102 Creation of 3-dimensional artificial niches for ex vivo culture of ovarian cells

Ovarian failure is the most common cause of infertility and it affects ∼1% of young women. Although numerous strategies have been proposed, the assisted reproduction techniques and hormone replacement therapies developed until now are not fully effective in recovering ovarian function and do not provide a definitive solution for female fertility restoration. There is therefore an urgent need for novel and efficient therapeutic alternatives. In this context, the development of an “artificial ovary” may represent a promising and safe tool to obtain transplantable “structures” that can be used for re-establishing reproductive activities. Here we describe a novel approach for decellularising porcine whole ovaries and successfully repopulating them with ovarian cells. Whole ovaries were subjected to a four-step decellularisation protocol that involved a freeze–thaw cycle, followed by sequential incubations in 0.5% sodium dodecyl sulfate for 3 h, 1% Triton X-100 for 9 h, and 2% deoxycholate for 12 h. At the end of the process, extracellular matrix (ECM)-based ovarian 3-dimensional (3D) scaffolds that maintained shape and homogeneity without any deformation were generated. Histological and 4′,6-diamidino-2-phenylindole (DAPI) staining as well as DNA quantification demonstrated a significant decrease in DNA content, compared with untreated ovaries. Histochemical characterisations revealed the preservation of key ECM components; namely, collagen, elastin, and glycosaminoglycan (GAGs), with full retention of their microarchitecture, after the decellularisation process, which was also confirmed by stereological quantification for the major matrix constituents. Functional assessment of the ECM-based 3D scaffolds was carried out by repopulating them with porcine ovarian cells. The results obtained allowed us to rule out any cytotoxic effects exerted by the generated bioscaffolds that, when re-seeded with ovarian cells, encouraged rapid cell adhesion and migration. Haematoxylin & eosin and DAPI staining as well as cell density analysis and DNA quantification demonstrated an increase in cell number throughout the culture period and showed the ability of the decellularised ECM-based ovarian 3D scaffold to encourage cell adherence, proliferation, and differentiation. The results obtained suggest that the creation of an “artificial ovary” may constitute an ideal niche for ex vivo culture of ovarian cells and may offer an advantageous solution for hormone and fertility restoration.

This research was funded by Carraresi Foundation, PSR2017 and PSR2020. Authors are members of the COST Action CA16119 In vitro 3-D total cell guidance and fitness (CellFit) and of the Trans-COST Actions Task-Force on Covid-19.