Department of Ophthalmology, Eberhard-Karls University Tuebingen, Schleichstrasse 12, Tuebingen, Germany.
Curr Eye Res. 2012 Mar;37(3):179-86. doi: 10.3109/02713683.2011.644382.
To evaluate the potential of decellularized bovine corneas (DBCs) as a carrier matrix for cultivating and transplanting human corneal endothelial cells (HCECs).
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(+)/HCO(3)(-)), collagen type VIII, collagen type IV and cytokeratin-3 (AE5) were analyzed by immunohistochemistry.
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(+)/HCO(3)(-), and Na(+)/K(+)-ATPase.
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.
评估脱细胞牛角膜（DBC）作为培养和移植人角膜内皮细胞（HCEC）的载体基质的潜力。
使用乙二胺四乙酸（EDTA，0.1%）、抑肽酶（10KIU/mL）和 0.3%十二烷基硫酸钠（SDS）对 10 只牛眼角膜的后弹力层进行脱细胞处理。使用苏木精-伊红（HE）和 4,6-二脒基-2-苯基吲哚（DAPI）染色来确认牛细胞的缺失。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。从人供体眼收获 HCEC，并在 DBC 的 Descemet 膜上播种。孵育 6 小时后以及第 1、4、7、10 和 14 天评估细胞形态。通过免疫组织化学分析分析紧密连接蛋白-1（ZO-1）、连接蛋白-43（CX-43）、钠钾三磷酸腺苷酶（Na+/K+-ATPase）、钠离子碳酸氢盐（Na+/HCO3-）、VIII 型胶原、IV 型胶原和细胞角蛋白-3（AE5）的表达。
HE 染色和 DAPI 染色显示，牛细胞已从基质和 Descemet 膜中基本去除。观察到 DNA 显著减少（脱细胞前平均值为 365.3±88.6ng/mg，脱细胞后平均值为 23.2±7.9ng/mg，p<0.001）。HCEC 在 DBC 上形成连续的、存活的、主要为多边形的单层，平均细胞密度为 2380±179 个细胞/mm2。免疫组织化学分析显示 AE5、VIII 型胶原、ZO-1、CX-43、Na+/HCO3-和 Na+/K+-ATPase 呈阳性染色。
HCEC 在 DBC 上的表型特性表明，HCEC 片能够在体外维持完整的屏障和离子泵功能。因此，DBC 可能是 HCEC 体外扩增的有前途的支架。这种异种基质可用于治疗孤立性角膜内皮疾病。
