Microfluidic and Static Organotypic Culture Systems to Support Ex Vivo Spermatogenesis From Prepubertal Porcine Testicular Tissue: A Comparative Study.
Kanbar, Marc; de Michele, Francesca; Poels, Jonathanet al.
[en] Background: In vitro maturation of immature testicular tissue (ITT) cryopreserved for fertility preservation is a promising fertility restoration strategy. Organotypic tissue culture proved successful in mice, leading to live births. In larger mammals, including humans, efficiently reproducing spermatogenesis ex vivo remains challenging. With advances in biomaterials technology, culture systems are becoming more complex to better mimic in vivo conditions. Along with improving culture media components, optimizing physical culture conditions (e.g., tissue perfusion, oxygen diffusion) also needs to be considered. Recent studies in mice showed that by using silicone-based hybrid culture systems, the efficiency of spermatogenesis can be improved. Such systems have not been reported for ITT of large mammals. Methods: Four different organotypic tissue culture systems were compared: static i.e., polytetrafluoroethylene membrane inserts (OT), agarose gel (AG) and agarose gel with polydimethylsiloxane chamber (AGPC), and dynamic i.e., microfluidic (MF). OT served as control. Porcine ITT fragments were cultured over a 30-day period using a single culture medium. Analyses were performed at days (d) 0, 5, 10, 20 and 30. Seminiferous tubule (ST) integrity, diameters, and tissue core integrity were evaluated on histology. Immunohistochemistry was used to identify germ cells (PGP9.5, VASA, SYCP3, CREM), somatic cells (SOX9, INSL3) and proliferating cells (Ki67), and to assess oxidative stress (MDA) and apoptosis (C-Caspase3). Testosterone was measured in supernatants using ELISA. Results: ITT fragments survived and grew in all systems. ST diameters, and Sertoli cell (SOX9) numbers increased, meiotic (SYCP3) and post-meiotic (CREM) germ cells were generated, and testosterone was secreted. When compared to control (OT), significantly larger STs (d10 through d30), better tissue core integrity (d5 through d20), higher numbers of undifferentiated spermatogonia (d30), meiotic and post-meiotic germ cells (SYCP3: d20 and 30, CREM: d20) were observed in the AGPC system. Apoptosis, lipid peroxidation (MDA), ST integrity, proliferating germ cell (Ki67/VASA) numbers, Leydig cell (INSL3) numbers and testosterone levels were not significantly different between systems. Conclusions: Using a modified culture system (AGPC), germ cell survival and the efficiency of porcine germ cell differentiation were moderately improved ex vivo. We assume that further optimization can be obtained with concomitant modifications in culture media components.
Disciplines :
Engineering, computing & technology: Multidisciplinary, general & others Reproductive medicine (gynecology, andrology, obstetrics) Biotechnology Life sciences: Multidisciplinary, general & others
Author, co-author :
Kanbar, Marc; Andrology Lab, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, Belgium ; Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Brussels, Belgium
de Michele, Francesca; Andrology Lab, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, Belgium ; Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Brussels, Belgium
Poels, Jonathan; Andrology Lab, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, Belgium ; Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Brussels, Belgium
Van Loo, Stéphanie ; Université de Liège - ULiège > Département d'aérospatiale et mécanique > Microfluidique
Giudice, Maria Grazia; Andrology Lab, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, Belgium ; Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Brussels, Belgium
Gilet, Tristan ; Université de Liège - ULiège > Département d'aérospatiale et mécanique > Microfluidique
Wyns, Christine; Andrology Lab, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, Belgium ; Department of Gynecology-Andrology, Cliniques Universitaires Saint-Luc, Brussels, Belgium
Language :
English
Title :
Microfluidic and Static Organotypic Culture Systems to Support Ex Vivo Spermatogenesis From Prepubertal Porcine Testicular Tissue: A Comparative Study.
This work was supported by the Fonds National de la Recherche Scientifique de Belgique (Grants Télevie Nos. 7.4535.19F and 7.6521.21) and the Fondation Salus Sanguinis.
Abe T. Nishimura H. Sato T. Suzuki H. Ogawa T. Suzuki T. (2020). Transcriptome Analysis Reveals Inadequate Spermatogenesis and Immediate Radical Immune Reactions during Organ Culture In Vitro Spermatogenesis. Biochem. Biophysical Res. Commun. 530 (4), 732–738. 10.1016/j.bbrc.2020.06.161
Abofoul-Azab M. AbuMadighem A. Lunenfeld E. Kapelushnik J. Shi Q. Pinkas H. et al. (2018). Development of Postmeiotic Cells In Vitro from Spermatogonial Cells of Prepubertal Cancer Patients. Stem Cells Dev. 27 (15), 1007–1020. 10.1089/scd.2017.0301
Almeida F. F. L. Leal M. C. França L. R. (2006). Testis Morphometry, Duration of Spermatogenesis, and Spermatogenic Efficiency in the Wild Boar (Sus scrofa Scrofa)1. Biol. Reprod. 75 (5), 792–799. 10.1095/biolreprod.106.053835
Baert Y. Ruetschle I. Cools W. Oehme A. Lorenz A. Marx U. et al. (2020). A Multi-Organ-Chip Co-culture of Liver and Testis Equivalents: a First Step toward a Systemic Male Reprotoxicity Model. Hum. Reprod. 35 (5), 1029–1044. 10.1093/humrep/deaa057
Bankhead P. Loughrey M. B. Fernández J. A. Dombrowski Y. McArt D. G. Dunne P. D. et al. (2017). QuPath: Open Source Software for Digital Pathology Image Analysis. Sci. Rep. 7 (1), 16878. 10.1038/s41598-017-17204-5
Bigaeva E. Gore E. Simon E. Zwick M. Oldenburger A. de Jong K. P. et al. (2019). Transcriptomic Characterization of Culture-Associated Changes in Murine and Human Precision-Cut Tissue Slices. Arch. Toxicol. 93 (12), 3549–3583. 10.1007/s00204-019-02611-6
Bonneau M. Carrié-Lemoine J. Prunier A. Garnier D. H. Terqui M. (1987). Age-related Changes in Plasma LH and Testosterone Concentration Profiles and Fat 5α-Androstenone Content in the Young Boar. Animal Reproduction Sci. 15 (3), 241–258. 10.1016/0378-4320(87)90046-7
Cambria E. Brunner S. Heusser S. Fisch P. Hitzl W. Ferguson S. J. et al. (2020). Cell-Laden Agarose-Collagen Composite Hydrogels for Mechanotransduction Studies. Front. Bioeng. Biotechnol. 8, 346. 10.3389/fbioe.2020.00346
Chuah Y. J. Koh Y. T. Lim K. Menon N. V. Wu Y. Kang Y. (2015). Simple Surface Engineering of Polydimethylsiloxane with Polydopamine for Stabilized Mesenchymal Stem Cell Adhesion and Multipotency. Sci. Rep. 5, 18162. 10.1038/srep18162
Colenbrander B. de Jong F. H. Wensing C. J. G. (1978). Changes in Serum Testosterone Concentrations in the Male Pig during Development. Reproduction 53 (2), 377–380. 10.1530/jrf.0.0530377
de Michele F. Poels J. Vermeulen M. Ambroise J. Gruson D. Guiot Y. et al. (2018). Haploid Germ Cells Generated in Organotypic Culture of Testicular Tissue from Prepubertal Boys. Front. Physiol. 9 (1413), 1413. 10.3389/fphys.2018.01413
de Michele F. Poels J. Weerens L. Petit C. Evrard Z. Ambroise J. et al. (2017). Preserved Seminiferous Tubule Integrity with Spermatogonial Survival and Induction of Sertoli and Leydig Cell Maturation after Long-Term Organotypic Culture of Prepubertal Human Testicular Tissue. Hum. Reprod. 32 (1), 32–45. 10.1093/humrep/dew300
Del Vento F. Vermeulen M. Ucakar B. Poels J. des Rieux A. Wyns C. (2019). Significant Benefits of Nanoparticles Containing a Necrosis Inhibitor on Mice Testicular Tissue Autografts Outcomes. Ijms 20 (23), 5833. 10.3390/ijms20235833
Dores C. Rancourt D. Dobrinski I. (2015). Stirred Suspension Bioreactors as a Novel Method to Enrich Germ Cells from Pre-pubertal Pig Testis. Andrology 3 (3), 590–597. 10.1111/andr.12031
Fayomi A. P. Orwig K. E. (2018). Spermatogonial Stem Cells and Spermatogenesis in Mice, Monkeys and Men. Stem Cell Res. 29, 207–214. 10.1016/j.scr.2018.04.009
Fayomi A. P. Peters K. Sukhwani M. Valli-Pulaski H. Shetty G. Meistrich M. L. et al. (2019). Autologous Grafting of Cryopreserved Prepubertal Rhesus Testis Produces Sperm and Offspring. Science 363 (6433), 1314–1319. 10.1126/science.aav2914
França L. R. Silva V. A. Jr.Chiarini-Garcia H. Garcia S. K. Debeljuk L. (2000). Cell Proliferation and Hormonal Changes during Postnatal Development of the Testis in the Pig. Biol. Reprod. 63 (6), 1629–1636. 10.1095/biolreprod63.6.1629
Goossens E. Jahnukainen K. Mitchell R. van Pelt A. Pennings G. Rives N. et al. (2020). Fertility Preservation in Boys: Recent Developments and New Insights. Hum. Reprod. Open 2020 (3), hoaa016. 10.1093/hropen/hoaa016
Griswold S. L. Behringer R. R. (2009). Fetal Leydig Cell Origin and Development. Sex. Dev. 3 (1), 1–15. 10.1159/000200077
Groenen M. A. Archibald A. L. Uenishi H. Tuggle C. K. Takeuchi Y. Rothschild M. F. et al. (2012). Analyses of Pig Genomes Provide Insight into Porcine Demography and Evolution. Nature 491 (7424), 393–398. 10.1038/nature11622
Guo J. Grow E. J. Mlcochova H. Maher G. J. Lindskog C. Nie X. et al. (2018). The Adult Human Testis Transcriptional Cell Atlas. Cell Res. 28 (12), 1141–1157. 10.1038/s41422-018-0099-2
Heckmann L. Langenstroth-Röwer D. Wistuba J. Portela J. M. D. van Pelt A. M. M. Redmann K. et al. (2020). The Initial Maturation Status of Marmoset Testicular Tissues Has an Impact on Germ Cell Maintenance and Somatic Cell Response in Tissue Fragment Culture. Mol. Hum. Reprod. 26 (6), 374–388. 10.1093/molehr/gaaa024
Hermann B. P. Sukhwani M. Winkler F. Pascarella J. N. Peters K. A. Sheng Y. et al. (2012). Spermatogonial Stem Cell Transplantation into Rhesus Testes Regenerates Spermatogenesis Producing Functional Sperm. Cell Stem Cell 11 (5), 715–726. 10.1016/j.stem.2012.07.017
Ivell R. Wade J. D. Anand-Ivell R. (2013). INSL3 as a Biomarker of Leydig Cell Functionality. Biol. Reproduction 88 (6), 147. 10.1095/biolreprod.113.108969
Jensen K. Krusenstjerna-Hafstrøm R. Lohse J. Petersen K. H. Derand H. (2017). A Novel Quantitative Immunohistochemistry Method for Precise Protein Measurements Directly in Formalin-Fixed, Paraffin-Embedded Specimens: Analytical Performance Measuring HER2. Mod. Pathol. 30 (2), 180–193. 10.1038/modpathol.2016.176
Kanbar M. de Michele F. Giudice M. G. Desmet L. Poels J. Wyns C. (2021). Long-term Follow-Up of Boys Who Have Undergone a Testicular Biopsy for Fertility Preservation. Hum. Reprod. 36 (1), 26–39. 10.1093/humrep/deaa281
Khong Y. M. Zhang J. Zhou S. Cheung C. Doberstein K. Samper V. et al. (2007). Novel Intra-tissue Perfusion System for Culturing Thick Liver Tissue. Tissue Eng. 13 (9), 2345–2356. 10.1089/ten.2007.0040
Kojima K. Nakamura H. Komeya M. Yamanaka H. Makino Y. Okada Y. et al. (2018). Neonatal Testis Growth Recreated In Vitro by Two‐dimensional Organ Spreading. Biotechnol. Bioeng. 115 (12), 3030–3041. 10.1002/bit.26822
Komeya M. Kimura H. Nakamura H. Yokonishi T. Sato T. Kojima K. et al. (2016). Long-term Ex Vivo Maintenance of Testis Tissues Producing Fertile Sperm in a Microfluidic Device. Sci. Rep. 6, 21472. 10.1038/srep21472
Komeya M. Yamanaka H. Sanjo H. Yao M. Nakamura H. Kimura H. et al. (2019). In Vitro spermatogenesis in Two‐dimensionally Spread Mouse Testis Tissues. Reprod. Med. Biol. 18 (4), 362–369. 10.1002/rmb2.12291
Koskenniemi J. J. Virtanen H. E. Toppari J. (2017). Testicular Growth and Development in Puberty. Curr. Opin. Endocrinol. Diabetes Obes. 24 (3), 215–224. 10.1097/MED.0000000000000339
Kurek M. Åkesson E. Yoshihara M. Oliver E. Cui Y. Becker M. et al. (2021). Spermatogonia Loss Correlates with LAMA 1 Expression in Human Prepubertal Testes Stored for Fertility Preservation. Cells 10 (2), 241. 10.3390/cells10020241
Liang D. Bhatta S. Gerzanich V. Simard J. M. (2007). Cytotoxic Edema: Mechanisms of Pathological Cell Swelling. Foc 22 (5), 1–9. 10.3171/foc.2007.22.5.3
Lovett M. Lee K. Edwards A. Kaplan D. L. (2009). Vascularization Strategies for Tissue Engineering. Tissue Eng. Part B Rev. 15 (3), 353–370. 10.1089/ten.TEB.2009.0085
Matsumura T. Sato T. Abe T. Sanjo H. Katagiri K. Kimura H. et al. (2021). Rat In Vitro Spermatogenesis Promoted by Chemical Supplementations and Oxygen-Tension Control. Sci. Rep. 11 (1), 3458. 10.1038/s41598-021-82792-2
McDonald J. C. Duffy D. C. Anderson J. R. Chiu D. T. Wu H. Schueller O. J. A. et al. (2000). Fabrication of Microfluidic Systems in Poly(dimethylsiloxane). Electrophoresis 21 (1), 27–40. 10.1002/(SICI)1522-2683
Medrano J. V. Vilanova-Pérez T. Fornés-Ferrer V. Navarro-Gomezlechon A. Martínez-Triguero M. L. García S. et al. (2018). Influence of Temperature, Serum, and Gonadotropin Supplementation in Short- and Long-Term Organotypic Culture of Human Immature Testicular Tissue. Fertil. Steril. 110 (6), 1045–1057. 10.1016/j.fertnstert.2018.07.018
Meurens F. Summerfield A. Nauwynck H. Saif L. Gerdts V. (2012). The Pig: a Model for Human Infectious Diseases. Trends Microbiol. 20 (1), 50–57. 10.1016/j.tim.2011.11.002
Mulder R. L. Font-Gonzalez A. Hudson M. M. van Santen H. M. Loeffen E. A. H. Burns K. C. et al. (2021). Fertility Preservation for Female Patients with Childhood, Adolescent, and Young Adult Cancer: Recommendations from the PanCareLIFE Consortium and the International Late Effects of Childhood Cancer Guideline Harmonization Group. Lancet Oncol. 22 (2), e45–e56. 10.1016/S1470-2045(20)30594-5
Oliver E. Stukenborg J. B. (2020). Rebuilding the Human Testis In Vitro. Andrology 8 (4), 825–834. 10.1111/andr.12710
Perrard M.-H. Sereni N. Schluth-Bolard C. Blondet A. d'Estaing S. G. Plotton I. et al. (2016). Complete Human and Rat Ex Vivo Spermatogenesis from Fresh or Frozen Testicular Tissue. Biol. Reproduction 95 (4), 89. 10.1095/biolreprod.116.142802
Place T. L. Domann F. E. Case A. J. (2017). Limitations of Oxygen Delivery to Cells in Culture: An Underappreciated Problem in Basic and Translational Research. Free Radic. Biol. Med. 113, 311–322. 10.1016/j.freeradbiomed.2017.10.003
Portela J. M. D. de Winter-Korver C. M. van Daalen S. K. M. Meißner A. de Melker A. A. Repping S. et al. (2019a). Assessment of Fresh and Cryopreserved Testicular Tissues from (Pre)pubertal Boys during Organ Culture as a Strategy for In Vitro Spermatogenesis. Hum. Reprod. 34 (12), 2443–2455. 10.1093/humrep/dez180
Portela J. M. D. Mulder C. L. van Daalen S. K. M. de Winter-Korver C. M. Stukenborg J.-B. Repping S. et al. (2019b). Strains Matter: Success of Murine In Vitro Spermatogenesis Is Dependent on Genetic Background. Dev. Biol. 456 (1), 25–30. 10.1016/j.ydbio.2019.08.007
Practice Committee of the American Society for Reproductive Medicine. Electronic address (2019). Fertility Preservation in Patients Undergoing Gonadotoxic Therapy or Gonadectomy: a Committee Opinion. Fertil. Steril. 112 (6), 1022–1033. 10.1016/j.fertnstert.2019.09.013
Price P. Goldsborough M. Tilkins M. (1998). Embryonic Stem Cell Serum Replacement International Patent Application. WO98/30679.
Reda A. Hou M. Winton T. R. Chapin R. E. Söder O. Stukenborg J.-B. (2016). In Vitrodifferentiation of Rat Spermatogonia into Round Spermatids in Tissue Culture. Mol. Hum. Reprod. 22 (9), 601–612. 10.1093/molehr/gaw047
Richer G. Baert Y. Goossens E. (2020). In‐vitro Spermatogenesis through Testis Modelling: Toward the Generation of Testicular Organoids. Andrology 8 (4), 879–891. 10.1111/andr.12741
Sato T. Katagiri K. Gohbara A. Inoue K. Ogonuki N. Ogura A. et al. (2011). In Vitro production of Functional Sperm in Cultured Neonatal Mouse Testes. Nature 471 (7339), 504–507. 10.1038/nature09850
Saulnier J. Oblette A. Delessard M. Dumont L. Rives A. Rives N. et al. (2021). Improving Freezing Protocols and Organotypic Culture: A Histological Study on Rat Prepubertal Testicular Tissue. Ann. Biomed. Eng. 49 (1), 203–218. 10.1007/s10439-020-02535-8
Schneider C. A. Rasband W. S. Eliceiri K. W. (2012). NIH Image to ImageJ: 25 Years of Image Analysis. Nat. Methods 9 (7), 671–675. 10.1038/nmeth.2089
Sharma S. Klaverkamp R.-S. Wistuba J. Schlatt S. (2022). Limited Spermatogenic Differentiation of Testicular Tissue from Prepubertal Marmosets (Callithrix jacchus) in an In Vitro Organ Culture System. Mol. Cell. Endocrinol. 539, 111488. 10.1016/j.mce.2021.111488
Stukenborg J.-B. Schlatt S. Simoni M. Yeung C.-H. Elhija M. A. Luetjens C. M. et al. (2009). New Horizons for In Vitro Spermatogenesis? an Update on Novel Three-Dimensional Culture Systems as Tools for Meiotic and Post-meiotic Differentiation of Testicular Germ Cells. Mol. Hum. Reprod. 15 (9), 521–529. 10.1093/molehr/gap052
Swindle M. Smith A. C. (1998). Comparative Anatomy and Physiology of the Pig. Scand. J. Laboratory Animal Sci. 25, 11–21.
Van Straaten H. W. M. Wensing C. J. G. (1978). Leydig Cell Development in the Testis of the Pig. Biol. Reprod. 18 (1), 86–93. 10.1095/biolreprod18.1.86
Vermeulen M. Del Vento F. Kanbar M. Pyr Dit Ruys S. Vertommen D. Poels J. et al. (2019). Generation of Organized Porcine Testicular Organoids in Solubilized Hydrogels from Decellularized Extracellular Matrix. Ijms 20 (21), 5476. 10.3390/ijms20215476
Vollert I. Seiffert M. Bachmair J. Sander M. Eder A. Conradi L. et al. (2014). In-vitro Perfusion of Engineered Heart Tissue through Endothelialized Channels. Tissue Eng. Part A 20 (3-4), 131025032956001–131025032956863. 10.1089/ten.TEA.2013.0214
Vreugdenhil P. K. Belzer F. O. Southard J. H. (1991). Effect of Cold Storage on Tissue and Cellular Glutathione. Cryobiology 28 (2), 143–149. 10.1016/0011-2240(91)90016-h
Wyns C. Kanbar M. Giudice M. G. Poels J. (2020). Fertility Preservation for Prepubertal Boys: Lessons Learned from the Past and Update on Remaining Challenges towards Clinical Translation. Hum. Reprod. Update 27, 433–459. 10.1093/humupd/dmaa050
Wyns C. Kanbar M. (2022). “In Vitro Spermatogenesis,” in Female and Male Fertility Preservation. Editors Grynberg M. Patrizio P. (Cham: Springer International Publishing), 587–607. 10.1007/978-3-030-47767-7_44
Yamanaka H. Komeya M. Nakamura H. Sanjo H. Sato T. Yao M. et al. (2018). A Monolayer Microfluidic Device Supporting Mouse Spermatogenesis with Improved Visibility. Biochem. Biophysical Res. Commun. 500 (4), 885–891. 10.1016/j.bbrc.2018.04.180
Yokonishi T. Sato T. Komeya M. Katagiri K. Kubota Y. Nakabayashi K. et al. (2014). Offspring Production with Sperm Grown In Vitro from Cryopreserved Testis Tissues. Nat. Commun. 5, 4320. 10.1038/ncomms5320
