Utilization of a decellularized testis scaffold and seminal exosomes for in vitro spermatogenesis from spermatogonial stem cells

Abstract

The present invention provides a method for culturing spermatogonial stem cells (SSCs) The process involves the development of a bioartificial testis by repopulating a decellularized testicular extracellular matrix (ECM) with isolated SSCs and culturing them in the presence of seminal exosomes (SEs). The invention encompasses a method for the complete in vitro reconstitution of spermatogenesis. This is achieved through the co-culture of SSCs on a biomechanically and biochemically optimized decellularized testicular ECM scaffold, further supplemented with seminal exosomes as a key bioactive component.

Description

[0001] Utilization of a Decellularized Testis Scaffold and Seminal Exosomes for In Vitro Spermatogenesis from Spermatogonial Stem Cells Field of the Invention The present invention pertains to the fields of regenerative medicine, urology, and reproductive biology Background of the Invention Male factor infertility is a significant medical condition affecting a substantial proportion of couples worldwide, with non-obstructive azoospermia (NOA) representing one of the most severe forms due to the absence of sperm in the ejaculate (Rambhatla, Shah et al. 2024). For prepubertal boys undergoing gonadotoxic therapies, the option of sperm cryopreservation does not exist, leaving them with no fertility preservation options (Jensen, Dong et al.2022). Existing strategies for fertility restoration in azoospermic men, such as testicular sperm extraction (TESE) followed by intracytoplasmic sperm injection (ICSI), are only successful if viable sperm are present, which is not the case for many (Cirigliano, Falcone et al. 2023). In vitro spermatogenesis from SSCs has been pursued using various 3D culture systems, including soft agarose culture, organoids, and hydrogels (Salem, Khadivi et al. 2023). However, these systems fail to recapitulate the intricate cytoarchitecture and cell-ECM interactions of the testicular niche, leading to arrested meiosis, aberrant DNA methylation, and extremely low yields of elongated spermatids (Fath-Bayati, Naserpour et al. 2023). The use of decellularized testis scaffolds has been explored, but conventional methods using ionic detergents like SDS severely compromise the integrity of the basement membrane of seminiferous tubules, deplete critical ECM-bound growth factors like glial cell line-derived neurotrophic factor (GDNF), and fail to preserve the biomechanical properties essential for SSC homing and differentiation(Hamzeh, Movahedin et al.2025). Furthermore, existing protocols neglect the pivotal role of the seminal microenvironment, which is rich in exosomes that carry regulatory cargo essential for germ cell maturation and protection from oxidative stress (Yang, Guo et al.2017). The present invention provides a comprehensive solution by introducing an optimized, gentle decellularization regimen that maintains the scaffold's ultrastructure and bioactivity. It uniquely integrates a novel SE-based supplementation system that delivers a precise combination of pro-spermatogenic factors, thereby overcoming the biochemical deficiencies of previous artificial niches and enabling the complete and synchronized differentiation of SSCs into functional spermatozoa. Summary of the Invention The invention addresses a profound and growing clinical need: the preservation of fertility for the many prepubertal children, adolescents, and young adults who face cancer diagnoses (Goossens, Jahnukainen et al.2020). As fertility rates decline across both human populations and wildlife species, the development and implementation of effective fertility preservation strategies have become an essential (Salomón Yunga-Ayavaca, Ximena Quinche-Morocho et al. 2023). While survival rates have dramatically improved, life-saving treatments like chemotherapy and radiation often result in permanent infertility (Yang, Strohl et al.2024). For these survivors, the ability to have a biological family is a critical aspect of their long-term quality of life and recovery. However, prepubertal boys present a unique challenge; as they have not yet begun spermatogenesis, they lack mature sperm for cryopreservation, which is the standard option for post-pubertal patients (Hussien, Hassan et al.). Consequently, the only available fertility preservation strategy for these children is the cryopreservation of testicular tissue biopsies containing spermatogonial stem cells (SSCs), with the hope that future technologies will be able to use this tissue to restore their fertility (Orlić, Alsamara et al.2025). Description The invention addresses a profound and growing clinical need: the preservation of fertility for the many prepubertal children, adolescents, and young adults who face cancer diagnoses (Goossens, Jahnukainen et al.2020). As fertility rates decline across both human populations and wildlife species, the development and implementation of effective fertility preservation strategies have become an essential (Salomón Yunga-Ayavaca, Ximena Quinche-Morocho et al. 2023). While survival rates have dramatically improved, life-saving treatments like chemotherapy and radiation often result in permanent infertility (Yang, Strohl et al.2024). For these survivors, the ability to have a biological family is a critical aspect of their long-term quality of life and recovery. However, prepubertal boys present a unique challenge; as they have not yet begun spermatogenesis, they lack mature sperm for cryopreservation, which is the standard option for post-pubertal patients (Hussien, Hassan et al.). Consequently, the only available fertility preservation strategy for these children is the cryopreservation of testicular tissue biopsies containing spermatogonial stem cells (SSCs), with the hope that future technologies will be able to use this tissue to restore their fertility (Orlić, Alsamara et al.2025). This invention provides that crucial future technology. It discloses a novel, integrated system for the complete in vitro reconstitution of spermatogenesis. This is achieved by co-culturing a SSCs on a biomechanically and biochemically optimized decellularized testicular extracellular matrix (ECM) scaffold, which is further supplemented with seminal exosomes (SEs) as a key bioactive component. This platform directly addresses the critical unmet need for restoring fertility in azoospermic patients, particularly prepubertal cancer survivors who have banked testicular tissue (Orlić, Alsamara et al. 2025). Current in vitro spermatogenesis platforms are severely limited by the absence of a physiological niche that provides the necessary structural, paracrine, and metabolic support for the complete and complex, multi-stage process of germ cell differentiation (Cho and Easley 2023). The invention overcomes these fundamental limitations through several key innovations: 1. A proprietary decellularization protocol that meticulously preserves the unique three-dimensional microarchitecture of the native testicular ECM, including its intricate vasculature and seminiferous tubule outlines. This structure is not merely structural; it provides essential biomechanical cues and spatial organization that guide cell fate. 2. The protocol is designed to retain crucial endogenous growth factors (such as GDNF, FGF2, and BMP4) bound to the matrix, creating a innate biochemical environment conducive to spermatogenesis. 3. The scaffold is dynamically recellularized with a SSCs and is primed with a proprietary cocktail of extracellular vesicles (exosomes) extracted from testicular tissue. These vesicles are rich in a multitude of factors (proteins, lipids, miRNAs) essential for driving the entire process of spermatogenesis, including SSC self-renewal, meiotic progression, and the final transformation during spermiogenesis. 4. In certain embodiments, the scaffold is combined with exosome-loaded microparticles or hydrogel carriers to enable controlled and staged release of seminal exosome cargo across SSC self-renewal, meiosis, and spermiogenesis. This integrated platform represents a paradigm shift from mere SSC culture in a dish to the creation of a true, bioengineered testis analog. This system is capable of generating functional haploid gametes (spermatozoa) in vitro, which can then be used in assisted reproductive technologies (ART) like IVF or ICSI to enable biological parenthood. For the first time, it offers a tangible and robust solution to transform banked testicular tissue from prepubertal boys into a source of life and hope for their future families. The present invention provides a method for culturing SSC. The process involves the creation of a bioartificial testis by repopulating a decellularized testicular matrix with isolated SSCs and culturing them in the presence of seminal exosomes. The method begins with the decellularization of testicular tissue. Tissue is perfused via the testicular artery using a sequential protocol: first with 0.1% (v / v) Triton X-100 and 0.05% (w / v) SDS for 48 hours, followed by a solution of 1% (v / v) Tween-20 and 0.1% (v / v) peracetic acid for 12 hours for sterilization and residual detergent removal. A final enzymatic treatment with DNase / RNase (50 U / mL) at 37°C for 6 hours ensures complete nuclear material removal. Validation includes histology (H&E, DAPI), quantification of residual DNA (<50 ng / mg dry tissue), and mass spectrometry confirming >85% retention of key ECM proteins (laminin-α5, collagen IV, fibronectin) and growth factors (GDNF, BMP4). Cryopreserved Spermatogonial Stem Cells (SSCs) from stem cell banks are used. Seminal Exosomes (SEs) are isolated from semen samples and subjected to differential ultracentrifugation at 100,000 × g for 70 minutes at 4°C to pellet the exosomes. The exosome pellet is washed in a large volume of sterile, ice-cold phosphate-buffered saline (PBS) and ultracentrifuged again under identical conditions to ensure purity. The purified SEs are resuspended in PBS and characterized by nanoparticle tracking analysis (NTA) to confirm a size distribution of 50–150 nm, transmission electron microscopy (TEM) for morphological assessment (cup-shaped vesicles), and western blot analysis for positive markers (CD63, CD9, TSG101) and the absence of the negative marker calnexin. Recellularization is achieved by co-injecting the purified SSCs (1x10⁵ cells / cm³ scaffold) and SEs (10⁹ particles / mL) into the decellularized scaffold supplemented with seminal EVs. The construct is cultured in testis-specific conditions (34°C, 5% CO₂). In certain embodiments, a perfusion bioreactor with independent control of seminiferous tubule and interstitial flow is employed, thereby replicating physiological nutrient and waste exchange conditions neglected in previous models. Key Technical Advantages: 1. Improvement of Spermatogenesis: The system supports the spermatogenic process, with flow cytometry analysis showing a 15% yield of haploid (1C) cells after 60 days of culture, compared to <1% in 3D organoid controls (p<0.001). 2. Market-Grade Processing: This decellularized testicular scaffold is market-ready and produced under GMP-compliant conditions with <0.5 EU / mL endotoxin levels in final constructs. Differences This invention is the first to integrate seminal exosomes as a critical bioactive component within a decellularized testis scaffold. The exosomes act as nano-carriers that provide timed release of key regulatory molecules directly to the germ cells, mimicking the in vivo paracrine signaling of the seminal plasma. Furthermore, our perfusion bioreactor is uniquely designed to simulate the differential pressure and flow dynamics between the seminiferous tubules and the interstitial space, a critical factor for nutrient exchange and waste removal that has been entirely neglected in previous systems. Protection Scope The decellularized testicular scaffold is defined by a specific ECM composition, retaining ≥80% of its original GDNF and FGF2 content, a tubular pore architecture with diameters of 80 ± 20 μm, and a biomechanical stiffness of 5.2 ± 0.8 kPa. The final construct is characterized by its ability to produce sperm like structure in vitro and by the staged release of seminal exosome cargo (including miR-34c-5p, miR- 449a, and miR-202) enabling phase-specific spermatogenic progression. Impact This technology offers a revolutionary therapeutic option for the thousands of men rendered infertile by cancer treatments, genetic conditions, or trauma. By generating functional sperm from a simple testicular biopsy, it eliminates the need for donor sperm and the associated ethical and psychological concerns. For pediatric oncology patients, this represents the first real hope for biological parenthood. The platform also has significant applications in toxicology and drug screening, providing a human-specific model to assess the impact of environmental toxins or pharmaceuticals on spermatogenesis. The use of autologous cells and a natural ECM scaffold minimizes the risk of immune rejection and ensures a high degree of biological safety, paving the way for rapid clinical translation. Furthermore, the inclusion of a perfusion bioreactor system and cryopreservation strategy enables standardized GMP manufacturing, quality control, and global clinical distribution, enhancing translational feasibility. Brief Description of Drawings [Fig 1]: Summary of the testicular decellularization protocol and functionalization with seminal extracellular vesicles. The intact testis is decellularized via perfusion with detergent solutions. Subsequently, extracellular vesicles are isolated from seminal plasma using ultracentrifugation and incorporated into the decellularized testicular scaffold to create a bioactive platform for spermatogonial stem cell culture.

[0002] References: 1. Cho, I. K. and C. A. J. R. M. Easley (2023). "Recent developments in in vitro spermatogenesis and future directions." 4(3): 215-232. 2. Cirigliano, L., M. Falcone, M. Gül, M. Preto, C. Ceruti, N. Plamadeala, F. Peretti, I. Ferro, M. Scavone and P. J. M. Gontero (2023). "Onco-TESE (Testicular Sperm Extraction): insights from a tertiary center and comprehensive literature analysis." 59(7): 1226. 3. Fath-Bayati, L., L. Naserpour, M. Khoshandam, R. Jannatifar and H. J. I. J. o. R. B. Fazaeli (2023). "Recent advances in developing 3D culture systems of spermatogonial stem cell preservation and differentiation: A narrative review." 21(9): 681. 4. Goossens, E., K. Jahnukainen, R. Mitchell, A. Van Pelt, G. Pennings, N. Rives, J. Poels, C. Wyns, S. Lane and K. J. H. r. o. Rodriguez-Wallberg (2020). "Fertility preservation in boys: recent developments and new insights." 2020(3): hoaa016. 5.- Hamzeh, M., M. Movahedin, F. Ganji and A. J. I. J. o. B. M. Ghiaseddin (2025). "Structural, mechanical, and cytocompatibility characteristics of hybrid scaffolds from chitosan / decellularized testicular ECM." 284: 137908. 6.- Hussien, E. A., E. E. N. Hassan, A. A. E. Abdellatif and M. S. J. T. E. J. o. H. M. Gaber "Fertility Preservation in Cancer Patients." 100: 2727-2733. 7.- Jensen, C. F., L. Dong, M. Gul, M. Fode, S. Hildorf, J. Thorup, E. Hoffmann, D. Cortes, J. Fedder and C. Y. J. N. R. U. Andersen (2022). "Fertility preservation in boys facing gonadotoxic cancer therapy." 19(2): 71-83. 8.- Orlić, A., I. Alsamara, M. J. C. T. Paric and R. Communications (2025). "Improving Fertility Preservation for Male Cancer Patients: A Scoping Review of Barriers and Facilitators." 100976. Rambhatla, A., R. Shah, I. Ziouziou, P. Kothari, G. Salvio, M. Gul, T. Hamoda, P. Kavoussi, W. Atmoko and T. J. T. W. J. o. M. s. H. Toprak (2024). "Global practice patterns and variations in the medical and surgical management of non-obstructive azoospermia: results of a world-wide survey, guidelines and expert recommendations." 43(1): 92. 9. Salem, M., F. Khadivi, P. Javanbakht, S. Mojaverrostami, M. Abbasi, N. Feizollahi, Y. Abbasi, E. Heidarian, F. J. S. C. R. Rezaei Yazdi and Therapy (2023). "Advances of three-dimensional (3D) culture systems for in vitro spermatogenesis." 14(1): 262. 10. Salomón Yunga-Ayavaca, E., R. Ximena Quinche-Morocho and F. J. R. C. d. l. F. d. V. Javier Angulo-Cubillán (2023). "Fertilidad mejorada en ganado lechero con baja condición corporal al usar gonadotrofina coriónica equina en inseminación artificial a tiempo fijo." 33(1). 11. Yang, C., W. b. Guo, W. S. Zhang, J. Bian, J. k. Yang, Q. z. Zhou, M. k. Chen, W. Peng, T. Qi and C. y. J. A. Wang (2017). "Comprehensive proteomics analysis of exosomes derived from human seminal plasma." 5(5): 1007-1015. 12. Yang, E. H., H. B. Strohl and H. I. J. C. Su (2024). "Fertility preservation before and after cancer treatment in children, adolescents, and young adults." 130(3): 344-355.

Claims

Claims 1. A method for in vitro production of spermatozoa from spermatogonial stem cells (SSCs), comprising the steps of: (1) Decellularization of testicular tissue: ^ (a) Perfusing whole testicular tissue sequentially with: o 0.1% (v / v) Triton X-100 + 0.05% (w / v) SDS for 48 hours, o 1% (v / v) Tween-20 + 0.1% (v / v) peracetic acid for 12 hours, o DNase / RNase solution (50 U / mL) at 37°C for 6 hours; ^ (b) Validating decellularization by residual DNA content (<50 ng / mg dry tissue) and retention of >80% of native GDNF and laminin-α5 via ELISA and mass spectrometry; (2) Preparation of cell and exosome suspension: ^ (a) Isolating SSCs from a testicular biopsy via enzymatic digestion and positive selection for CD90+ / GFRα1+ cells; ^ (b) Isolating seminal exosomes (SEs) from semen by differential ultracentrifugation. (3) Recellularization and culture: ^ (a) Seeding the suspension into the decellularized scaffold via vascular and tubular infusion; ^ (b) Culturing in 34°C and 5% CO₂ with stage-specific media: SSC Maintenance Media: StemPro-34 SFM + 20 ng / mL GDNF + 10 ng / mL FGF2 for 7 days; Meiotic Induction Media: α-MEM + 10⁻⁶ M retinoic acid + 30 ng / mL BMP4 for 21 days; Spermiogenic Media: HTF + 1 mM db-cAMP + 50 ng / mL testosterone for 32 days;(4) Quality control: ^ Confirming haploid cell yield (>10% via flow cytometry) and functional competence by oocyte activation assay.

2. A bioengineered testicular construct obtained by the method of claim 1, characterized by: ^ (a) A decellularized scaffold with <50 ng / mg residual DNA and preserved seminiferous tubule architecture; ^ (b) SSC seeding density of ≥1x10⁵ cells / cm³; ^ (c) Sustained release of SE-derived miRNAs over 60 days.

3. Use of the bioengineered testicular construct of claim 2 in a method for treating male infertility, comprising: ^ (a) Generating autologous spermatozoa in vitro ^ (b) Evaluation sperm viability and functionality 4. Use of the bioengineered testicular construct of claim 2 as an ex vivo model for screening the gonadotoxic or protective effects of pharmaceutical compounds or environmental toxins.

5. The seminal exosome (SE) preparation of claim 1, characterized by its miRNA cargo (miR-34c-5p at >1000 copies / exosome) and its use in promoting meiotic progression of germ cells in vitro.

6. A perfusion bioreactor apparatus for maintaining the construct of claim 2, comprising separate fluidic circuits that independently regulate flow and pressure across seminiferous tubules and interstitial compartments, thereby mimicking physiologic nutrient and waste exchange.

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