PURPOSE: 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 cryo-medium containing 50% glycerol at -80 degrees 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. METHODS: Amniotic membrane from different donors was stored in cell culture media containing 50% glycerol for different time periods, on average 4 months (group 1), 15 months (group 2), and 24 months (group 3), at -80 degrees C. Samples of the tissue and cryo-medium were examined for bacterial and fungal contamination. Tissue samples were incubated in 0.5 ml/cm(2) serum-free medium at 37 degrees C. The medium was changed after 1, 2, and 3 days. The proteins released by AM were TCA-precipitated and the presence of the proteins TIMP-1 and IL-1ra was 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. RESULTS: 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 48 hr and 72 hr. 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. CONCLUSIONS: Long-term storage of amniotic membrane in cell culture media with 50% glycerol does not significantly impair sterility, histology, or biological properties of AM.
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机译：目的：冷冻保存的羊膜（AM）由于其抗血管生成，抗炎和促进伤口愈合的能力而被广泛用于眼科。一种保存组织的常用方法是在-80摄氏度下在含有50％甘油的冷冻培养基中进行保存。本研究的目的是研究保存时间对冷冻保存物的无菌性以及组织学和生物学特性的影响。上午。方法：将不同供体的羊膜在不同时间段内分别保存在含50％甘油的细胞培养基中，平均时间为-80度，分别为4个月（第1组），15个月（第2组）和24个月（第3组）。 C.检查组织和冷冻培养基的样品中细菌和真菌的污染。组织样品在37摄氏度的0.5 ml / cm（2）无血清培养基中孵育。1、2和3天后更换培养基。将由AM释放的蛋白质进行TCA沉淀，并使用Western印迹分析蛋白质TIMP-1和IL-1ra的存在，并通过图像分析进行半定量。通过苏木精伊红染色和免疫组织化学在AM冷冻切片中检查羊膜上皮和基底膜成分胶原IV，胶原VII，层粘连蛋白，层粘连蛋白5和纤连蛋白的完整性。结果：所有检查样品均未显示细菌或真菌污染。在所有时间段内孵育的所有培养基样品中均发现了可溶性蛋白TIMP-1和IL-1ra。孵育一天后可检测到被检测的蛋白质，但在48小时和72小时后的第二次和第三次洗涤中，染色信号显着降低。三个特定组之间的蛋白质印迹信号强度差异在统计学上不显着。所有样品的上皮均完好无损。所有样品的基底膜均显示胶原IV，胶原VII，层粘连蛋白，层粘连蛋白5和纤连蛋白的相似分布。结论：在含有50％甘油的细胞培养基中长期保存羊膜不会明显损害AM的无菌性，组织学或生物学特性。
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