Valtink Monika, Gruschwitz Rita, Funk Richard H W, Engelmann Katrin
Tissue Engineering Laboratories, Biotechnology Center, University of Technology, Dresden, Germany.
Cells Tissues Organs. 2008;187(4):286-94. doi: 10.1159/000113406. Epub 2008 Jan 14.
Access to primary human corneal endothelial cells (HCEC) is limited and donor-derived differences between cultures exacerbate the issue of data reproducibility, whereas cell lines can provide sufficient numbers of homogenous cells for multiple experiments. An immortalized HCEC population was adapted to serum-free culture medium and repeated cloning was performed. Clonally grown cells were propagated under serum-free conditions and growth curves were recorded. Cells were characterized immunocytochemically for junctional proteins, collagens, Na,K-ATPase and HCEC-specific 9.3.E-antigen. Ultrastructure was monitored by scanning and transmission electron microscopy. Two clonal cell lines, HCEC-B4G12 and HCEC-H9C1, could be isolated and expanded, which differed morphologically: B4G12 cells were polygonal, strongly adherent and formed a strict monolayer, H9C1 cells were less adherent and formed floating spheres. The generation time of B4G12 cells was 62.26 +/- 14.5 h and that of H9C1 cells 44.05 +/- 5.05 h. Scanning electron microscopy revealed that B4G12 cells had a smooth cell surface, while H9C1 cells had numerous thin filopodia. Both cell lines expressed ZO-1 and occludin adequately, and little but well detectable amounts of connexin-43. Expression of HCEC-specific 9.3.E-antigen was found commensurately in both cell lines, while expression of Na,K-ATPase alpha1 was higher in H9C1 cells than in B4G12 cells. B4G12 cells expressed collagen IV abundantly and almost no collagen III, while H9C1 cells expressed both collagens at reasonable amounts. It is concluded that the clonal cell line B4G12 represents an ideal model of differentiated HCEC, while H9C1 may reflect features of developing or transitional HCEC.
获取原代人角膜内皮细胞（HCEC）存在限制，且不同培养物之间源自供体的差异加剧了数据可重复性问题，而细胞系可为多次实验提供足够数量的同质细胞。将永生化的HCEC群体适应无血清培养基并进行重复克隆。克隆生长的细胞在无血清条件下传代培养并记录生长曲线。通过免疫细胞化学方法对细胞进行连接蛋白、胶原蛋白、钠钾ATP酶和HCEC特异性9.3.E抗原的表征。通过扫描电子显微镜和透射电子显微镜监测超微结构。可以分离并扩增出两种克隆细胞系，即HCEC - B4G12和HCEC - H9C1，它们在形态上有所不同：B4G12细胞呈多边形，粘附性强，形成紧密的单层，H9C1细胞粘附性较弱，形成漂浮球体。B4G12细胞的代时为62.26±14.5小时，H9C1细胞的代时为44.05±5.05小时。扫描电子显微镜显示，B4G12细胞的细胞表面光滑，而H9C1细胞有许多细的丝状伪足。两种细胞系均充分表达ZO - 1和闭合蛋白，且有少量但可检测到的连接蛋白43。在两种细胞系中均相应地发现了HCEC特异性9.3.E抗原的表达，而钠钾ATP酶α1在H9C1细胞中的表达高于B4G12细胞。B4G12细胞大量表达IV型胶原蛋白，几乎不表达III型胶原蛋白，而H9C1细胞以合理的量表达这两种胶原蛋白。结论是，克隆细胞系B4G12代表了分化型HCEC的理想模型，而H9C1可能反映了发育中或过渡型HCEC的特征。
