpose: To evaluate the potential of decellularized bovine corneas (DBCs) as a carrier matrix for cultivating and transplanting human corneal endothelial cells (HCECs).Methods: Posterior lamellae of ten bovine corneas were decellularized using ethylene diamin tetra-acetic acid (EDTA, 0.1%), aprotinin (10 KIU/mL) and 0.3% sodium dodecyl sulphate (SDS). Hematoxylin-eosin (HE) and 4,6-diamidino-2-phenylindole (DAPI) staining was done to confirm the absence of bovine cells. Quantitative analysis was performed to determine levels of desoxyribonucleic acid (DNA) using a DNA Purification Kit. HCECs were harvested from human donor eyes and seeded on the Descemet's membrane of the DBCs. Cell morphology was assessed after 6 h of incubation, and at days 1, 4, 7, 10 and 14. Expression of zonula occludens-1 (ZO-1), connexin-43 (CX-43), Na+/K+-adenosine triphosphatase (Na+/K+-ATPase), natrium hydrogen carboanhydrase (Na+/HCO3-), collagen type VIII, collagen type IV and cytokeratin-3 (AE5) were analyzed by immunohistochemistry.Results: HE staining and DAPI staining showed that bovine cells were substantially removed from the stroma and Descemet's membrane. A significant DNA reduction (mean before decelluraziation 365.3 +/- 88.6 ng/mg, mean after decelluarization 23.2 +/- 7.9 ng/mg, p < 0.001) was observed. HCECs formed a continuous, viable, predominantly polygonal monolayer with a mean cell density of 2380 +/- 179 cells/mm(2) on DBCs. Immunohistochemistry analysis demonstrated positive staining for AE5, collagen type VIII, ZO-1, CX-43, Na+/HCO3-, and Na+/K+-ATPase.Conclusions: Phenotypical properties of HCECs on DBCs imply that the HCEC sheets are capable of maintaining an intact barrier and ionic pump function in vitro. DBCs might, therefore, be a promising scaffold for ex vivo expansion of HCECs. This xenogeneic substrate might be used for therapy of isolated corneal endothelial diseases.