May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Eye Bank Storage of Cultured Corneal Epithelium: Sterility Testing of Storage Media, Long-Term Preservation, and Deepithelialization of the Amniotic Membrane Substrate
Author Affiliations & Notes
  • S. Raeder
    Ulleval University Hospital, Oslo, Norway
    Dept of Ophthalmology,
  • T. Paaske Utheim
    Ulleval University Hospital, Oslo, Norway
    Dept of Ophthalmology,
  • Ø. Aass Utheim
    Ulleval University Hospital, Oslo, Norway
    Dept of Ophthalmology,
  • M. de la Paz
    Centro de Oftalmologia Barraquer, Barcelona, Spain
  • J. R. Eidet
    Ulleval University Hospital, Oslo, Norway
    Dept of Ophthalmology,
  • T. Lyberg
    Ulleval University Hospital, Oslo, Norway
    Center for Clinical Research,
  • Footnotes
    Commercial Relationships  S. Raeder, Patent application, P; T. Paaske Utheim, Patent application, P; Ø. Aass Utheim, None; M. de la Paz, None; J.R. Eidet, None; T. Lyberg, None.
  • Footnotes
    Support  Southern Eastern Norway Regional Health Authority
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 5717. doi:https://doi.org/
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      S. Raeder, T. Paaske Utheim, Ø. Aass Utheim, M. de la Paz, J. R. Eidet, T. Lyberg; Eye Bank Storage of Cultured Corneal Epithelium: Sterility Testing of Storage Media, Long-Term Preservation, and Deepithelialization of the Amniotic Membrane Substrate. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5717. doi: https://doi.org/.

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

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Abstract

Purpose: : In recent studies we have examined the feasibility of eye bank storage of cultured human limbal epithelial cells (HLEC) for transplantation. This study aimed at investigating microbiological sterility, long-term preservation, and effects of deepithelialization of the amniotic membrane substrate of cultured HLEC subject to eye bank storage.

Methods: : HLEC were expanded from cadaver explants on intact amniotic membranes (iAM) or denuded amniotic membranes (dAM) for 3 weeks and stored in a closed container in organ culture medium at 23°C. Sterility of the storage medium was tested using the Bactec blood bottle method. A laser confocal microscope and digital imaging were used to distinguish live (calcein AM-positive) from dead (ethidium homodimer-1-positive) cells in HLEC cultures on iAM following 2-and 3-week OC storage. Quantitative transmission electron microscopy analysis and scanning electron microscopy was performed on HLEC cultures on iAM and dAM following 1-week storage.

Results: : None of the 46 blood culture bottles (Bactec Plus Aerobic/F bottles (n=23) and Bactec Lytic/10 Anaerobic/F bottles (n=23)) were contaminated, giving a contamination rate of 0%. Basal layer viability of cultured limbal epithelial cells was 85.6% ± 13.5% (range 0.25-97%) after 2-weeks storage versus 52.7% ± 13.1% (range 0.26-81%) after 3-weeks storage (P<0.001). Following 1-week storage, the number of desmosomes per µm in HLEC cultured on iAM (1.39 ± 0.77 (range 0.49 - 2.51)) was not significantly different from HLEC cultured on dAM (0.98 ± 0.45 (range 0.02-1.37)), and scanning electron microscopy demonstrated tightly apposed superficial cells in both groups.

Conclusions: : These data demonstrates that cultured HLEC may be easily controlled for microbial contamination during eye bank storage and that cultured corneal epithelium demonstrates high cell survival rates for up to 2-weeks storage. With respect to desmosomal attachments denuding of the amniotic membrane did not have any significant impact on the structural integrity of cultured HLEC following storage.

Keywords: cornea: epithelium • cornea: storage • transplantation 
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