Hypothermic Storage of Amnion and Chorion Membranes Retain Native Tissue Characteristics

Placental tissues have been utilized to treat wounds since the early 1900s. Historically, placental tissues have been processed in various ways, including cryopreservation, dehydration, and hypothermic storage.

The processing method utilized has been shown to alter tissue characteristics, including extracellular matrix structure, composition of cytokines and growth factors, cell viability, and thickness. In this study, we evaluated the impact of a fresh hypothermic storage process (Allofresh) on both a fresh amnion product (HSAM) and a fresh chorion product (HSCM) compared to unprocessed placental tissue. Specifically, we have measured qualitative structural differences, quantified tissue thickness, and evaluated growth factors and cytokines.

Tissue from amnion and chorion membranes from two independent donors were prepared as 1) unprocessed placental membranes or 2) processed into HSAM or HSCM. Of note, HSCM processing includes a debridement step, which removes a portion of the trophoblast membrane. Qualitative structural assessments for HSAM, HSCM, and unprocessed amnion and chorion membranes were made using scanning electron microscopy (SEM) as well as hematoxylin and eosin (H&E) and Masson’s trichrome staining. To quantify differences in graft thickness, multiple areas from each slide were sampled and quantified using ImageJ.

To characterize growth factors and cytokine content, HSAM and HSCM were extracted overnight using TPER at 4°C; supernatants were then analyzed using Quantibody arrays (RayBiotech).Both HSAM and HSCM demonstrated tissue architecture characteristic of native, unprocessed amnion and chorion tissues, respectively. HSAM tissue structure maintained intact epithelial and stromal layers, while HSCM retained intact reticular, basement membrane, and partial trophoblast layers. Upon quantification of tissue thickness, we found native amnion grafts and HSAM resulted in comparable graft thickness. As expected, due to the debridement step during HSCM processing, tissue thickness was reduced compared to native chorion, but all retained layers were comparable.

HSAM and HSCM retained growth factors and cytokines characteristic of fresh amnion and chorion tissues, including bFGF, EG-VEGF, HGF, IGF-I, IGFBP-1, IL-1Ra, PDGF-ββ, TGF-α, TGF-β1, TGF-β3, TIMP-1, and TIMP-2.These results demonstrate that HSAM and HSCM retain native tissue characteristics, including architectural structure, thickness, and growth factors and cytokines found in fresh, unprocessed amnion and chorion.