The Novel Cryopreservation Technique for Preparation of Decellularized Human Amniotic Membrane as an AppropriateExtracellular Matrix for the Regenerative Medical Purposes

Background and Aim: The human amniotic membrane (HAM) is appliedfor various regenerative purposes in the reconstruction of damagedepithelial skin and/or ocular surface as an appropriate biologicalextracellular matrix (ECM). The fresh HAM also called fetus layer, is widelyused by surgeons as a surgical hand-friendly scaffold for the renovation ofthe cellular expansion. Decellularization of HAM is currently performedby an enzymatic procedure (Trypsin-EDTA) that affects the cellularexpansion. In the represented study, a novel biomaterial free procedurefor the preparation of denuded HAM is clearly expressed.Methods: The whole placenta was obtained from HCV, HIV, and syphilisseronegative cesarean delivery from the maternity ward of Imam RezaHospital, Mashhad. Then the placenta was washed three times by balancesalt solution BSS) in order to remove the blood residues. The thick chorionwas separated completely from transparent amnion. The separatedamniotic layer was kept in cryopreservation media for 48 hours at -80°C.Then, the transparent amniotic membrane was vigorously washed withsterile injectable NaCl 0.9% three times, consecutively. At the final step,the denuded HAM was expanded on the sterile nitrocellulose paper,epithelial side up, sealed in the appropriate cover and stored at -80°C.All procedures performed under sterile cabinet laminar flow class II.Results: The obtained Denuded HAM was used for explant culture oflimbal stem cells. The data illustrate the amount of P 63 positive limbalstem cells have raised on our cryopreserved denuded HAM compare tothe commercial enzymatic denuded HAM (P=0.005).Conclusion: Based on our results and data obtained from other studies,the novel cryopreservation method applied for decellularization of the human amniotic membrane support the cellular proliferation while itretains and supports the limbal cell stemness which plays the key role inregenerative medicine compared to the commercial enzymatic denudedHAM. To sum up, the grafts of cryopreserved human amniotic membraneseems to be the appropriate extracellular matrix biological membrane forepithelial layer reconstruction.

کلیدواژه ها:

Human amniotic membrane ، Cryopreservation ، Cell expansion ، Limbal stem cells ، Decellularization