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

The human amniotic membrane (AM) is emerging as an in- teresting biomaterial for regenerative medicine due to its biological and mechanical proprieties. The beneficial effects of the AM are probably related to its bioactive factors pro- duced by local cells and stored in the stromal matrix. How- ever, the search for a preservation method capable of pre- serving AM properties remains a challenge. The aim of this study was to evaluate important features of 2 anatomical re- gions of the human AM (reflected and placental amnion) af- ter 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 solu- tion for 30 days (cryopreserved AM). After the storage peri- od, the structural integrity of the membrane was assessed by histological and Picrosirius polarization analysis, cellular vi- ability 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 viabili- ty, mainly in the placental amnion region of the AM, but pre- served 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 pre- serving the structural integrity and soluble protein content, indicating the feasibility of this method in preserving AM for its use in regenerative medicine.