Effects of Preservation Methods in the Composition of the Placental and Reflected Regions of the Human Amniotic Membrane

Abstract

The human amniotic membrane (AM) is emerging as an interesting biomaterial for regenerative medicine due to its biological and mechanical proprieties. The beneficial effects of the AM are probably related to its bioactive factors produced by local cells and stored in the stromal matrix. However, the search for a preservation method capable of preserving AM properties remains a challenge. The aim of this study was to evaluate important features of 2 anatomical regions of the human AM (reflected and placental amnion) after different preservation methods. For this purpose, human placentas were harvested and processed for AM isolation and storage at 2 different conditions: room temperature for 18 h in DMEM (fresh AM) and −80°C in DMEM/glycerol solution for 30 days (cryopreserved AM). After the storage period, the structural integrity of the membrane was assessed by histological and Picrosirius polarization analysis, cellular viability analysis was performed using the MTT assay, and the soluble proteins were quantified with the Qubit Protein Assay Kit. Both preservation protocols reduced the cell viability, mainly in the placental amnion region of the AM, but preserved the morphology of epithelial and stromal layers, as well as the organization and distribution of collagen fibers. There was a reduction in soluble proteins only in fresh AM. Importantly, the cryopreserved AM group presented the same concentration as the control group. In conclusion, the cryopreservation using DMEM/glycerol was ideal for preserving the structural integrity and soluble protein content, indicating the feasibility of this method in preserving AM for its use in regenerative medicine.