May 2006
Volume 47, Issue 13
ARVO Annual Meeting Abstract  |   May 2006
Two Distinct Subclones Derived From an Immortalized Human Corneal Endothelial Cell Line Under Serum–Free Conditions
Author Affiliations & Notes
  • M. Valtink
    University, Dresden, Germany
  • R.H. W. Funk
    University, Dresden, Germany
  • K. Engelmann
    Ophthalmology, Klinikum Chemnitz, Chemnitz, Germany
  • Footnotes
    Commercial Relationships  M. Valtink, None; R.H.W. Funk, None; K. Engelmann, None.
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 3016. doi:
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      M. Valtink, R.H. W. Funk, K. Engelmann; Two Distinct Subclones Derived From an Immortalized Human Corneal Endothelial Cell Line Under Serum–Free Conditions . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3016.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : A previously established immortalized human corneal endothelial cell (HCEC) line shows a heterogenous morphology. The aim was to adapt the cell line to serum–free culture conditions and to isolate and characterize clonal cell lines derived from this cell line.

Methods: : Immortalized HCEC were adapted to serum–free culture medium over 3 passages before cloning. Serum–free adapted cells were seeded at clonal density (0.3 cells/well) in 96 well–plates and cultured under serum–free conditions. Clonally grown cell clusters were trypsinized to single cell suspensions and subcloned likewise. The developed subclones were serially passaged using serum–free medium. Cells were also grown on Thermanox cover slips, fixed with glutaraldehyde and embedded in epon for electron microscopy (TEM) or embedded in LR White for immunogold staining against HCEC –specific antigen recognized by mab 9.3.E, occludin, and Na,K–ATPase.

Results: : Two clonally grown cell lines could be developed, HCEC–B4G12 and HCEC–H9C1. B4G12 comprises of strongly adherent, polygonal cells and establishes a strict and closed monolayer that does not give rise to floating daughter cells. H9C1 cells grow only weakly adherent with single cells that aggregate to sphere–like, floating clusters. TEM revealed numerous membrane protrusions in H9C1 cells in contrast to B4G12 cells. Both subclones express the 9.3.E–antigen, occludin and Na,K–ATPase. Expression was higher but restricted to the cytoplasm in H9C1 cells, while in B4G12 cells most immunogold signals were located closer to the cell membrane.

Conclusions: : Cell lines represent ideal models for basic research in cell biology. In order to precisely characterize cellular features and functions, homogeneity of such cell lines is inevitable. The heterogeneity of the here used mother cell line may represent the entirety of HCEC including peripheral and central cells. The two distinct clonal cell lines derived from it may reflect model lines for central and peripheral HCEC, this has to be examined further.These subclones may be useful to study (a) adherence, (b) influence of proliferative processes or (c) metabolic functions of HCEC. Such investigations may help gain more knowledge about the differences in peripheral vs central HCEC.

Keywords: cornea: endothelium • cornea: basic science • proliferation 

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