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Use of alginate hydrogel to improve long-term 3D culture of spermatogonial stem cells: stemness gene expression and structural features

Published online by Cambridge University Press:  13 October 2021

Masoud Hemadi
Cellular and Molecular Research Center, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
Vahideh Assadollahi
Cancer and Immunology Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran
Ghasem Saki
Physiology Research Center, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
Afshin Pirnia
Medical Technology Research Center, Research Institute of Health Technology, Kermanshah University of Medical Sciences, Kermanshah, Iran
Masoud Alasvand
Cancer and Immunology Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran
Abolfazl Zendehdel
Geriatric Department, Ziayian Hospital, Tehran University of Medical Sciences, Tehran, Iran
Mohammadreza Gholami*
Medical Technology Research Center, Research Institute of Health Technology, Kermanshah University of Medical Sciences, Kermanshah, Iran
Author for correspondence: Mohammadreza Gholami. Medical Technology Research Center, Institute of Health Technology, Kermanshah University of Medical Sciences, Kermanshah Zip code: 67148-69914, Iran. Tel:/Fax: +98 8334276477. E-mail:


The quality and quantity of a spermatogonial stem-cell (SSC) culture can be measured in less time using a 3D culture in a scaffold. The present study investigated stemness gene expression and the morphological and structural characterization of SSCs encapsulated in alginate. SSCs were harvested from BALB/c neonatal mice testes through two-step mechanical and enzymatic digestion. The spermatogonial populations were separated using magnetic-activated cell sorting (MACS) using an anti-Thy1 antibody and c-Kit. The SSCs then were encapsulated in alginate hydrogel. After 2 months of SSC culturing, the alginate microbeads were extracted and stained to evaluate their histological properties. Real-time polymerase chain reaction (PCR) was performed to determine the stemness gene expression. Scanning electron microscopy (SEM) was performed to evaluate the SSC morphology, density and scaffold structure. The results showed that encapsulated SSCs had decreased expression of Oct4, Sox2 and Nanos2 genes, but the expression of Nanog, Bcl6b and Plzf genes was not significantly altered. Histological examination showed that SSCs with pale nuclei and numerous nucleolus formed colonies. SEM evaluation revealed that the alginate scaffold structure preserved the SSC morphology and density for more than 60 days. Cultivation of SSCs on alginate hydrogel can affect Oct4, Sox2 and Nanos2 expression.

Research Article
© The Author(s), 2021. Published by Cambridge University Press

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