Department of Ophthalmology, Eberhard-Karls University, 72076 Tuebingen, Germany.
Acta Ophthalmol. 2012 Mar;90(2):e125-31. doi: 10.1111/j.1755-3768.2011.02261.x. Epub 2011 Dec 2.
To evaluate the potential use of decellularized porcine corneas (DPCs) as a carrier matrix for cultivating human corneal cells in tissue engineering.
Corneal cells were isolated from human corneoscleral rims. Porcine corneas were decellularized using hypotonic tris buffer, ethylene diamine tetra-acetic acid (EDTA, 0.1%), aprotinin (10 KIU/ml) and 0.3% sodium dodecyl sulphate. Haematoxylin-eosin (HE) and 4,6-diamidino-2-phenylindole (DAPI) staining were performed to confirm removal of the corneal cells. Quantitative analysis was performed to determine levels of desoxyribonucleic acid (DNA) using DNA Purification Kit (Fermentas, St. Leon-Rot, Germany). Alcian blue staining was carried out to analyse the structure of the extracellular matrix (ECM). Corneal stromal cells were injected into the DPCs; limbal corneal epithelial cells and corneal endothelial cells were seeded onto the anterior and posterior surfaces of the DPCs, respectively. Evaluation was undertaken at days 14 and 30. The phenotypical properties of the cultivated corneal cells were investigated using Immunolocalization of type I collagen, keratocan, lumican, cytokeratin 3 (AE5) and type VIII collagen.
Haematoxylin-eosin and DAPI staining showed efficient elimination of porcine corneal cells, whereas alcian blue confirmed gross preservation of the ECM. The quantitative analysis of the DNA content showed a significant reduction (mean before decellularization: 75.45 ± 13.71 ng/mg; mean after decellularization: 9.87 ± 2.04 ng/mg, p < 0.001). All three types of corneal cells were efficiently cultured and expanded on the DPCs.
Decellularized porcine corneas might serve as a potential scaffold for tissue engineering of the cornea, possibly providing xenogenic substrate for corneal transplantation.
评估脱细胞猪角膜（DPC）作为培养人眼角膜细胞的组织工程载体基质的潜力。
从人眼角膜缘分离角膜细胞。使用低渗三缓冲液、乙二胺四乙酸（EDTA，0.1%）、抑肽酶（10 KIU/ml）和 0.3%十二烷基硫酸钠对猪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，以确认去除角膜细胞。使用 DNA 纯化试剂盒（德国 Fermentas，圣莱昂-罗特）进行定量分析，以确定脱氧核糖核酸（DNA）水平。进行阿利新蓝染色，以分析细胞外基质（ECM）的结构。将角膜基质细胞注入 DPC 中；将角膜缘上皮细胞和角膜内皮细胞分别接种到 DPC 的前表面和后表面。在第 14 天和第 30 天进行评估。使用 I 型胶原、角膜蛋白聚糖、亮氨酸、角蛋白 3（AE5）和 VIII 型胶原的免疫定位法，研究培养的角膜细胞的表型特性。
HE 和 DAPI 染色显示出有效去除猪角膜细胞，而阿利新蓝染色证实 ECM 大体上得以保留。DNA 含量的定量分析显示出显著减少（脱细胞前平均值：75.45±13.71ng/mg；脱细胞后平均值：9.87±2.04ng/mg，p<0.001）。所有三种类型的角膜细胞都能在 DPC 上高效培养和扩增。
脱细胞猪角膜可能作为角膜组织工程的潜在支架，可能为角膜移植提供异种基质。
