Abstract

SA.20.01

The effect of long-term storage on the biological and histological properties of cryopreserved amniotic membrane

Henning Thomasen1, Mikk Pauklin1, Bernhard Nölle2, Gerd Geerling3, Jan Vetter4, Klaus-Peter Steuhl5, Daniel Meller6
1Zentrum für Augenheilkunde, Universitätsklinikum Essen, Essen; 2Klinik für Ophthalmologie, Campus Kiel, Universitätsklinikum Schleswig-Holstein, Kiel; 3Universitäts-Augenklinik Würzburg; 4Universitäts-Augenklinik Mainz; 5Vorderer Augenabschnitt, Zentrum für Augenheilkunde, Universitätsklinikum Essen, Essen; 6Hornhautlabor, Zentrum für Augenheilkunde, Universitätsklinikum Essen, Essen

Objective
Cryopreserved amniotic membrane (Cryo-AM) is widely used in ophthalmology because of its anti-angiogenic, anti-inflammatory and wound healing promoting capabilities. A common method to conserve the tissue is the storage in cryomedia containing 50% at very low temperatures such as -80°C. The aim of this study was to examine the influence of storage time on the sterility as well as the histological and biological properties of Cryo-AM.
Methods
Amniotic membrane from different donors was stored in cryo-media containing 50% glycerol for 4 months, 14 months and 24 months at -80°C. Samples of the tissue and cryomedia were examined for bacterial and fungal contamination. Tissue samples were placed in serum-free media at 37°C (0.5ml/cm2). The medium was changed after 1, 2 and 3 days. The proteins released by Cryo-AM were TCA-precipitated and the precence of the proteins TIMP-1, TGF-b1, CTGF and IL-1ra was analyzed using western blotting. The basement membrane components collagen IV, collagen VII, laminin, laminin 5 and fibronectin were examined immunohistochemically in cryosections of the tissue.
Results
None of the examined samples showed bacterial or fungal contamination. The soluble proteins TIMP-1, CTGF and IL-1ra were found in all samples in media incubated for about a day, however, TGF-b1 was not found in all samples. The western blot signal diminished after longer incubation periods. The basement membranes of all samples showed a similar distribution of Collagen IV, Collagen VII, Laminin, Laminin 5 and Fibronectin.
Conclusions
Long-term storage in glycerol does not significantly impair sterility, histology or biological properties of amniotic membrane.

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