Advancing in vitro spermatogenesis: a comparative review of static and dynamic culture systems for reproductive biology and infertility treatment

Avances en la espermatogénesis in vitro: una revisión comparativa de los sistemas de cultivo estáticos y dinámicos para la biología reproductiva y el tratamiento de la infertilidad

Infertility is an important issue among couples worldwide affecting an estimated 50–80 million people globally. The World Health Organization estimates that male factors are responsible for approximately 20–30% of all infertility cases. In vitro spermatogenesis (IVS) is the experimental approach that has been developed for mimicking seminiferous tubule-like functional structures in vitro. Cell culture is one of the most important techniques needed in biology-based science, and the standard methods are usually 2-dimensional (2D) cultures such as T-flasks, tissue culture well plates, Petri dishes and well plates designed for spheroids formation. The conventional 2D cell culture has several restrictions. Then 3-dimensional (3D) cultures were created, and these static culture mediums also had limitations. Considering some of the limitations of conventional culture, static culture environments have been changed to dynamic culture medium systems. Techniques such as microfluidics and bioreactors have been developed to achieve more physiologically relevant tissue substitutes and show good potential to provide an effective approach for IVS. This review offers a comprehensive comparison of static and dynamic culture methods for IVS, their fundamental principles, advantages and limitations, as well as their potential in facilitating reproductive biology and infertility treatment.

Resumen

La infertilidad es un problema importante entre las parejas en todo el mundo, que afecta a un estimado de 50 a 80 millones de personas a nivel global. La Organización Mundial de la Salud estima que los factores masculinos son responsables de aproximadamente el 20–30% de todos los casos de infertilidad. La espermatogénesis in vitro (EIV) es el enfoque experimental que se ha desarrollado para imitar estructuras funcionales similares a los túbulos seminíferos in vitro. El cultivo celular es una de las técnicas más importantes necesarias en las ciencias biológicas. Los métodos estándar suelen ser cultivos bidimensionales (2D), como matraces T, placas de cultivo tisular, placas de Petri y placas diseñadas para la formación de esferoides. El cultivo celular 2D convencional tiene varias limitaciones. Posteriormente, se crearon cultivos tridimensionales (3D), pero estos medios de cultivo estáticos también presentaban limitaciones. Considerando algunas de las limitaciones de los cultivos convencionales, los entornos de cultivo estáticos han evolucionado hacia sistemas de medios de cultivo dinámicos. Se han desarrollado técnicas como la microfluídica y los biorreactores para lograr sustitutos tisulares más fisiológicamente relevantes, mostrando un buen potencial para proporcionar un enfoque efectivo para la EIV. Esta revisión ofrece una comparación exhaustiva de los métodos de cultivo estáticos y dinámicos para la EIV, sus principios fundamentales, ventajas y limitaciones, así como su potencial en el avance de la biología reproductiva y el tratamiento de la infertilidad.

In vitro spermatogenesis; Male infertility; Static culture; Three-dimensional culture; Dynamic culture; Microfluidics; Bioreactors

Palabras Clave

Espermatogénesis in vitro; Infertilidad masculina; Cultivo estático; Cultivo tridimensional; Cultivo dinámico; Microfluídica; Biorreactores

[1] Roshandel E, Mehravar M, Nikoonezhad M, Alizadeh AM, Majidi M, Salimi M, et al. Cell-based therapy approaches in treatment of non-obstructive azoospermia. Reproductive Sciences. 2023; 30: 1482–1494.

[2] Lu C, Yang M, Rossi RM, Wang A, Feitosa WB, Diaz FJ, et al. Deletion of the mouse X-linked Prame gene causes germ cell reduction in spermatogenesis. Molecular Reproduction and Development. 2020; 87: 666–679.

[3] Wiwanitkit V. Difference in physiogenomics between male and female infertility. Andrologia. 2008; 40: 158–160.

[4] Di Persio S, Neuhaus N. Human spermatogonial stem cells and their niche in male (in) fertility: novel concepts from single-cell RNA-sequencing. Human Reproduction. 2023; 38: 1–13.

[5] Martinez MP, Elbardisi H, Majzoub A, Arafa M. Epidemiology of genetic disorders in male infertility. In Zitzmann M, O’Bryan MK (eds.) Genetics of male infertility (pp. 73–94). 1st edn. Springer: Germany. 2020.

[6] Sá R, Ferraz L, Barros A, Sousa M. The Klinefelter syndrome and testicular sperm retrieval outcomes. Genes. 2023; 14: 647.

[7] Sethi S, Singh R. Y-chromosome deletion testing in infertility. In Editor B, Editor C (eds.) Genetic testing in reproductive medicine (pp. 17–29). 1st edn. Springer: Germany. 2024.

[8] Nguyen AV, Julian S, Weng N, Flannigan R. Advances in human in vitro spermatogenesis: a review. Molecular Aspects of Medicine. 2024; 100: 101320.

[9] Georgakopoulos I, Kouloulias V, Ntoumas GN, Desse D, Koukourakis I, Kougioumtzopoulou A, et al. Radiotherapy and testicular function: a comprehensive review of the radiation-induced effects with an emphasis on spermatogenesis. Biomedicines. 2024; 12: 1492.

[10] Duffin K, Mitchell RT, Brougham MF, Hamer G, van Pelt AM, Mulder CL. Impacts of cancer therapy on male fertility: past and present. Molecular Aspects of Medicine. 2024; 100: 101308.

[11] Gizer M, Önen S, Korkusuz P. The evolutionary route of in vitro human spermatogenesis: what is the next destination? Stem Cell Reviews and Reports. 2024; 20: 1406–1419.

[12] Karoii DH, Mahforoozmahalleh ZH. The role of long noncoding RNAs in gene expression and epigenetic regulation in spermatogonial stem cells and therapeutic implications. In Piccaluga PP, Visani G, Khattab S (eds.) Stem cell transplantation (pp. 1–24). Springer: Germany. 2024.

[13] Khalimova YS. Features of sperm development: spermatogenesis and fertilization. American Journal of Bioscience and Clinical Integrity. 2024; 1: 90–98.

[14] Umoh EA. Spermatogenesis. 1st edn. Springer: Cham. 2024.

[15] Honaramooz A. Theoretical and experimental strategies for preservation and restoration of male fertility. In Robinson JL, Nagy PA (eds.) Human reproductive and prenatal genetics (pp. 411–438). Elsevier: Amsterdam. 2023.

[16] Bashiri Z, Gholipourmalekabadi M, Khadivi F, Salem M, Afzali A, Cham TC, et al. In vitro spermatogenesis in artificial testis: current knowledge and clinical implications for male infertility. Cell and Tissue Research. 2023; 394: 393–421.

[17] Grin L, Girsh E, Harlev A. Male fertility preservation—methods, indications and challenges. Andrologia. 2021; 53: e13635.

[18] Cui Y. Advancements in testicular organoid research: insights for fertility preservation [doctoral thesis]. Karolinska Institutet. 2024.

[19] Sadeghi MR. A new perspective for the future of male infertility treatment and research. Journal of Reproduction & Infertility. 2020; 21: 1.

[20] Cho IK, Easley CA. Recent developments in in vitro spermatogenesis and future directions. Reproductive Medicine. 2023; 4: 215–232.

[21] Sá R, Neves R, Fernandes S, Alves C, Carvalho F, Silva J, et al. Cytological and expression studies and quantitative analysis of the temporal and stage-specific effects of follicle-stimulating hormone and testosterone during cocultures of the normal human seminiferous epithelium. Biology of Reproduction. 2008; 79: 962–975.

[22] Sá R, Graça I, Silva J, Malheiro I, Carvalho F, Barros A, et al. Quantitative analysis of cellular proliferation and differentiation of the human seminiferous epithelium in vitro. Reproductive Sciences. 2012; 19: 1063–1074.

[23] Licata JP, Schwab KH, Har-El YE, Gerstenhaber JA, Lelkes PI. Bioreactor technologies for enhanced organoid culture. International Journal of Molecular Sciences. 2023; 24: 11427.

[24] Urzì O, Gasparro R, Costanzo E, De Luca A, Giavaresi G, Fontana S, et al. Three-dimensional cell cultures: the bridge between in vitro and in vivo models. International Journal of Molecular Sciences. 2023; 24: 12046.

[25] Kapałczyńska M, Kolenda T, Przybyła W, Zajączkowska M, Teresiak A, Filas V, et al. 2D and 3D cell cultures—a comparison of different types of cancer cell cultures. Archives of Medical Science. 2018; 14: 910–919.

[26] Canadas RF, Marques AP, Reis RL, Oliveira JM. Bioreactors and microfluidics for osteochondral interface maturation. Advances in Experimental Medicine and Biology. 2018; 1059: 395–420.

[27] Serrano JC, Gillrie MR, Li R, Ishamuddin SH, Moeendarbary E, Kamm RD. Microfluidic—based reconstitution of functional lymphatic microvasculature: elucidating the role of lymphatics in health and disease. Advanced Science. 2024; 11: e2302903.

[28] Naeemi S, Sabetkish S, Kiani MJ, Dehghan A, Kajbafzadeh AM. Ex-vivo and in-vivo expansion of spermatogonial stem cells using cell-seeded microfluidic testis scaffolds and animal model. Cell and Tissue Banking. 2023; 24: 153–166.

[29] Komeya M, Hayashi K, Nakamura H, Yamanaka H, Sanjo H, Kojima K, et al. Pumpless microfluidic system driven by hydrostatic pressure induces and maintains mouse spermatogenesis in vitro. Scientific Reports. 2017; 7: 1–8.

[30] Sakib S, Voigt A, de Lima EMLN, Su L, Ungrin M, Rancourt D, et al. The proliferation of pre-pubertal porcine spermatogonia in stirred suspension bioreactors is partially mediated by the wnt/β-catenin pathway. International Journal of Molecular Sciences. 2021; 22: 13549.

[31] Amirkhani Z, Movahedin M, Baheiraei N, Ghiaseddin A. Mini bioreactor can support in vitro spermatogenesis of mouse testicular tissue. Cell Journal. 2022; 24: 277.

[32] Priyadarshini BM, Dikshit V, Zhang Y. 3D-printed bioreactors for in vitro modeling and analysis. International Journal of Bioprinting. 2020; 6: 267.

[33] 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. Journal of Tissue Engineering. 2021; 12: 20417314211060590.

[34] Isah T, Umar S, Mujib A, Sharma MP, Rajasekharan P, Zafar N, et al. Secondary metabolism of pharmaceuticals in the plant in vitro cultures: strategies, approaches, and limitations to achieving higher yield. Plant Cell, Tissue and Organ Culture. 2018; 132: 239–265.

[35] Komeya M, Kimura H, Nakamura H, Yokonishi T, Sato T, Kojima K, et al. Long-term ex vivo maintenance of testis tissues producing fertile sperm in a microfluidic device. Scientific Reports. 2016; 6: 21472.

[36] Sá R, Sousa M, Cremades N, Alves C, Silva J, Barros A. In vitro maturation of spermatozoa. In Agarwal A, Rizk B, Gupta S (eds.) Male Infertility (pp. 453–480). CRC Press: America. 2007.

[37] Bhaskar R, Gupta MK. Testicular tissue engineering: an emerging solution for in vitro spermatogenesis. In Pal K, Chauhan AK (eds.) Biopolymer-based formulations (pp. 835–858). Elsevier: Amsterdam. 2020.

[38] Martinovitch P. The development in vitro of the mammalian gonad. Ovary and ovogenesis. Proceedings of the Royal Society B: Biological Sciences. 1938; 125: 232–249.

[39] Trowell O. The culture of mature organs in a synthetic medium. Experimental Cell Research. 1959; 16: 118–147.

[40] Steinberger E, Steinberger A, Perloff WH. Initiation of spermatogenesis in vitro. Endocrinology. 1964; 74: 788–792.

[41] Steinberger A. Growth of rat testes fragments in organ culture. Federation Proceedings. 1963; 22: 1033–1042.

[42] Steinberger A, Steinberger E. Stimulatory effect of vitamins and glutamine on the differentiation of germ cells in rat testes organ culture grown in chemically defined media. Experimental Cell Research. 1966; 44: 429–435.

[43] Haneji T, Nishimune Y. Hormones and the differentiation of type A spermatogonia in mouse cryptorchid testes incubated in vitro. Journal of Endocrinology. 1982; 94: 43–50.

[44] Boitani C, Politi MG, Menna T. Spermatogonial cell proliferation in organ culture of immature rat testis. Biology of Reproduction. 1993; 48: 761–767.

[45] Suzuki S, Sato K. The fertilising ability of spermatogenic cells derived from cultured mouse immature testicular tissue. Zygote. 2003; 11: 307–316.

[46] Sato T, Katagiri K, Gohbara A, Inoue K, Ogonuki N, Ogura A, et al. In vitro production of functional sperm in cultured neonatal mouse testes. Nature. 2011; 471: 504–507.

[47] Medrano JV, Rombaut C, Simon C, Pellicer A, Goossens E. Human spermatogonial stem cells display limited proliferation in vitro under mouse spermatogonial stem cell culture conditions. Fertility and Sterility. 2016; 106: 1539–1549.e8.

[48] Yokonishi T, Sato T, Katagiri K, Komeya M, Kubota Y, Ogawa T. In vitro reconstruction of mouse seminiferous tubules supporting germ cell differentiation. Biology of Reproduction. 2013; 89: 15.

[49] Feng X, Matsumura T, Yamashita Y, Sato T, Hashimoto K, Odaka H, et al. In vitro spermatogenesis in isolated seminiferous tubules of immature mice. PLOS ONE. 2023; 18: e0283773.

[50] Zheng Y, Tian X, Zhang Y, Qin J, An J, Zeng W. In vitro propagation of male germline stem cells from piglets. Journal of Assisted Reproduction and Genetics. 2013; 30: 945–952.

[51] Steinberger A, Steinberger E. In vitro culture of rat testicular cells. Experimental Cell Research. 1966; 44: 443–452.

[52] Dietrich A, Scholten R, Vink A, Oud J. Testicular cell suspensions of the mouse in vitro. Andrologia. 1983; 15: 236–246.

[53] Tres LL, Kierszenbaum AL. Viability of rat spermatogenic cells in vitro is facilitated by their coculture with Sertoli cells in serum-free hormone-supplemented medium. Proceedings of the National Academy of Sciences. 1983; 80: 3377–3381.

[54] Nagao Y. Viability of meiotic prophase spermatocytes of rats is facilitated in primary culture of dispersed testicular cells on collagen gel by supplementing epinephrine or norepinephrine: evidence that meiotic prophase spermatocytes complete meiotic divisions in vitro. In vitro Cellular & Developmental Biology. 1989; 25: 1088–1098.

[55] Rassoulzadegan M, Paquis-Flucklinger V, Bertino B, Sage J, Jasin M, Miyagawa K, et al. Transmeiotic differentiation of male germ cells in culture. Cell. 1993; 75: 997–1006.

[56] Hofmann MC, Hess RA, Goldberg E, Millan JL. Immortalized germ cells undergo meiosis in vitro. Proceedings of the National Academy of Sciences. 1994; 91: 5533–5537.

[57] Marh J, Tres LL, Yamazaki Y, Yanagimachi R, Kierszenbaum AL. Mouse round spermatids developed in vitro from preexisting spermatocytes can produce normal offspring by nuclear injection into in vivo-developed mature oocytes. Biology of Reproduction. 2003; 69: 169–176.

[58] Hasegawa H, Terada Y, Ugajin T, Yaegashi N, Sato K. A novel culture system for mouse spermatid maturation which produces elongating spermatids capable of inducing calcium oscillation during fertilization and embryonic development. Journal of Assisted Reproduction and Genetics. 2010; 27: 565–570.

[59] Tesarik J, Greco E, Rienzi L, Ubaldi F, Guido M, Cohen-Bacrie P, et al. Differentiation of spermatogenic cells during in-vitro culture of testicular biopsy samples from patients with obstructive azoospermia: effect of recombinant follicle stimulating hormone. Human Reproduction. 1998; 13: 2772–2781.

[60] Tesarik J, Mendoza C, Anniballo R, Greco E. In-vitro differentiation of germ cells from frozen testicular biopsy specimens. Human Reproduc-tion. 2000; 15: 1713–1716.

[61] Tanaka A, Nagayoshi M, Awata S, Mawatari Y, Tanaka I, Kusunoki H. Completion of meiosis in human primary spermatocytes through in vitro coculture with Vero cells. Fertility and Sterility. 2003; 79: 795–801.

[62] Wang P, Suo LJ, Shang H, Li Y, Li GX, Li QW, et al. Differentiation of spermatogonial stem cell-like cells from murine testicular tissue into haploid male germ cells in vitro. Cytotechnology. 2014; 66: 365–372.

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

[64] Oatley JM, Brinster RL. The germline stem cell niche unit in mammalian testes. Physiological Reviews. 2012; 92: 577–595.

[65] Kanatsu-Shinohara M, Shinohara T. Spermatogonial stem cell self-renewal and development. Annual Review of Cell and Developmental Biology. 2013; 29: 163–187.

[66] Griswold MD. Spermatogenesis: the commitment to meiosis. Physiolog-ical Reviews. 2016; 96: 1–17.

[67] de Rooij DG, Russell LD. All you wanted to know about spermatogonia but were afraid to ask. Journal of Andrology. 2000; 21: 776–798.

[68] Yokonishi T, McKey J, Ide S, Capel B. Sertoli cell ablation and replace-ment of the spermatogonial niche in mouse. Nature Communications. 2020; 11: 1–11.

[69] Shinohara T, Avarbock MR, Brinster RL. β1- and α6-integrin are surface markers on mouse spermatogonial stem cells. Proceedings of the National Academy of Sciences. 1999; 96: 5504–5509.

[70] Liu B, Shen LJ, Zhao TX, Sun M, Wang JK, Long CL, et al. Automobile exhaust-derived PM2.5 induces blood-testis barrier damage through ROS-MAPK-Nrf2 pathway in sertoli cells of rats. Ecotoxicology and Environmental Safety. 2020; 189: 110053.

[71] Kurek M. Laminins in stemness and germ cell development in human [doctoral thesis]. Karolinska Institutet. 2019.

[72] Cremades N, Bernabeu R, Barros A, Sousa M. In-vitro maturation of round spermatids using co-culture on vero cells. Human Reproduction. 1999; 14: 1287–1293.

[73] Sá R, Miranda C, Carvalho F, Barros A, Sousa M. Expression of stem cell markers: OCT4, KIT, ITGA6, and ITGB1 in the male germinal epithelium. Systems Biology in Reproductive Medicine. 2013; 59: 233–243.

[74] Sousa M, Cremades N, Alves C, Silva J, Barros A. Developmental potential of human spermatogenic cells co-cultured with Sertoli cells. Human Reproduction. 2002; 17: 161–172.

[75] Shima Y, Miyabayashi K, Haraguchi S, Arakawa T, Otake H, Baba T, et al. Contribution of Leydig and Sertoli cells to testosterone production in mouse fetal testes. Molecular Endocrinology. 2013; 27: 63–73.

[76] Li X, Tian E, Wang Y, Wen Z, Lei Z, Zhong Y, et al. Stem Leydig cells: current research and future prospects of regenerative medicine of male reproductive health. Seminars in Cell & Developmental Biology. 2022; 121: 63–70.

[77] Chen LY, Brown PR, Willis WB, Eddy EM. Peritubular myoid cells participate in male mouse spermatogonial stem cell maintenance. Endocrinology. 2014; 155: 4964–4974.

[78] Jokar J, Abdulabbas HT, Alipanah H, Ghasemian A, Ai J, Rahimian N, et al. Tissue engineering studies in male infertility disorder. Human Fertility. 2023; 26: 1617–1635.

[79] Chen LY, Willis WD, Eddy EM. Targeting the Gdnf gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development. Proceedings of the National Academy of Sciences. 2016; 113: 1829–1834.

[80] Kim YH, Oh MG, Bhang DH, Kim BJ, Jung SE, Kim SM, et al. Testicular endothelial cells promote self-renewal of spermatogonial stem cells in rats. Biology of Reproduction. 2019; 101: 360–367.

[81] Bhang DH, Kim BJ, Kim BG, Schadler K, Baek KH, Kim YH, et al. Testicular endothelial cells are a critical population in the germline stem cell niche. Nature Communications. 2018; 9: 1–16.

[82] Davidoff M, Middendorff R, Koeva Y, Pusch W, Jezek D, Müller D. Glial cell line-derived neurotrophic factor (GDNF) and its receptors GFRalpha-1 and GFRalpha-2 in the human testis. Italian Journal of Anatomy and Embryology. 2001; 106: 173–180.

[83] 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. International Journal of Molecular Sciences. 2018; 19: 286.

[84] Hayashi K, Ohta H, Kurimoto K, Aramaki S, Saitou M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell. 2011; 146: 519–532.

[85] Eslahi N, Hadjighassem MR, Joghataei MT, Mirzapour T, Bakhtiyari M, Shakeri M, et al. The effects of poly L-lactic acid nanofiber scaffold on mouse spermatogonial stem cell culture. International Journal of Nanomedicine. 2013; 8: 4563.

[86] Fennema E, Rivron N, Rouwkema J, van Blitterswijk C, de Boer J. Spheroid culture as a tool for creating 3D complex tissues. Trends in Biotechnology. 2013; 31: 108–115.

[87] Komeya M, Sato T, Ogawa T. In vitro spermatogenesis: a century—long research journey, still half way around. Reproductive Medicine and Biology. 2018; 17: 407–420.

[88] Ballios BG, Cooke MJ, van der Kooy D, Shoichet MS. A hydrogel-based stem cell delivery system to treat retinal degenerative diseases. Biomaterials. 2010; 31: 2555–2564.

[89] Robinson M, Sparanese S, Witherspoon L, Flannigan R. Human in vitro spermatogenesis as a regenerative therapy—where do we stand? Nature Reviews Urology. 2023; 20: 461–479.

[90] Hamburger AW, Salmon SE. Primary bioassay of human tumor stem cells. Science. 1977; 197: 461–463.

[91] Janson IA, Putnam AJ. Extracellular matrix elasticity and topography: material—based cues that affect cell function via conserved mechanisms. Journal of Biomedical Materials Research Part A. 2015; 103: 1246–1258.

[92] Al-Lamki RS, Bradley JR, Pober JS. Human organ culture: updating the approach to bridge the gap from in vitro to in vivo in inflammation, cancer, and stem cell biology. Frontiers in Medicine. 2017; 4: 148.

[93] Bhatia S, Naved T, Sardana S. Organ culture. In Bhatia S (ed.) Introduction to pharmaceutical biotechnology (pp. 1–28). 1st edn. IOP Publishing: UK. 2019.

[94] Huh D, Hamilton GA, Ingber DE. From 3D cell culture to organs-on-chips. Trends in Cell Biology. 2011; 21: 745–754.

[95] Pfannkuche JJ, Guo W, Cui S, Ma J, Lang G, Peroglio M, et al. Intervertebral disc organ culture for the investigation of disc pathology and regeneration—benefits, limitations, and future directions of bioreactors. Connective Tissue Research. 2020; 61: 304–321.

[96] Ganjibakhsh M, Mehraein F, Koruji M, Aflatoonian R, Farzaneh P. Three-dimensional decellularized amnion membrane scaffold as a novel tool for cancer research; cell behavior, drug resistance and cancer stem cell content. Materials Science and Engineering: C. 2019; 100: 330–340.

[97] Astashkina A, Mann B, Grainger DW. A critical evaluation of in vitro cell culture models for high-throughput drug screening and toxicity. Pharmacology & Therapeutics. 2012; 134: 82–106.

[98] Gupta N, Liu JR, Patel B, Solomon DE, Vaidya B, Gupta V. Microfluidics—based 3D cell culture models: utility in novel drug dis-covery and delivery research. Bioengineering & Translational Medicine. 2016; 1: 63–81.

[99] Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, et al. Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Archives of Toxicology. 2013; 87: 1315–1530.

[100] Kim S, Kim HJ, Jeon NL. Biological applications of microfluidic gradient devices. Integrative Biology. 2010; 2: 584–603.

[101] Gerecht-Nir S, Cohen S, Itskovitz‐Eldor J. Bioreactor cultivation enhances the efficiency of human embryoid body (hEB) formation and differentiation. Biotechnology and Bioengineering. 2004; 86: 493–502.

[102] Pazos P, Fortaner S, Prieto P. Long-term in vitro toxicity models: comparisons between a flow-cell bioreactor, a static-cell bioreactor and static cell cultures. Alternatives to Laboratory Animals. 2002; 30: 515–523.

[103] Haycock JW. 3D cell culture: a review of current approaches and techniques. Methods in Molecular Biology. 2011; 695: 1–15.

[104] Sanyal S. Culture and assay systems used for 3D cell culture. Corning. 2014; 9: 1–18.

[105] Mukherjee N, Saris DB, Schultz FM, Berglund LJ, An KN, O’Driscoll SW. The enhancement of periosteal chondrogenesis in organ culture by dynamic fluid pressure. Journal of Orthopaedic Research. 2001; 19: 524–530.

[106] Sharma S, Venzac B, Burgers T, Le Gac S, Schlatt S. Microfluidics in male reproduction: is ex vivo culture of primate testis tissue a future strategy for ART or toxicology research? Molecular Human Reproduction. 2020; 26: 179–192.

[107] Seidi S, Eftekhari A, Khusro A, Heris RS, Sahibzada MUK, Gajdács M. Simulation and modeling of physiological processes of vital organs in organ-on-a-chip biosystem. Journal of King Saud University-Science. 2022; 34: 101710.

[108] Duval K, Grover H, Han LH, Mou Y, Pegoraro AF, Fredberg J, et al. Modeling physiological events in 2D vs. 3D cell culture. Physiology. 2017; 32: 266–277.

[109] Young EW, Beebe DJ. Fundamentals of microfluidic cell culture in controlled microenvironments. Chemical Society Reviews. 2010; 39: 1036–1048.

[110] van den Berg A, Mummery CL, Passier R, van der Meer AD. Personalised organs-on-chips: functional testing for precision medicine. Lab on a Chip. 2019; 19: 198–205.

[111] Mehling M, Tay S. Microfluidic cell culture. Current Opinion in Biotechnology. 2014; 25: 95–102.

[112] Xing Z, Xue Y, Finne-Wistrand A, Yang ZQ, Mustafa K. Copolymer cell/scaffold constructs for bone tissue engineering: co-culture of low ratios of human endothelial and osteoblast-like cells in a dynamic culture system. Journal of Biomedical Materials Research Part A. 2013; 101: 1113–1120.

[113] Yuan H, Xing K, Hsu HY. Trinity of three-dimensional (3D) scaffold, vibration, and 3D printing on cell culture application: a systematic review and indicating future direction. Bioengineering. 2018; 5: 57.

[114] Huang X, Huang Z, Gao W, Gao W, He R, Li Y, et al. Current advances in 3D dynamic cell culture systems. Gels. 2022; 8: 829.

[115] Urciuolo F, Imparato G, Netti P. In vitro strategies for mimicking dynamic cell—ECM reciprocity in 3D culture models. Frontiers in Bioengineering and Biotechnology. 2023; 11: 1197075.

[116] Baker BM, Chen CS. Deconstructing the third dimension—how 3D culture microenvironments alter cellular cues. Journal of Cell Science. 2012; 125: 3015–3024.

[117] Bareither R, Pollard D. A review of advanced small—scale parallel bioreactor technology for accelerated process development: current state and future need. Biotechnology Progress. 2011; 27: 2–14.

[118] Zhao L, Fu HY, Zhou W, Hu WS. Advances in process monitoring tools for cell culture bioprocesses. Engineering in Life Sciences. 2015; 15: 459–468.

[119] Huang M, Fan S, Xing W, Liu C. Microfluidic cell culture system studies and computational fluid dynamics. Mathematical and Computer Modelling. 2010; 52: 2036–2042.

[120] Diaz P, Dullea A, Chu KY, Zizzo J, Loloi J, Reddy R, et al. Future of male infertility evaluation and treatment: brief review of emerging technology. Urology. 2022; 169: 9–16.

[121] Patrício D, Santiago J, Mano JF, Fardilha M. Organoids of the male reproductive system: challenges, opportunities, and their potential use in fertility research. WIREs Mechanisms of Disease. 2023; 15: e1590.

[122] Arkoun B, Dumont L, Milazzo JP, Way A, Bironneau A, Wils J, et al. Retinol improves in vitro differentiation of pre-pubertal mouse spermatogonial stem cells into sperm during the first wave of spermatogenesis. PLOS ONE. 2015; 10: e0116660.

[123] Dumont L, Oblette A, Rondanino C, Jumeau F, Bironneau A, Liot D, et al. Vitamin A prevents round spermatid nuclear damage and promotes the production of motile sperm during in vitro maturation of vitrified pre-pubertal mouse testicular tissue. Molecular Human Reproduction. 2016; 22: 819–832.

[124] Isoler-Alcaraz J, Fernández-Pérez D, Larriba E, Del Mazo J. Cellular and molecular characterization of gametogenic progression in ex vivo cultured prepuberal mouse testes. Reproductive Biology and Endocrinology. 2017; 15: 1–16.

[125] Nakamura N, Merry GE, Inselman AL, Sloper DT, Del Valle PL, Sato T, et al. Evaluation of culture time and media in an in vitro testis organ culture system. Birth Defects Research. 2017; 109: 465–474.

[126] Ogawa T. In vitro spermatogenesis: the dawn of a new era in the study of male infertility. International Journal of Urology. 2012; 19: 282–283.

[127] Halldorsson S, Lucumi E, Gómez-Sjöberg R, Fleming RM. Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices. Biosensors and Bioelectronics. 2015; 63: 218–231.

[128] Alias AB, Huang HY, Yao DJ. A review on microfluidics: an aid to assisted reproductive technology. Molecules. 2021; 26: 4354.

[129] Ozcan P, Takmaz T, Yazici MGK, Alagoz OA, Yesiladali M, Sevket O, et al. Does the use of microfluidic sperm sorting for the sperm selection improve in vitro fertilization success rates in male factor infertility?Journal of Obstetrics and Gynaecology Research. 2021; 47: 382–388.

[130] Ramsoomair CK, Alver CG, Flannigan R, Ramasamy R, Agarwal A. Spermatogonial stem cells and in vitro spermatogenesis: how far are we from a human testis on a chip? European Urology Focus. 2023; 9: 46–48.

[131] Nieto D, Couceiro R, Aymerich M, Lopez-Lopez R, Abal M, Flores-Arias MT. A laser-based technology for fabricating a soda-lime glass based microfluidic device for circulating tumour cell capture. Colloids and Surfaces B: Biointerfaces. 2015; 134: 363–369.

[132] Becker H, Gaertner C. Microreplication technologies for polymer-based µ-TAS applications. Lab on a Chip. 2003; 3: 21–35.

[133] Funano SI, Ota N, Tanaka Y. A simple and reversible glass—glass bonding method to construct a microfluidic device and its application for cell recovery. Lab on a Chip. 2021; 21: 2244–2254.

[134] Leester-Schädel M, Lorenz T, Jürgens F, Richter C. Fabrication of microfluidic devices. In Jensen KF (ed.) Microsystems for pharmatech-nology (pp. 23–57). 1st edn. Royal Society of Chemistry: London. 2016.

[135] Bahadorimehr A, Majlis BY. Fabrication of glass-based microfluidic devices with photoresist as mask. Electronics and Electrical Engineering. 2011; 116: 45–48.

[136] Adams AA, Okagbare PI, Feng J, Hupert ML, Patterson D, Göttert J, et al. Highly efficient circulating tumor cell isolation from whole blood and label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor. Journal of the American Chemical Society. 2008; 130: 8633–8641.

[137] Becker H, Dietz W. Microfluidic devices for µ-TAS applications fabricated by polymer hot embossing. In Frazier AB, Ahn CH (eds.) Microfluidic devices and systems. SPIE 3515 (pp. 49–56). SPIE: Bellingham, WA, USA. 1998.

[138] Zhou J, Khodakov DA, Ellis AV, Voelcker NH. Surface modification for PDMS—based microfluidic devices. Electrophoresis. 2012; 33: 89–104.

[139] Ogończyk D, Węgrzyn J, Jankowski P, Dąbrowski B, Garstecki P. Bonding of microfluidic devices fabricated in polycarbonate. Lab on a Chip. 2010; 10: 1324–1327.

[140] Chang CW, Cheng YJ, Tu M, Chen YH, Peng CC, Liao WH, et al. A polydimethylsiloxane—polycarbonate hybrid microfluidic device capable of generating perpendicular chemical and oxygen gradients for cell culture studies. Lab on a Chip. 2014; 14: 3762–3772.

[141] Becker H, Locascio LE. Polymer microfluidic devices. Talanta. 2002; 56: 267–287.

[142] Derzsi L, Jankowski P, Lisowski W, Garstecki P. Hydrophilic polycar-bonate for generation of oil in water emulsions in microfluidic devices. Lab on a Chip. 2011; 11: 1151–1156.

[143] Cui F, Jafarishad H, Zhou Z, Chen J, Shao J, Wen Q, et al. Batch fabrication of electrochemical sensors on a glycol-modified polyethylene terephthalate-based microfluidic device. Biosensors and Bioelectronics. 2020; 167: 112521.

[144] Cho H, Kim J, Jeon CW, Han KH. A disposable microfluidic device with a reusable magnetophoretic functional substrate for isolation of circulating tumor cells. Lab on a Chip. 2017; 17: 4113–4123.

[145] Liu AL, He FY, Hu YL, Xia XH. Plastified poly (ethylene terephthalate)(PET)-toner microfluidic chip by direct-printing integrated with electro-chemical detection for pharmaceutical analysis. Talanta. 2006; 68: 1303–1308.

[146] Wang T, Wang J, Wang S, Wang X, Yang W, Li M, et al. Construction of hydrophobic-hydrophilic microfluidic patterns with controlled wettabil-ity on PVDF filter paper surface using maskless microplasma scanning. Surfaces and Interfaces. 2023; 37: 102695.

[147] Meymivand A, Shahhosseini S, Kashani MN, HMTShirazi R, Yamini Y. Exploring the impact of polyvinylidene fluoride membrane physical properties on the enrichment efficacy of microfluidic electro-membrane extraction of acidic drugs. Journal of Chromatography A. 2024; 1725: 464909.

[148] Harris N, Hill M, Beeby S, Shen Y, White N, Hawkes J, et al. A silicon microfluidic ultrasonic separator. Sensors and Actuators B: Chemical. 2003; 95: 425–434.

[149] Ren K, Zhou J, Wu H. Materials for microfluidic chip fabrication. Accounts of Chemical Research. 2013; 46: 2396–2406.

[150] Luque A, Quero JM, Hibert C, Flückiger P, Gañán-Calvo AM. Integrable silicon microfluidic valve with pneumatic actuation. Sensors and Actuators A: Physical. 2005; 118: 144–1451.

[151] Skottvoll FS, Escobedo-Cousin E, Mielnik MM. The role of silicon technology in organ‐on‐chip: current status and future perspective. Advanced Materials Technologies. 2024; 10: 2401254.

[152] Becker H, Heim U. Hot embossing as a method for the fabrication of polymer high aspect ratio structures. Sensors and Actuators A: Physical. 2000; 83: 130–135.

[153] Chang WY, Chu CH, Lin YC. A flexible piezoelectric sensor for microfluidic applications using polyvinylidene fluoride. IEEE Sensors Journal. 2008; 8: 495–500.

[154] Pullano SA, Mahbub I, Islam SK, Fiorillo AS. PVDF sensor stimulated by infrared radiation for temperature monitoring in microfluidic devices. Sensors. 2017; 17: 850.

[155] Huh D, Matthews BD, Mammoto A, Montoya-Zavala M, Hsin HY, Ingber DE. Reconstituting organ-level lung functions on a chip. Science. 2010; 328: 1662–1668.

[156] Bhatia SN, Ingber DE. Microfluidic organs-on-chips. Nature Biotech-nology. 2014; 32: 760–772.

[157] Yamanaka H, Komeya M, Nakamura H, Sanjo H, Sato T, Yao M, et al. A monolayer microfluidic device supporting mouse spermatogenesis with improved visibility. Biochemical and Biophysical Research Communica-tions. 2018; 500: 885–891.

[158] Naeemi S, Kajbafzadeh A, Eidi A, Khanbabaee R, Sadri-Ardekani H. Microfluidics and improving the cell culture channel: evaluation of spermatogonial stem cells using microfluidic chips. Journal of Comparative Pathobiology. 2021; 18: 3555–3564.

[159] Harink B, Le Gac S, Truckenmüller R, van Blitterswijk C, Habibovic P. Regeneration-on-a-chip? The perspectives on use of microfluidics in regenerative medicine. Lab on a Chip. 2013; 13: 3512–3528.

[160] Kilcoyne KR, Mitchell RT. Effect of environmental and pharmaceutical exposures on fetal testis development and function: a systematic review of human experimental data. Human Reproduction Update. 2019; 25: 397–421.

[161] Le Gac S, Ferraz M, Venzac B, Comizzoli P. Understanding and assisting reproduction in wildlife species using microfluidics. Trends in Biotechnology. 2021; 39: 584–597.

[162] Thapa S, Heo YS. Microfluidic technology for in vitro fertilization (IVF). JMST Advances. 2019; 1: 1–11.

[163] Sharma S, Venzac B, Burgers T, Schlatt S, Le Gac S. Testis-on-chip platform to study ex vivo primate spermatogenesis and endocrine dynamics. Organs-on-a-Chip. 2022; 4: 100023.

[164] Kanbar M, De Michele F, Poels J, Van Loo S, Giudice MG, Gilet T, et al. Microfluidic and static organotypic culture systems to support ex vivo spermatogenesis from prepubertal porcine testicular tissue: a comparative study. Frontiers in Physiology. 2022; 13: 884122.

[165] Pittman RN. Regulation of Tissue Oxygenation. 1st edn. Morgan & Claypool Life Sciences: San Rafael. 2011.

[166] Goldman SM, Barabino GA. Spatial engineering of osteochondral tissue constructs through microfluidically directed differentiation of mesenchymal stem cells. BioResearch Open Access. 2016; 5: 109–117.

[167] Lewis ML, Moriarity DM, Campbell PS. Use of microgravity bioreactors for development of an in vitro rat salivary gland cell culture model. Journal of Cellular Biochemistry. 1993; 51: 265–273.

[168] Helsel AR, Oatley MJ, Oatley JM. Glycolysis-optimized conditions enhance maintenance of regenerative integrity in mouse spermatogonial stem cells during long-term culture. Stem Cell Reports. 2017; 8: 1430–1441.

[169] Dores C, Rancourt D, Dobrinski I. Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis. Andrology. 2015; 3: 590–597.

[170] Sen A, Kallos MS, Behie LA. Expansion of mammalian neural stem cells in bioreactors: effect of power input and medium viscosity. Developmental Brain Research. 2002; 134: 103–113.

[171] Zhang X, Li L, Bai Y, Shi R, Wei H, Zhang S. Mouse undifferentiated spermatogonial stem cells cultured as aggregates under simulated microgravity. Andrologia. 2014; 46: 1013–1021.

[172] Perrard MH, Sereni N, Schluth-Bolard C, Blondet A, SG DE, Plotton I, et al. Complete human and rat ex vivo spermatogenesis from fresh or frozen testicular tissue. Biology of Reproduction. 2016; 95: 89.

[173] Basaria S. Male hypogonadism. The Lancet. 2014; 383: 1250–1263.

[174] Forbes CM, Flannigan R, Schlegel PN. Spermatogonial stem cell transplantation and male infertility: current status and future directions. Arab Journal of Urology. 2018; 16: 171–180.

[175] Green DM, Kawashima T, Stovall M, Leisenring W, Sklar CA, Mertens AC, et al. Fertility of male survivors of childhood cancer: a report from the childhood cancer survivor study. Journal of Clinical Oncology. 2010; 28: 332–339.

[176] Mulder RL, Font-Gonzalez A, Green DM, Loeffen EAH, Hudson MM, Loonen J, et al. Fertility preservation for male patients with childhood, adolescent, and young adult cancer: recommendations from the PanCareLIFE consortium and the international late effects of childhood cancer guideline harmonization group. The Lancet Oncology. 2021; 22: e57–e67.

[177] Phillips SM, Padgett LS, Leisenring WM, Stratton KK, Bishop K, Krull KR, et al. Survivors of childhood cancer in the United States: prevalence and burden of morbidity. Cancer Epidemiology, Biomarkers & Prevention. 2015; 24: 653–663.

[178] Salem M, Khadivi F, Javanbakht P, Mojaverrostami S, Abbasi M, Feizollahi N, et al. Advances of three-dimensional (3D) culture systems for in vitro spermatogenesis. Stem Cell Research & Therapy. 2023; 14: 262.

[179] Shen J, Wang X, Yang C, Ren G, Wang L, Piao S, et al. Development and evaluation of a microfluidic human testicular tissue chip: a novel in vitro platform for reproductive biology and pharmacology studies. Lab on a Chip. 2025; 25: 577–589.

[180] Kashaninejad N, Shiddiky MJA, Nguyen NT. Advances in microfluidics-based assisted reproductive technology: from sperm sorter to reproductive system‐on‐a‐chip. Advanced Biosystems. 2018; 2: 1700197.

[181] Horvath-Pereira BdO, Almeida GHDR, Silva Júnior LNd, do Nasci-mento PG, Horvath Pereira BdO, Fireman JVBT, et al. Biomaterials for testicular bioengineering: how far have we come and where do we have to go? Frontiers in Endocrinology. 2023; 14: 1085872.

[182] Zeller P, Legendre A, Jacques S, Fleury M, Gilard F, Tcherkez G, et al. Hepatocytes cocultured with Sertoli cells in bioreactor favors Sertoli barrier tightness in rat. Journal of Applied Toxicology. 2017; 37: 287–295.

[183] Sabetkish S, Kajbafzadeh AM, Sabetkish N. Recellularization of testicular feminization testis in C57bl6 as a natural bioreactor for creation of cellularized seminiferous tubules: an experimental study. Cell and Tissue Banking. 2021; 22: 287–295.

[184] Bhaskar R, Gupta MK, Han SS. Tissue engineering approaches for the in vitro production of spermatids to treat male infertility: a review. European Polymer Journal. 2022; 174: 111318.

[185] Forouzandegan M, Sadeghmousavi S, Heidari A, Khaboushan AS, Kajbafzadeh AM, Zolbin MM. Harnessing the potential of tissue engineering to target male infertility: insights into testicular regeneration. Tissue and Cell. 2025; 93: 102658.

[186] Baert Y, Ruetschle I, Cools W, Oehme A, Lorenz A, Marx U, et al. A multi-organ-chip co-culture of liver and testis equivalents: a first step toward a systemic male reprotoxicity model. Human Reproduction. 2020; 35: 1029–1044.

[187] Wu T, Wu Y, Yan J, Zhang J, Wang S. Microfluidic chip as a promising evaluation method in assisted reproduction: a systematic review. Bioengineering & Translational Medicine. 2024; 9: e10625.

[188] Ghanbari E, Khazaei M, Ghahremani-Nasab M, Mehdizadeh A, Yousefi M. Novel therapeutic approaches of tissue engineering in male infertility. Cell and Tissue Research. 2020; 380: 31–42.