Optimizing the cell seeding protocol to human decellularized ovarian scaffold: application of dynamic system for bio-engineering

Background: Decellularized tissue scaffolds provide an extracellular matrix controlling the stem cells differentiation toward specific lineages. Mesenchymal stem cells application for artificial ovary production may enhance ex vivo functions of the ovary. On the other hand, the scaffold needs interaction and integration with cells.Objective: Therefore, develop of ovarian engineered constructs (OVECs) requires the use of best method for well seeding of the cells into the ovarian and the other type of scaffolds. The main goal of the present study was to develop an optimized culture system for the efficient seeding of peritoneum mesenchymal stem cells (PMSCs) into human decellularized ovarian scaffold.Materials and Methods: In experimental study, three methods were used for cellular seeding including rotational (spinner flask) and static (conventional and injection) seeding cultures. OVECs were evaluated with Hematoxylin and Eosin staining and viability analyses for the seeded PMSCs. Then, immunohistochemistry analysis was performed in best method of cellular seeding for primordial germ cell-like cells, mesenchymal stem cells and proliferation markers. Also, stereology analysis was made for the number of penetrated cells in depth of the OVECs.Results: Our results revealed that rotational seeding increases the permeability of PMSCs into the scaffold and survival rate of the seeded PMSCs comparing to the other methods, at the same time. On the other hand, the rotational seeded PMSCs had well capability of proliferation with ki67 expression and differentiation to ovarian specific cells with expression of primordial germ cell line markers without mesenchymal stem cells markers production. Furthermore, stereology showed a well distribution of PMSCs along the outer surfaces of the OVECs with further distribution at the central part of scaffold. Average total cell values were determined 2142187 cells/mm3 on each OVEC.Conclusion: The rotational seeding method is more appropriate approach for cell seeding into decellularized tissues compared to static seeding.

کلیدواژه ها:

Peritoneum mesenchymal stem cells ، Rotational seeding ، Tissue engineered ovary ، Stereology