Hypothermic Storage of Amniotic Membranes Maintains Native Characteristics of Unprocessed Amnion Tissue

Introduction: Various processing techniques are utilized to manufacture placental allografts, including cryopreservation, dehydration, lyophilization, and hypothermic storage. Processing is known to impact native tissue composition and characteristics, compared to native tissues. In this study, the impact of fresh hypothermic processing and storage (AlloFresh™) on an amnion membrane (HSAM°) was compared to unprocessed, fresh amniotic membrane (uAM).Methods:Amniotic tissue from three donors were processed into two groups: HSAM, which was stored at 1-10°C for up to 42 days until use, and uAM, which was utilized within 24 hours. Qualitative structural assessments were made using scanning electron microscopy (SEM). Additionally, hematoxylin and eosin (H&E), Masson’s trichrome, and immunohistochemistry staining were utilized for extracellular matrix (ECM) comparisons. To quantify graft thickness, multiple views were sampled and quantified using ImageJ. An in vitro simulated wound fluid (SWF) model was developed to evaluate ECM degradation for up to 17 days. Degradation was assessed quantitatively by changes in mass and qualitatively using SEM. The ability of HSAM to function as a scaffold was evaluated using an in vitro fibroblast attachment & proliferation model for up to 14 days.Results:HSAM demonstrated tissue architecture characteristic of uAM, including intact epithelial and stromal layers. Immunohistochemistry revealed that HSAM retained similar expression of collagens I and III, hepatocyte growth factor, insulin-like growth factor 1, and transforming growth factor beta 1. Quantification of tissue thickness revealed no significant differences between HSAM and uAM. When evaluating in vitro degradation, there was a significant difference in tissue mass at day 0. SEM imaging revealed a gradual loss of cell continuity on the epithelial layer and loss of ECM fibers on the stromal layer for both uAM and HSCM. Human dermal fibroblasts seeded onto HSAM attached and proliferated on HSAM, as expected, compared to non-seeded controls. Imaging at 14 days revealed that seeded fibroblasts attached and deposited their own ECM (collagen type I), confirming the functionality of HSAM as a scaffold.Discussion: These results demonstrate that HSAM maintains the native characteristics of unprocessed amniotic membranes, and this preserved ECM and structure functions as a scaffold in vitro.References: