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