Generation of haploid spermatids on silk fibroin-Alg-laminin-based porous 3D scaffolds
Generation of haploid spermatids on silk fibroin-Alg-laminin-based porous 3D scaffolds
zahra bashiri,1,*morteza koruji,2
1. Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran. 2. Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
Introduction: In vitro production of sperm is one of the most important options for fertility preservation in azoospermic men and prepubertal boys with cancer. Therefore, in this study, a biocompatible porous scaffold based on silk fibroin-Alg containing laminin was developed to differentiate mouse spermatogonia stem cells (SSCs).
Methods: After extraction and characterization of silk fibroin using SDS-PAGE analysis, stable porous 3D scaffolds were successfully prepared from combined solutions through a freeze-dried method. Then, structural and biological properties, biocompatibility, water absorption, degradability, and mechanical behavior of biomimetic scaffolds were characterized. Neonatal mice testicular cells were seeded on 3D scaffolds after confirmation of nature and their differentiation efficiency was evaluated using Real Time‐PCR, flow cytometry, immunohistochemistry techniques, and H & E staining. The function of Leydig and Sertoli cells was also assessed using ELISA.
Results: Blend matrices showed uniform porous microstructure with interconnected network, which significantly maintained long-term weight and better mechanical properties than pure structures. The results of molecular analysis after 21 days of culture showed that the expression of differential markers (Acrosin, Scp3 and Prm1) in the 3D system containing laminin was significantly higher than other groups. The hormonal analysis confirmed the function of Leydig and Sertoli cells for the synthesis of testosterone and inhibin.
Conclusion: The usage of a 3D system containing laminin could lead to the differentiation of SSCs and the progression of meiosis to the stage of haploid spermatid that pave the way for new human infertility treatments in the future.