Repeated Freezing Procedures Preserve Structural and Functional Properties of Amniotic Membrane for Application in Ophthalmology

Abstract

For decades, the unique regenerative properties of the human amniotic membrane (hAM) have been successfully utilized in ophthalmology. As a directly applied biomaterial, the hAM should be available in a ready to use manner in clinical settings. However, an extended period of time is obligatory for performing quality and safety tests. Hence, the low temperature storage of the hAM is a virtually inevitable step in the chain from donor retrieval to patient application. At the same time, the impact of subzero temperatures carries an increased risk of irreversible alterations of the structure and composition of biological objects. In the present study, we performed a comprehensive analysis of the hAM as a medicinal product; this is intended for a novel strategy of application in ophthalmology requiring a GMP production protocol including double freezing–thawing cycles. We compared clinically relevant parameters, such as levels of growth factors and extracellular matrix proteins content, morphology, ultrastructure and mechanical properties, before and after one and two freezing cycles. It was found that epidermal growth factor (EGF), transforming growth factor beta 1 (TGF-β1), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), hyaluronic acid, and laminin could be detected in all studied conditions without significant differences. Additionally, histological and ultrastructure analysis, as well as transparency and mechanical tests, demonstrated that properties of the hAM required to support therapeutic efficacy in ophthalmology are not impaired by dual freezing.

Proteome Composition of Bovine Amniotic Membrane and Its Potential Role in Corneal Healing

“…There have been no comprehensive proteomics studies comparing the effects of the various techniques in preserving all the desirable features of AM in human and veterinary medicine. A recent study using identical AM cryopreservation protocol to the present study reported no detrimental effects of dual freezing-thawing cycles on protein concentrations (i.e., EGF, TGF-β1, HGF, bFGF, hyaluronic acid, and laminin), histological and ultrastructure, transparency, and biomechanical properties of the hAM kept in the freezer in a short period of time 34 ; however, AM kept for periods longer than seven days at −80°C presented low concentrations of cytokines. 17 , 28 The findings of this recent study suggest that the short period of the deep-freezing method used herein does not significantly affect the protein composition of AM.…”

Surgical Application of Human Amniotic Membrane and Amnion-Chorion Membrane in the Oral Cavity and Efficacy Evaluation: Corollary With Ophthalmological and Wound Healing Experiences

“…It was interesting to note that the cryopreservation process did not affect some of its biomechanical properties ( Fenelon et al, 2020 ). Similarly, repeated freezing procedures impacted cell viability but not histological and ultrastructure analysis ( Pogozhykh et al, 2020 ).…”

Human Amniotic Membrane: A review on tissue engineering, application, and storage

“…22 In routine clinical practice, various factors play a role in safe storage, such as the storage temperature and its duration. 96 In this regard, the performance of hAM under different cryopreservation times has been researched by Wagner et al performing studies at 0.5, 1, 3, and 6 months after cryopreservation with hAM stored at −80 C with glycerol, such as CPA, and glycerol-free. The results showed higher cell viability of samples cryopreserved in glycerol for up to 3 months of storage.…”