June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
The effects of zinc supplements on primary human foetal retinal pigment epithelium in culture
Author Affiliations & Notes
  • Po-Jung Pao
    Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, Lonodon, United Kingdom
    Department of Structural and Molecular Biology, University College London, London, United Kingdom
  • Safiya Bishar Abdirahman
    Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, Lonodon, United Kingdom
  • Talha Soorma
    Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, Lonodon, United Kingdom
  • Lajos Csincsik
    Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, Lonodon, United Kingdom
  • Stephen J Perkins
    Department of Structural and Molecular Biology, University College London, London, United Kingdom
  • Stefanie M Hauck
    Research Unit Protein Science, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany
  • Richard Thompson
    Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD
  • Imre Lengyel
    Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, Lonodon, United Kingdom
  • Footnotes
    Commercial Relationships Po-Jung Pao, None; Safiya Abdirahman, None; Talha Soorma, None; Lajos Csincsik, None; Stephen Perkins, None; Stefanie Hauck, None; Richard Thompson, None; Imre Lengyel, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 2347. doi:
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      Po-Jung Pao, Safiya Bishar Abdirahman, Talha Soorma, Lajos Csincsik, Stephen J Perkins, Stefanie M Hauck, Richard Thompson, Imre Lengyel; The effects of zinc supplements on primary human foetal retinal pigment epithelium in culture. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2347.

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

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Abstract

Purpose: The retinal pigment epithelium (RPE)/choroid complex contains very high concentrations of zinc. With age, zinc levels decline and this has been associated with pathological conditions such as age-related macular degeneration (AMD). Cultured RPE cells are widely used as a model for studying the pathogenesis of AMD. However, due to the presence of the high zinc-binding capacity proteins like serum albumin in the culture media, RPE cells might grow in a zinc-deficient environment slowing their growth and differentiation. To test this hypothesis we cultured primary human RPE cells in a zinc-enriched environment.

Methods: Commercially available primary human foetal RPE cells were seeded onto permeable cell-culture inserts (Millipore). RPE cells were maintained in culture medium supplemented with different concentrations of zinc (0, 75, 100, 125 and 150 µM). Cellular differentiation was monitored by measuring trans-epithelial resistance (TER), cellular pigmentation, expression of RPE-specific genes and analysing protein secretion. Once fixed, differentiation was assessed by scanning (SEM) and transmission (TEM) electron microscopy and immunohistochemistry. Toxicity of added zinc was assessed by measuring lactate dehydrogenase (LDH) release.

Results: RPE cells developed a confluent monolayer with TER > 200 Ωcm2 within 4 weeks with cobble-stone morphology. In the presence of 100 µM and 125 µM added zinc (corresponding to ~300-400 nM bio-available zinc) TER was significantly higher than control (300 Ωcm2 and 400 Ωcm2 respectively, versus 200 Ωcm2). Cells under these conditions were highly pigmented and developed dense microvilli. They also expressed and secreted proteins that were characteristic of RPE cells in vivo. Measurement of LDH release showed no significant cell death in the presence of zinc.

Conclusions: Our results show that elevated zinc levels are necessary to generate better differentiated RPE cells in culture.

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