Immortalization of human corneal endothelial cells using electroporation protocol optimized for human corneal endothelial and human retinal pigment epithelial cells

Bednarz, J; Teifel, Michael; Friedl, Peter; Engelmann, Katrin

doi:10.1034/j.1600-0420.2000.078002130.x

Cited by 106 publications

(78 citation statements)

References 24 publications

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“…The results of the present study are in contrast to those in the report by Kernt et al (16), namely, that voriconazole of concentrations up to 10 mg/ml had no significant toxicity on HCECs, trabecular meshwork cells, or RPE cells when administered for 24 h. However, the immortalized simian virus 40 (SV40)-transfected HCECs and RPE cells used in their study have an extended life span and enhanced proliferation capacity; these characteristics might, at least in part, account for the stronger resistance to damage from voriconazole than that seen with normal, nonmodified cells (2). The report by Gao et al (8) that voriconazole concentrations of Ն50 g/ml caused focal necrosis in rat retina also supports our assumption.…”

Section: Discussionmentioning

confidence: 82%

Cytotoxicity of Voriconazole on Cultured Human Corneal Endothelial Cells

Han

Shin

Hyon

et al. 2011

Antimicrob Agents Chemother

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The purpose of the present study was to evaluate the toxicity of voriconazole on cultured human corneal endothelial cells (HCECs). HCECs were cultured and exposed to various concentrations of voriconazole (5.0 to 1,000 g/ml). Cell viability was measured using a Cell Counting Kit-8 (CCK-8) and live/dead viability/ cytotoxicity assays. Cell damage was assessed using phase-contrast microscopy after 24

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Section: Discussionmentioning

confidence: 82%

Cytotoxicity of Voriconazole on Cultured Human Corneal Endothelial Cells

Han

Shin

Hyon

et al. 2011

Antimicrob Agents Chemother

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“…18 Cells were grown in T25 culture flasks in cell growth medium containing 8% fetal bovine serum. The culture medium of subconfluent cells was replaced with serum-free medium (OptiMEM-1; Invitrogen-Life Technologies, Carlsbad, CA) alone or supplemented with H 2 O 2 (200 mol/L) and incubated for 2 hours at 37°C.…”

Section: Human Corneal Endothelial Cell Culturementioning

confidence: 99%

Evidence of Oxidative Stress in the Pathogenesis of Fuchs Endothelial Corneal Dystrophy

Jurkunas

Bitar

Funaki

et al. 2010

The American Journal of Pathology

261

263

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Fuchs endothelial corneal dystrophy (FECD) is a progressive, blinding disease characterized by corneal endothelial (CE) cell apoptosis. Corneal transplantation is the only measure currently available to restore vision in these patients. Despite the identification of some genetic factors, the pathophysiology of FECD remains unclear. In this study, we observed a decrease in the antioxidant response element-driven antioxidants in FECD corneal endothelium. We further demonstrated that nuclear factor erythroid 2-related factor 2, a transcription factor known to bind the antioxidant response element and activate antioxidant defense, is down-regulated in FECD endothelium. Importantly, we detected significantly higher levels of oxidative DNA damage and apoptosis in FECD endothelium compared with normal controls and pseudophakic bullous keratopathy (iatrogenic CE cell loss) specimens. A marker of oxidative DNA damage, 8-hydroxy-2-deoxyguanosine, colocalized to mitochondria, indicating that the mitochondrial genome is the specific target of oxidative stress in FECD. Oxidative DNA damage was not detected in pseudophakic bullous keratopathy corneas, whereas it colocalized with terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling-positive cells in FECD samples. Ex vivo, oxidative stress caused characteristic morphological changes and apoptosis of CE, suggestive of findings that characterize FECD in vivo. Together, these data suggest that suboptimal nuclear factor erythroid 2-related factor 2-regulated defenses may account for oxidant-antioxidant imbalance in FECD , which in turn leads to oxidative DNA damage and apoptosis. This study provides evidence that oxidative stress plays a key role in FECD pathogenesis. Corneal endothelium (CE) is a monolayer of cells situated in the anterior chamber surface of the cornea; its primary function is to maintain the cornea in a state of deturgescence through sodium-activated ATPase pumping of water, thus, transparency. Fuchs endothelial corneal dystrophy (FECD) is the most common cause of endogenous corneal endothelial degeneration and is characterized by alterations in corneal endothelial cell morphology, progressive loss of CE cells, and concomitant accumulation of extracellular deposits in the basement membrane that eventually lead to corneal edema and opacity. Because CE does not divide in vivo, loss of endothelial cells seen in FECD is permanent. Corneal transplantation is the only treatment modality that can restore lost vision-rendering FECD the second most common cause of corneal transplants performed on the elderly (Ͼ60 years old) in the United States.2 Lack of knowledge of the mechanism of CE degeneration in FECD precludes the development of pharmacotherapeutics for this common and blinding condition.FECD has been termed a disorder of aging; it is a bilateral and slowly progressive disorder, typically appearing after the age of 60 years. FECD is usually a sporadic condition, but it can be inherited as an autosomal dominant trait.3-5 FECD is characterized by endo...

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“…Therefore, cell lines that show a substantial similarity with the primary cell counterparts regarding cell type-specific characteristics can be used as an alternative, for example, in order to establish protocols for cellular therapies. For that reason, our group established an immortalized cell population of human corneal endothelial cells (HCEC) [Bednarz et al, 2000]. These cells form a functional monolayer when transplanted onto denuded donor corneas [Aboalchamat et al, 1999], have recently been characterized regarding electrophysiological properties [Mergler et al, 2003] and apoptotic mechanism [Thuret et al, 2003], and were successfully used to construct human corneal organotypic equivalents [Reichl et al, 2004;Zorn-Kruppa et al, 2005] or transferable cell sheets [Nitschke et al, 2007].…”

Section: Introductionmentioning

confidence: 99%

Two Clonal Cell Lines of Immortalized Human Corneal Endothelial Cells Show either Differentiated or Precursor Cell Characteristics

Valtink

Gruschwitz

Funk

et al. 2008

Cells Tissues Organs

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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 α1 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.

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Immortalization of human corneal endothelial cells using electroporation protocol optimized for human corneal endothelial and human retinal pigment epithelial cells

Cited by 106 publications

References 24 publications

Cytotoxicity of Voriconazole on Cultured Human Corneal Endothelial Cells

Cytotoxicity of Voriconazole on Cultured Human Corneal Endothelial Cells

Evidence of Oxidative Stress in the Pathogenesis of Fuchs Endothelial Corneal Dystrophy

Two Clonal Cell Lines of Immortalized Human Corneal Endothelial Cells Show either Differentiated or Precursor Cell Characteristics

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