Iranian Journal of Basic Medical Sciences

Iranian Journal of Basic Medical Sciences

Modular 3D culture platform combining methylcellulose, testicular ECM, and Sertoli cells for in vitro spermatogenesis

Articles in Press

Document Type : Original Article

Authors
Narjes Feizollahi 1
Ehsan Heidarian 2
Mehdi Forouzesh 3
Maryam Salem 4, 5
Soheil Kianirad 6
Nasrin Ghanami Gashti 7
Masoome Jabarpour 8
Rasool Setareyi 9
Ayob Jabari 10, 11
Farnaz Khadivi 12, 13
Morteza Koruji 14, 15
Mohammad Naji 2
Mehdi Abbasi 1
1 Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
2 Urology and Nephrology Research Center, Research Institute for Urology and Nephrology, Shahid Beheshti University of Medical Sciences,Tehran, Iran
3 Legal Medicine Research Center, Legal Medicine Organization, Tehran, Iran
4 Research Laboratory for Embryology and Stem Cells, Department of Anatomical Sciences, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran
5 Infertility Department, Alavi Hospital, Ardabil University of Medical Science, Ardabil, Iran
6 Department of Chemical and Petroleum Engineering, Sharif University of Technology, Tehran, Iran
7 Bernal Institute and School of Engineering, University of Limerick, Limerick, Ireland
8 Department of Infertility, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran
9 Department of Life Science Engineering, Faculty of New Science and Technology, University of Tehran, Tehran, Iran
10 Department of Obstetrics and Gynecology, Molud Infertility Treatment Center, Zahedan University of Medical Sciences, Zahedan, Iran
11 Clinical Immunology Research Center, Zahedan University of Medical Sciences, Zahedan, Iran
12 Department of Anatomy, School of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran
13 Medical Plants Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran
14 Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Tehran, Iran
15 Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
10.22038/ijbms.2026.93648.20214
Abstract
Objective(s): We aimed to develop and characterize a 3D culture platform combining methylcellulose (MC) with decellularized testicular extracellular matrix (ECM) and Sertoli cell co-culture to support human spermatogonial stem cell (SSC) survival and differentiation.
Materials and Methods: Human testicular cells were isolated and characterized by immunofluorescence and flow cytometry. Sheep testicular tissue was decellularized using detergents and characterized by several histology techniques. ECM was solubilized and mixed with MC at various ratios to make hybrid hydrogels. Hydrogels were characterized for pore size, porosity, mechanical properties, gelation kinetics, and degradation kinetics using scanning electron microscopy, rheological analysis, turbidity analysis, and mass loss assays. Human SSCs and Sertoli cells were cultured in 3D hydrogels for four weeks under differentiation conditions. Cell viability was assessed using the MTT assay, and gene expression of SSC, meiotic, post-meiotic, and apoptotic markers was evaluated by RT-PCR.
Results: After four weeks of proliferation, PLZF-positive cells increased from 19% to 73%. Decellularization reduced DNA content by 96% while preserving key matrix components. Hybrid hydrogels displayed interconnected porous structures with pore sizes of about 65–181 µm. All functional hydrogels supported cell viability; however, ECM-rich hydrogels significantly up-regulated PRM2 compared to 2D controls, indicating enhanced post-meiotic differentiation. MC-only scaffolds displayed elevated pro-apoptotic BAX expression compared to ECM-rich matrices, suggesting that bioactive ECM ligands confer cytoprotection.
Conclusion: The modular MC/ECM hybrid hydrogel provides a tunable, physiologically relevant 3D culture system that is superior to conventional approaches in supporting SSC maintenance and differentiation, offering promise for fertility preservation strategies.
Keywords
Cell differentiation
Decellularized extracellular matrix
Hydrogels
Methylcellulose
Sertoli cells
Germline adult stem cells
Subjects
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Iranian Journal of Basic Medical Sciences

Articles in Press, Accepted Manuscript
Available Online from 14 July 2026