Morphological Comparison of Cryopreserved Rabbit Amniotic Membranes Stored either at –80° Celsius or in Liquid Nitrogen

Abstract

Abstract: : Purpose: Comparison of morphological changes in rabbit amniotic membranes (RAMs) stored either at –80°C or in liquid nitrogen. The investigation may be of interest for use of cryopreserved RAMs as a support in experimental transplantation of retinal pigment epithelial cells (RPE). Methods:Fresh RAMs were gradually frozen by 1°C per minute and stored either at –80°C or in liquid nitrogen. One or two years later both stored RAMs were thawed. One part of each RAMs was immediately fixed by Karnowsky and underwent further procedure for the examination with transmission electron microscopy (TEM). Second part of each RAMs was embedded in Tissue–Tek freezing medium (Sakura) and shock–frozen for immunohistochemical examination. Subsequently, sections were acetate/ethanol–fixed and stained with monoclonal mouse antibodies against fibronectin (clone FBN11, NeoMarkers) and pancytokeratin (clone Lu–5, NeoMarkers). The visualization and counterstaining were performed using AEC substrate system (Ultravision) and haemalaun. Results: TEM revealed marked difference in the epithelial layer and underlying basement membrane already after one year of storage. Whereas in RAMs stored in liquid nitrogen the epithelium and the basement membrane appeared to be good maintained, in RAMs stored at –80°C the epithelium was either absent or completely disintegrated and the basement membrane was only sparsely detected. Immunohistochemistry verified the general absence of pancytokeratin staining, as well as either complete absence or altered distribution of fibronectin in RAMs stored at –80°C. In contrast, in RAMs stored in liquid nitrogen the epithelium could be regularly visualized and fibronectin staining was usually detected in areas of epithelium and the basement membrane. Conclusions: Regarding the presence of epithelium, basement membrane and fibronectin RAMs stored at –80°C showed limited lifespan . The absence of epithelium appeared to be beneficial avoiding the necessity of denudation RAMs of epithelial layer. However, the absence of the basement membrane and fibronectin may cause problems with overgrowing the membrane surface with the regular RPE cell carpet and development of functional RPE–layer.

Keywords: transplantation • microscopy: electron microscopy • immunohistochemistry