Purchase this article with an account.
Henning Thomasen, Mikk Pauklin, Bernhard Noelle, Gerd Geerling, Jan Vetter, Philipp Steven, Klaus-Peter Steuhl, Daniel Meller; Impact Of Storage Time On Cryopreserved Amniotic Membrane. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2001.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Cryopreserved amniotic membrane (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 cell culture medium containing 50% glycerol at -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 cryopreserved AM.
Amniotic membrane from different donors which was stored in cell culture medium containing 50% glycerol for different time periods, on average 4 months (group 1), 15 months (group 2) and 24 months (group 3), at -80°C was analysed. Samples of the tissue and cryo-medium were examined for bacterial and fungal contamination. Tissue samples were incubated in 0.5ml/cm2 serum-free medium at 37°C. The medium was changed after 1, 2, and 3 days. The proteins released by AM were TCA-precipitated and the proteins TIMP-1 and IL-1ra were analyzed using Western blotting and semi quantified by means of image analysis. Integrity of the amniotic epithelium and the basement membrane components collagen IV, collagen VII, laminin, laminin 5 and fibronectin were examined by haematoxylin eosin stain and immunohistochemistry in cryosections of AM.
None of the examined samples showed bacterial or fungal contamination. The soluble proteins TIMP-1 and IL-1ra were found in all samples of medium incubated for all time periods. The examined proteins were detectable after one-day incubation but the staining signal diminished significantly in the second and third wash after 48h and 72h. Differences in the intensity of the Western Blot signal between the three particular groups were statistically not significant. The epithelia of all samples were intact. The basement membranes of all samples showed a similar distribution of collagen IV, collagen VII, laminin, laminin 5 and fibronectin.
Long-term storage of amniotic membrane in cell culture media with 50% glycerol does not significantly impair sterility, histology or biological properties of AM.
This PDF is available to Subscribers Only