May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Tissue Banking Methods for Amniotic Membrane Integrity Preservation: A Comparative Study
Author Affiliations & Notes
  • R. P. Casaroli-Marano
    Department of Cell Biology, University of Barcelona, Barcelona, Spain
    Transplant Services Foundation, Corporació Sanitària Clínic, Barcelona, Spain
  • E. M. Martínez-Conesa
    Department of Cell Biology, University of Barcelona, Barcelona, Spain
    Transplant Services Foundation, Corporació Sanitària Clínic, Barcelona, Spain
  • E. Agustí
    Transplant Services Foundation, Corporació Sanitària Clínic, Barcelona, Spain
  • N. Otero
    Transplant Services Foundation, Corporació Sanitària Clínic, Barcelona, Spain
  • A. Adán
    Instituto Clínic de Oftalmologia, Hospital Clínic de Barcelona, Barcelona, Spain
  • E. Trías
    Transplant Services Foundation, Corporació Sanitària Clínic, Barcelona, Spain
  • B. Miranda
    Transplant Services Foundation, Corporació Sanitària Clínic, Barcelona, Spain
  • Footnotes
    Commercial Relationships  R.P. Casaroli-Marano, None; E.M. Martínez-Conesa, None; E. Agustí, None; N. Otero, None; A. Adán, None; E. Trías, None; B. Miranda, None.
  • Footnotes
    Support  FIS Grant PI05/1016; FIS Grant PI05/1245; Grant 2007 by Societat Catalana d'Oftalmologia
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 2356. doi:https://doi.org/
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    • Get Citation

      R. P. Casaroli-Marano, E. M. Martínez-Conesa, E. Agustí, N. Otero, A. Adán, E. Trías, B. Miranda; Tissue Banking Methods for Amniotic Membrane Integrity Preservation: A Comparative Study. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2356. doi: https://doi.org/.

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

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Abstract

Purpose: : Preserved human amniotic membrane (AM) is currently being used for a wide spectrum of ocular surface disorders. Preservation methods of AM required for transplantation is a crucial point for optimize reconstructive results. The aim of this work is compare three different methods to maintain AM for ophthalmologic application.

Methods: : AM obtained under sterile conditions was spread uniformly on individually sterilized 0.22 µm nitrocellulose filters. Filters were placed in: a) RPMI medium/Albumin/DMSO and stored in liquid N2 at -196°C; b) DMEM medium/Glycerol and freeze at -80°C; and c) DMEM medium/Glycerol and preserved at -20°C. Specimens at each condition were progressively thawed at 1, 3, and 6 months of storage. AM pieces were fixed and prepared in paraffin for histological evaluation by light microscopy. Protein extracts were obtained after homogenization. SDS-PAGE electrophoresis and Western-blot analysis for were carried out. Fresh AM was used as control.

Results: : AM protein concentrations decreased approximately one-fold after freezing when compared to fresh control specimen. Comassie, Ponceau Red and Silver stains showed a selective impairment of high molecular weight proteins (> 90 kDa). Epithelial markers (keratin 1/5/10/14 and E-cadherin) were preserved in all conditions analyzed as well as specific basal membranes markers (laminin and type IV collagen), indicating integrity of AM epithelial surface. However, type III collagen - the main component of AM stroma - showed some degradation at higher temperature at 6 months of storage. Vimentin was normal for all situations. Histological examination exhibited some epithelial alterations in freezing pieces.

Conclusions: : Methods at very low temperature (-80°C and -196°C) were effective to maintain the integrity characteristics of AM in our study. Some degraded protein could be observed at 6 months of storage. Freezing method at -80°C seems to be easier and less expensive to preserve AM in tissue banking storage for therapeutic use.

Keywords: cornea: epithelium • regeneration • cornea: storage 
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