An efficient protocol for decellularization of the human endometrial fragments for clinical usage.

Abstract

The present study was aimed tocompare different decellularization protocols for human endometrial fragments. The freeze-thaw cycles in combination with treatment by Triton X-100 and four concentrations of sodium dodecyl sulfate (SDS; 0.1, 0.5, 1, and 1.5%) with two exposure times (24 and 72h) were applied for tissues decellularization. After analysis the morphology and DNA content of tissues the group with better morphology and lower DNA content was selected for further assessments. The nucleus by Acridine orange and extracellular matrix (ECM) using Masson's trichrome, Alcian blue, and periodic acid-Schiff staining were studied. The amount of tissues collagen types I and IV, fibronectin, glycosaminoglycans (GAGs), and elastin was analyzed by Raman spectroscopy. The ultrastructure and porosity of decellularized scaffold were studied by scanning electron microscopy (SEM). The MTT assay was applied for assessments of cytotoxicity of scaffold. The treated group with 1% SDS for 72h showed the morphology similar to native control in having the minimum level of DNA and well preserved ECM. Raman spectroscopy results demonstrated, the amount of collagen types I and IV, GAG, and fibronectin was not significantly different in decellularized scaffold compared with native group but the elastin protein level was significantly decreased (P < 0.001). SEM micrographs also showed a porous and fiber rich ECM in decellularized sample similar to the native control. This combined protocol for decellularization of human endometrial tissue is effective and it could be suitable for recellularization and clinical applications in the future.