Tissue Banking Methods for Amniotic Membrane Integrity Preservation: A Comparative Study

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