Amniotic membrane (AM) has been widely used as a biological dressing for many pathologies and illnesses worldwide, and products derived from this tissue have been commercially available in several countries. In Brazil, regulatory agencies have recently authorized its clinical use as a non-experimental therapy for burns, diabetic and venous stasis ulcers, and intrauterine adhesions. In this study, we present our pathway through validating the first available service in the country of AM cryopreservation, with a protocol for long-term storage in high-efficiency nitrogen cryogenic freezers and a specific way of packing the tissue for optimal clinical handling and efficient storage space utilization while preserving live cells and the tissue's biological properties. Using gauze as support, cryoprotectant dimethyl sulfoxide and product presentation as a multilayer roll exhibited the best cell viability results and maintained the tissue integrity and presence of stem/progenitor cells. Essential proteins involved in tissue regeneration and immune and antimicrobial control were detected from the secretome of cryopreserved tissue similar to fresh tissue. Furthermore, immunogenic markers, such as human leukocyte antigens, were detected at very low levels in the tissue, confirming their low immunogenicity. Finally, we demonstrate that the tissue can be kept under refrigerated conditions for up to 7 d for further use, maintaining sterility and considerable cell viability. Our cryopreservation and storage protocol kept the AM viable for at least 20 months. In conclusion, this study enabled us to determine a novel efficient protocol for long-term AM preservation for future clinical applications.
Keywords: DMSO; amniotic membrane; cryopreservation; secretome; stem cells; tissue bank.
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