Recent advances in developing 3D culture systems of spermatogonial stem cell preservation and differentiation: A narrative review


Male infertility has received vast attention in recent years and has no clear etiology in almost 40% of cases. Several methods have been suggested for preserving sperm and spermatogonial stem cells (SSCs) in both in vivo and in vitro conditions. The efficacy of these methods is related to their abilities, including providing an optimal environment for sperm preservation and long-term SSC culture for in vivo and in vitro differentiation of these cells. In this review article, a full MEDLINE/PubMed search was performed using the following search terms: "Spermatogonial Progenitor Cells, Stem Cells, Fertility Preservations, Sperm Freezing, Cell Differentiations, Tissue Scaffold, 3-Dimensional Cell Culture", which retrieved results from 1973-2022. Related articles were added to the bibliography of selected articles. Exclusion criteria included non-English language, abstract only, and unrelated articles. The production of functioning male germ cells is suggested by introducing modern bioengineered systems as a new hope for the maintenance of male fertility. Till now, few in vitro spermatogenesis investigations have provided appreciable amounts of mature gametes. Each method had benefits and disadvantages, but the 3-dimensional culture method had the greatest impact on the differentiation and preservation of SSCs. One of the critical elements of research is the preservation of sperm and the differentiation of SSCs. Several methods have been employed in this area. Various scaffolds providing an environment similar to an extracellular matrix and conditions for germ cell development and survival have been employed in recent research.

Key words: Tissue engineering, Male infertility, Scaffold, Stem cells, 3D cell culture, Differentiation.

[1] Afferri A, Allen H, Dierickx S, Bittaye M, Marena M, Pacey A, et al. Availability of services for the diagnosis and treatment of infertility in the Gambias public and private health facilities: A cross-sectional survey. BMC Health Serv Res 2022; 22: 1–11.

[2] Virant-Klun I, Imamovic-Kumalic S, Pinter B. From oxidative stress to male infertility: Review of the associations of endocrine-disrupting chemicals (Bisphenols, Phthalates, and Parabens) with human semen quality. Antioxidants 2022; 11: 1617.

[3] Carlsen E, Giwercman A, Keiding N, Skakkebaek NE. Evidence for decreasing quality of semen during past 50 years. BMJ 1992; 305: 609–613.

[4] Ilacqua A, Izzo G, Emerenziani GP, Baldari C, Aversa A. Lifestyle and fertility: The influence of stress and quality of life on male fertility. Reprod Biol Endocrinol 2018; 16: 115.

[5] Dolmans M-M, Manavella DD. Recent advances in fertility preservation. J Obstet Gynaecol Res 2019; 45: 266–279.

[6] Abdel-Naser MB, Zouboulis CC. Male fertility and skin diseases. Rev Endocr Metab Disord 2016; 17: 353–365.

[7] Das S, Roychoudhury Sh, Roychoudhury Sh, Agarwal A, Henkel R. Role of infection and leukocytes in male infertility. Adv Exp Med Biol 2022; 1358: 115–140.

[8] Abdel-Naser MB, Altenburg A, Zouboulis CC, Wollina U. Schistosomiasis (bilharziasis) and male infertility. Andrologia 2019; 51: e13165.

[9] Micol LA, Adenubi F, Williamson E, Lane Sh, Mitchell RT, Sangster P. The importance of the urologist in male oncology fertility preservation. BJU Int 2022; 130: 637– 645.

[10] Beveridge T. Anatomy of the infrarenal aortic plexus: Implications for nerve-sparing retroperitoneal lymph node dissection [Ph.D. thesis]. Canada: The University of Western Ontario; 2018.

[11] van Wolff M, Nawroth F. Indications for and against fertility preservation. In: van Wolff M, Nawroth F. Fertility preservation in oncological and non-oncological diseases. Switzerland: Springer; 2020. 25–29.

[12] Klitzman R. How much is a child worth? Providers’ and patients’ views and responses concerning ethical and policy challenges in paying for ART. PLoS One 2017; 12: e0171939.

[13] Sanner C, Coleman M. (Re) constructing family images: Stepmotherhood before biological motherhood. J Marriage Fam 2017; 79: 1462–1477.

[14] Zapzalka DM, Redmon JB, Pryor JL. A survey of oncologists regarding sperm cryopreservation and assisted reproductive techniques for male cancer patients. Cancer 1999; 86: 1812–1817.

[15] Redig AJ, Brannigan R, Stryker SJ, Woodruff TK, Jeruss JS. Incorporating fertility preservation into the care of young oncology patients. Cancer 2011; 117: 4–10.

[16] Yokonishi T, Ogawa T. Cryopreservation of testis tissues and in vitro spermatogenesis. Reprod Med Biol 2016; 15: 21–28.

[17] Sharma S, Wistuba J, Pock T, Schlatt S, Neuhaus N. Spermatogonial stem cells: Updates from specification to clinical relevance. Hum Reprod Update 2019; 25: 275– 297.

[18] Kubota H, Brinster RL. Spermatogonial stem cells. Biol Reprod 2018; 99: 52–74.

[19] Ibtisham F, Honaramooz A. Spermatogonial stem cells for in vitro spermatogenesis and in vivo restoration of fertility. Cells 2020; 9: 745.

[20] Seki Y, Yamaji M, Yabuta Y, Sano M, Shigeta M, Matsui Y, et al. Cellular dynamics associated with the genomewide epigenetic reprogramming in migrating primordial germ cells in mice. Development 2007; 134: 2627–2638.

[21] Wang P, Miao Y, Li X-H, Zhang N, Wang Q, Yue W, et al. Proteome landscape and spatial map of mouse primordial germ cells. Sci China Life Sci 2021; 64: 966– 981.

[22] Akyash F, Aflatoonian R, Yazd EF, Golzadeh J, Tahajjodi SS, Moore H, et al. Testicular sperm extraction derived cells conditioned medium as an in vitro niche supports germ cells development from human embryonic stem cells. In: Proceedings of the 35th Annual Meeting of the European Society for Human Reproduction and Embryology. Viena, Austria; 2019.

[23] Nikolic A, Volarevic V, Armstrong L, Lako M, Stojkovic M. Primordial germ cells: Current knowledge and perspectives. Stem Cells Int 2016; 2016: 1741072.

[24] Hayashi K, Saitou M. Perspectives of germ cell development in vitro in mammals. Anim Sci J 2014; 85: 617–626.

[25] Zhu Y, Hu H-L, Li P, Yang Sh, Zhang W, Ding H, et al. Generation of male germ cells from induced pluripotent stem cells (iPS cells): An in vitro and in vivo study. Asian J Androl 2012; 14: 574–579.

[26] Li L, Yang R, Yin C, Kee K. Studying human reproductive biology through single-cell analysis and in vitro differentiation of stem cells into germ cell-like cells. Hum Reprod Update 2020; 26: 670–688.

[27] Pirouz M, Klimke A, Kessel M. The reciprocal relationship between primordial germ cells and pluripotent stem cells. J Mol Med 2012; 90: 753–761.

[28] Wainwright EN, Wilhelm D. The game plan: Cellular and molecular mechanisms of mammalian testis development. Curr Top Dev Biol 2010; 90: 231–262.

[29] Piprek RP. Molecular and cellular machinery of gonadal differentiation in mammals. Int J Dev Biol 2010; 54: 779– 786.

[30] Mackay S. Gonadal development in mammals at the cellular and molecular levels. Int Rev Cytol 2000; 200: 47–99.

[31] Brennan J, Capel B. One tissue, two fates: Molecular genetic events that underlie testis versus ovary development. Nat Rev Genet 2004; 5: 509–521.

[32] Nef S, Stevant I, Greenfield A. Characterizing the bipotential mammalian gonad. Curr Top Dev Biol 2019; 134: 167–194.

[33] Chen L-Y, Willis WD, Eddy EM. Targeting the Gdnf gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development. Proc Natl Acad Sci USA 2016; 113: 1829–1834.

[34] Costoya JA, Hobbs RM, Barna M, Cattoretti G, Manova K, Sukhwani M, et al. Essential role of Plzf in maintenance of spermatogonial stem cells. Nat Genet 2004; 36: 653– 659.

[35] Kashfi A, Narenji Sani R, Ahmadi-hamedani M. The beneficial effect of equine chorionic gonadotropin hormone (eCG) on the in vitro co-culture of bovine spermatogonial stem cell with Sertoli cells. Comparat Clin Pathol 2019; 28: 701–704.

[36] Davis JC, Snyder EM, Hogarth CA, Small Ch, Griswold MD. Induction of spermatogenic synchrony by retinoic acid in neonatal mice. Spermatogenesis 2013; 3: e23180.

[37] Dovere L, Fera S, Grasso M, Lamberti D, Gargioli C, Muciaccia B, et al. The niche-derived glial cell linederived neurotrophic factor (GDNF) induces migration of mouse spermatogonial stem/progenitor cells. PLoS One 2013; 8: e59431.

[38] Kitadate Y, Jorg DJ, Tokue M, Maruyama A, Ichikawa R, Tsuchiya S, et al. Competition for mitogens regulates spermatogenic stem cell homeostasis in an open niche. Cell Stem Cell 2019; 24: 79–92.

[39] Masaki K, Sakai M, Kuroki Sh, Jo J-I, Hoshina K, Fujimori Y, et al. FGF2 has distinct molecular functions from GDNF in the mouse germline niche. Stem Cell Reports 2018; 10: 1782–1792.

[40] Salaritabar A, Berindan-Neagoe I, Darvish B, Hadjiakhoondi F, Manayi A, Devi KP, et al. Targeting Hedgehog signaling pathway: Paving the road for cancer therapy. Pharmacol Res 2019; 141: 466–480.

[41] Goossens E, Van Saen D, Tournaye H. Spermatogonial stem cell preservation and transplantation: From research to clinic. Hum Reprod 2013; 28: 897–907.

[42] Gies I, De Schepper J, Goossens E, Van Saen D, Pennings G, Tournaye H. Spermatogonial stem cell preservation in boys with Klinefelter syndrome: To bank or not to bank, that’s the question. Fertil Steril 2012; 98: 284–289.

[43] Pourhabibi Zarandi N, Galdon G, Kogan S, Atala A, Sadri-Ardekani H. Cryostorage of immature and mature human testis tissue to preserve spermatogonial stem cells (SSCs): A systematic review of current experiences toward clinical applications. Stem Cells Cloning 2018; 11: 23–38.

[44] Mohammadzadeh E, Mirzapour T, Nowroozi MR, Nazarian H, Piryaei A, Alipour F, et al. Differentiation of spermatogonial stem cells by soft agar threedimensional culture system. Artif Cells Nanomed Biotechnol 2019; 47: 1772–1781.

[45] Ashouri Movassagh S, Banitalebi Dehkordi M, Koruji M, Pourmand GhR, Farzaneh P, Ashouri Movassagh S, et al. In vitro spermatogenesis by three-dimensional culture of spermatogonial stem cells on decellularized testicular matrix. Galen Med J 2019; 8: e1565.

[46] Sen Ch, Freund D, Gomperts BN. Three-dimensional models of the lung: Past, present and future: A mini review. Biochem Soc Trans 2022; 50: 1045–1056.

[47] Sato T, Katagiri K, Kojima K, Komeya M, Yao M, Ogawa T. In vitro spermatogenesis in explanted adult mouse testis tissues. PloS One 2015; 10: e0130171.

[48] Mahmoud H. Concise review: Spermatogenesis in an artificial three-dimensional system. Stem Cells 2012; 30: 2355–2360.

[49] Eyni H, Ghorbani S, Nazari H, Hajialyani M, Razavi Bazaz S, Mohaqiq M, et al. Advanced bioengineering of male germ stem cells to preserve fertility. J Tissue Eng 2021; 12: 20417314211060590.

[50] Kopan R, Ilagan MXG. The canonical Notch signaling pathway: Unfolding the activation mechanism. Cell 2009; 137: 216–233.

[51] Jahnukainen K, Mitchell RT, Stukenborg J-B. Testicular function and fertility preservation after treatment for haematological cancer. Curr Opin Endocrinol Diabetes Obes 2015; 22: 217–223.

[52] Stukenborg J-B, Schlatt S, Simoni M, Yeung Ch-H, Abu Elhija M, Luetjens CM, et al. New horizons for in vitro spermatogenesis? An update on novel threedimensional culture systems as tools for meiotic and post-meiotic differentiation of testicular germ cells. Mol Hum Reprod 2009; 15: 521–529.

[53] Vasileva SA. Cultivation of mammals early male germ cells in a semi liquid medium. IOP Conf Series: Earth Environment Sci 2019; 315: 072014.

[54] Gummow BM. Multiple signaling pathways regulate steroidogenic factor-1 activity through induction of cofactor availability. US: University of Michigan; 2005.

[55] Salem M, Feizollahi N, Jabari A, Golmohammadi MG, Shirinsokhan A, Ghanami Gashti N, et al. Differentiation of human spermatogonial stem cells using a human decellularized testicular scaffold supplemented by platelet-rich plasma. Artif Organs 2023; 47: 840–853.

[56] Shaban S, El-Husseny MWA, Abushouk AI, Salem AMA, Mamdouh M, Abdel-Daim MM. Effects of antioxidant supplements on the survival and differentiation of stem cells. Oxid Med Cell Longev 2017; 2017: 5032102.

[57] Erkkilaa K, Hirvonen V, Wuokko E, Parvinen M, Dunkel L. N-acetyl-L-cysteine inhibits apoptosis in human male germ cells in vitro. J Clin Endocrinol Metab 1998; 83: 2523–2531.

[58] Kazemzadeh Sh, Mohammadpour Sh, Madadi S, Babakhani A, Shabani M, Khanehzad M. Melatonin in cryopreservation media improves transplantation efficiency of frozen-thawed spermatogonial stem cells into testes of azoospermic mice. Stem Cell Res Ther 2022; 13: 346.

[59] Bashiri Z, Gholipourmalekabadi M, Falak R, Amiri I, Asgari H, Chauhan NPS, et al. In vitro production of mouse morphological sperm in artificial testis bioengineered by 3D printing of extracellular matrix. Int J Biol Macromol 2022; 217: 824–841.

[60] Ethics Committee of the American Society for Reproductive Medicine. Fertility preservation and reproduction in cancer patients. Fertil Steril 2005; 83: 1622–1628.

[61] Ferro C, Florindo HF, Santos HA. Selenium nanoparticles for biomedical applications: From development and characterization to therapeutics. Adv Healthc Mater 2021; 10: 2100598.

[62] Pirnia A, Parivar K, Hemadi M, Yaghmaei P, Gholami M. Stemness of spermatogonial stem cells encapsulated in alginate hydrogel during cryopreservation. Andrologia 2017; 49: e12650.

[63] Zhao Y, Zhang P, Ge W, Feng Y, Li L, Sun Z, et al. Alginate oligosaccharides improve germ cell development and testicular microenvironment to rescue busulfan disrupted spermatogenesis. Theranostics 2020; 10: 3308–3324.

[64] Baert Y, Dvorakova-Hortova K, Margaryan H, Goossens E. Mouse in vitro spermatogenesis on alginate-based 3D bioprinted scaffolds. Biofabrication 2019; 11: 035011.

[65] Del Vento F, Poels J, Vermeulen M, Ucakar B, Giudice MG, Kanbar M, et al. Accelerated and improved vascular maturity after transplantation of testicular tissue in hydrogels supplemented with VEGF-and PDGF-loaded nanoparticles. Int J Mol Sci 2021; 22: 5779.

[66] Hemadi M, Assadollahi V, Saki G, Pirnia A, Alasvand M, Zendehdel A, et al. Use of alginate hydrogel to improve long-term 3D culture of spermatogonial stem cells: Stemness gene expression and structural features. Zygote 2022; 30: 312–318.

[67] Veisi M, Mansouri K, Assadollahi V, Jalili C, Pirnia A, Salahshoor MR, et al. Evaluation of co-cultured spermatogonial stem cells encapsulated in alginate hydrogel with Sertoli cells and their transplantation into azoospermic mice. Zygote 2022; 30: 344–351.

[68] Baert Y, Stukenborg J-B, Landreh M, De Kock J, Jornvall H, Soder O, et al. Derivation and characterization of a cytocompatible scaffold from human testis. Hum Reprod 2015; 30: 256–267.

[69] Borzouie Z, Hekmatimoghaddam SH, Jebali A, Aflatoonian B. The viability of human testis-derived cells on human serum albumin-based scaffold as an artificial male germ cell niche. Int J Fertil Steril 2020; 14: 150–153.

[70] Borzouie Z, Naghibzadeh M, Talebi AR, Pourrajab F, Jebali A, Nikukar H, et al. Development of an artificial male germ cell niche using electrospun poly vinyl alcohol/ human serum albumin/ gelatin fibers. Cell J 2019; 21: 300–306.

[71] Ghorbani S, Eyni H, Khosrowpour Z, Salari Asl L, Shabani R, Nazari H, et al. Spermatogenesis induction of spermatogonial stem cells using nanofibrous poly (llactic acid)/multi-walled carbon nanotube scaffolds and naringenin. Polymers Adv Technol 2019; 30: 3011–3025.

[72] Köse S, Yersal N, Onen S, Korkusuz P. Comparison of hematopoietic and spermatogonial stem cell niches from the regenerative medicine aspect. Adv Exp Med Biol 2018; 1107: 15–40.

[73] Albalushi H. In vitro models aiming for fertility preservation. Sweden: Karolinska Institutet; 2018.

[74] Grin L, Girsh E, Harlev A. Male fertility preservationmethods, indications and challenges. Andrologia 2021; 53: e13635.

[75] Kanbar M, Delwiche G, Wyns Ch. Fertility preservation for prepubertal boys: Are we ready for autologous grafting of cryopreserved immature testicular tissue? Ann Endocrinol 2022; 83: 210–217.

[76] Del Vento F, Vermeulen M, de Michele F, Giudice MG, Poels J, Des Rieux A, et al. Tissue engineering to improve immature testicular tissue and cell transplantation outcomes: One step closer to fertility restoration for prepubertal boys exposed to gonadotoxic treatments. Int J Mol Sci 2018; 19: 286.

[77] Bhaskar R, Gupta MK. Testicular tissue engineering: An emerging solution for in vitro spermatogenesis. In: Pal K, Banejee I, Sarkar P, Kim D, Deng W-P, Dubey NK. Biopolymer-based formulations. US: Elsevier; 2020. 835–858.

[78] O’Hara L, Smith LB. Androgen receptor roles in spermatogenesis and infertility. Best Pract Res Clin Endocrinol Metab 2015; 29: 595–605.

[79] Pui HP, Saga Y. Gonocytes-to-spermatogonia transition initiates prior to birth in murine testes and it requires FGF signaling. Mech Dev 2017; 144: 125–139.

[80] Jensen JR, Morbeck DE, Coddington III CC. Fertility preservation. Mayo Clin Proc 2011; 86: 45–49.

[81] Del-Pozo-Lerida S, Salvador C, Martinez-Soler F, Tortosa A, Perucho M, Gimenez-Bonafe P. Preservation of fertility in patients with cancer (review). Oncol Rep 2019; 41: 2607–2614.

[82] Deepinder F, Agarwal A. Approach to fertility preservation in adult and pre-pubertal males. In: Seli E, Agarwal A. Fertility preservation: Emerging technologies and clinical applications. Switzerland: Springer; 2012. 353–364.