26 Development of human amniotic membrane products for regenerative medicine applications.

Introduction: Human amniotic membrane (HAM) has important biological properties that make this tissue an ideal substrate for regenerative medicine applications, including treatment of ocular diseases and wound healing. NHSBT can successfully decellularise HAM for promoting enhancement of limbal stem cell expansion in vitro more efficiently than the cellular HAM. In this study we present new formulations of decellularised HAM as freeze-dried powder and derived natural hydrogel. The aim was to develop a variety of GMP-compliant allografts to treat ocular diseases.

Materials And Methods: Six HAM, obtained from elective caesarian deliveries, were dissected, decontaminated and subjected to an in-house developed decellularisation protocol including a mild SDS concentration as detergent and nuclease steps. Following decellularisation, the tissue was placed in a sterile tissue culture flask and freeze dried. The freeze-dried tissue was cut into pieces of ~1g each, dipped into liquid nitrogen, then ground with a pulverisette. Ground tissue was solubilised using porcine pepsin and 0.1M HCl (stirred for 48 hours, 25oC). At the end of solubilisation, the pre-gel solution was kept on ice to adjust the pH back to 7.4. Gelation was induced when the temperature of the solution was increased to 25oC and aliquots were used for both in vitro cytotoxicity (up to 48 hours) and biocompatibility (up to 7 days) testing (MG63 and HAM cells). Cells were added into the solution before gelling and on top after gelling.

Results: The pre-gel solution obtained from decellularised HAM appear homogenous without undigested powder, and it was able to gel within 20 minutes at RT. Gels with a concentration of 4-8mg/mL tissue powder retained shape (including in an aqueous environment). When added on top of gels, cells were observed to attach and proliferate over time. When added into gels, the cells were observed throughout the gels and appeared to be migrating through the gel.

Conclusion: Acellular HAM can be successfully freeze dried and converted into new formulations for topical application (powder and hydrogel). The new formulations could improve HAM delivery and provide a better scaffold for tissue regeneration. To our knowledge, this is the first time an amnion hydrogel formulation has been developed in GMP compliant setting for tissue banking purpose. Further studies will also investigate the ability of amnion hydrogel to promote stem cells differentiation into the three lineages (adipogenic, chondrogenic, osteogenic) in and/or on the gels.

References: Figueiredo GS . Acta Biomater 2017;61, 124-133.