Original Paper

Two Clonal Cell Lines of Immortalized Human Corneal Endothelial Cells Show either Differentiated or Precursor Cell Characteristics
Monika Valtink^a, Rita Gruschwitz^b, Richard H.W. Funk^b, Katrin Engelmann<sup>c

a</sup>Tissue Engineering Laboratories, Biotechnology Center and
^bInstitute of Anatomy, Medical Faculty Carl Gustav Carus, University of Technology, Dresden, and
^cDepartment of Ophthalmology, Klinikum Chemnitz gGmbH, Chemnitz, Germany

Address of Corresponding Author

Cells Tissues Organs 2008;187:286-294 (DOI: 10.1159/000113406)

  Key Words

• Human corneal endothelium
• Cell line
• Serum-free cultivation
• Clonal growth
• Differentiation

  Abstract

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 1 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.

Copyright © 2008 S. Karger AG, Basel

  Author Contacts

Ms. Monika Valtink
Tissue Engineering Laboratories at the BIOTEChnological Center, Medical Faculty
University of Technology Dresden, Tatzberg 47-51, DE-01307 Dresden (Germany)
Tel. +49 351 463 40207, Fax +49 351 463 40244
E-Mail monika.valtink@biotec.tu-dresden.de

  Article Information

Accepted after revision: October 26, 2007
Published online: January 14, 2008
Number of Print Pages : 9
Number of Figures : 4, Number of Tables : 1, Number of References : 35