Document Type : Original Article
Authors
1
.Department of Developmental Biology, University of Science and Culture, Tehran, Iran;.Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
2
.Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
3
.Department of Endocrinology and Female Infertility, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran ;4.Department of Gynecology and Obstetrics, Arash Women’s
4
6.Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
5
.Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran;7.Department of Anatomy, Faculty of Medical Science, Tarbiat Modares University, Tehr
Abstract
Objective
Decellularized tissue scaffolds provide an extracellular matrix to control stem cells differentiation toward specific lineages. The application of mesenchymal stem cells for artificial ovary production may enhance ex vivo functions of the ovary. On the other hand, the scaffold needs interaction and integration with cells. Thus, the development of ovarian engineered constructs (OVECs) requires the use of efficient methods for seeding of the cells into the ovarian and other types of scaffolds. The main goal of the present study was to develop an optimized culture system for efficient seeding of peritoneum mesenchymal stem cells (PMSCs) into human decellularized ovarian scaffold.
Materials and Methods
In this 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 using the best method of cellular seeding for primordial germ cell-like cells, mesenchymal stem cells and proliferation markers. Stereology analysis was also performed for the number of penetrated cells into the OVECs.
Results
Our results showed that rotational seeding increases the permeability of PMSCs into the scaffold and survival rate of the seeded PMSCs, comparing to the other methods. On the other hand, rotationally seeded PMSCs had a more favorable 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 more favorable distribution of PMSCs along the outer surfaces of the OVEC with further distribution at the central part of the scaffold. The average total cell values were determined 2142187 cells/mm3 on each OVEC.
Conclusion
The rotational seeding method is a more favorable approach to cell seeding into ovarian decellularized tissue than static seeding.
Keywords
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