Fabrication and characterization of decellularized placenta-derived 3D bio-scaffolds for spermatogonial stem cells culture

Background: Male infertility affects 7 % of the male population and 10 % of infertile men are azoospermic. Extracellular matrix (ECM) of tissues contains various ranges of growth factors, proteins, proteoglycans, hyaluronic acid and others. Decellularization of tissue and production of tissue engineering scaffolds from ECM components is one of the most reliable strategies in fabrication of scaffolds. Although various protocols have been developed to remove the cells from tissues with minimal degradation of ECM components, an optimal protocol is still needed to satisfy scientists.Objective: In this study, we decellularized human placenta and optimized a reliable protocol for spermatogonial stem cells (SSCs) in vitro culture.Materials and Methods: Human placenta was obtained from mothers undergoing cesarean after obtaining informed consent. The tissues were treated with sodium dodecyl sulphate (SDS) for 30 min. The decellularized tissues were casted, freeze and freeze dried to fabricate a porous scaffold. The removal of the cells from tissues was determined by H&E and 4’,6-diamidino-2-phenylindole (DAPI) staining, and DNA content assay. Alcian blue, Masson’s trichrome and Orcein staining was used to confirm that ECM remained intact after decellularization process. Morphology of porous scaffold was viewed under scanning electron microscopy (SEM). SSCs viability for scaffold were determined by MTT assay.Results: Histological analysis showed that the 0.5% SDS for 30 min was completely decellularized. Decellularization was further confirmed by DAPI staining and DNA content assay. Collaegen, elastin and glycosaminoglycans remained intact after decellularization process. MTT test showed no SSCs viability changes for the decellularized tissues.Conclusion: Our study proved a reliable and effective protocol for decellularization of placenta with minimal negative effects on ECM components. The decellularized placenta is suggested as a promising bio-scaffold tissue engineering application that could improve attachment of SSCs.

Keywords:

Decellularized Scaffold , Placenta , Extracellular matrix