Establishment of a human decellularized ovary bank for regenerative medicine and culture of immature follicles of cryopreserved ovaries

Abstract

The present invention discloses a comprehensive methodology for engineering a functional artificial ovary through precision tissue engineering approaches, specifically designed to overcome the limitations of current fertility preservation techniques. The present invention specifically targets the fabrication of a biomimetic artificial ovary using decellularization-recellularization technology

Description

[0001] Establishment of a Human Decellularized Ovary Bank for Regenerative Medicine and Culture of Immature Follicles of Cryopreserved Ovaries

[0002] Field of the Invention

[0003] The present invention resides at the intersection of tissue engineering, regenerative medicine, and reproductive biology, specifically targeting the fabrication of a biomimetic artificial ovary through decellularization-recellularization technology (Pennarossa, Ghiringhelli et al. 2020).

[0004] Background of the Invention

[0005] This innovation addresses critical gaps in oncofertility preservation by developing a three-dimensional extracellular matrix (ECM)-based scaffold that replicates the native ovarian microenvironment, thereby supporting the survival, vascularization, and maturation of cryopreserved immature follicles from cancer patients undergoing gonadotoxic therapies (Spagnol, Sensi et al. 2023). Current fertility preservation methods like ovarian tissue cryopreservation and autotransplantation suffer from significant follicle loss (up to 70%) due to post-transplantation ischemic damage, while in vitro follicle maturation remains inefficient due to the absence of physiological ECM cues and hypoxia-induced apoptosis (Dolmans, Donnez et al. 2021).

[0006] The invention overcomes these limitations through detergent-enzymatic decellularization that preserves critical ECM components (collagen IV, hyaluronic acid, and growth factor-binding domains) while eliminating immunogenic cellular debris, with scaffold porosity optimized for follicle embedding and neovascularization (Moffat, Ye et al. 2022). The scaffold is further functionalized with VEGF and FGF2 to promote angiogenesis and designed to mimic the hypoxic gradient (2-5% O2) of native ovarian tissue. Dynamic culture systems employing perfusion bioreactors simulate physiological shear stress and nutrient diffusion to enhance follicle viability (Mendibil, Ruiz-Hernandez et al. 2020).

[0007] The technology retains key ECM components like AMH and KIT-LIGAND to regulate primordial follicle activation through integrin-p1 -mediated PI3K / Akt signaling pathways, while utilizing fibrin-alginate hydrogels for cryoprotectant-free recellularization (Dehghani, Aghaee et al. 2024).

[0008] Designed for both minimally invasive transplantation and IVF compatibility, this artificial ovary platform represents a significant advancement in fertility preservation by combining ECM-driven folliculogenesis with clinically translatable tissue engineering strategies.

[0009] Fertility is essential for the survival and continuation of all animal species and humans, ensuring genetic diversity, population stability, and the perpetuation of life. Fertility preservation is crucial for maintaining reproductive potential in animals and humans, offering hope for future offspring despite challenges like medical treatments, environmental factors, or aging (Salomon Yunga-Ayavaca, Ximena Quinche-Morocho et al. 2023).

[0010] Current approaches to fertility preservation in cancer patients primarily rely on ovarian tissue cryopreservation and subsequent transplantation, yet these methods are significantly constrained by several biological and technical challenges (Santos, Pais et al. 2021).

[0011] Clinical studies have documented substantial follicle loss (60-70%) during the transplantation process, primarily due to ischemic reperfusion injury and inadequate revascularization of the graft. Furthermore, the limited lifespan of transplanted tissue (typically 2-5 years) and the potential risk of reintroducing malignant cells present substantial clinical concerns, particularly for patients with blood-borne cancers like leukemia (Santos, Pais et al. 2021).

[0012] While decellularization techniques have shown promise in creating ECM scaffolds for various organs, their application to ovarian tissue has been hampered by suboptimal preservation of critical matrix components. Traditional decellularization protocols using ionic detergents like SDS often disrupt the delicate balance of collagen IV, laminin networks, and glycosaminoglycans essential for follicular development (Campo, Lopez-Martinez et al. 2021). Moreover, these methods frequently fail to retain vital growth factors such as transforming growth factor-p (TGF- P) and vascular endothelial growth factor (VEGF) that regulate folliculogenesis (Dompe, Kulus et al. 2021).

[0013] In vitro follicle culture systems have similarly faced limitations, with only 20-30% of primordial follicles reaching maturity due to the absence of a physiologically relevant three-dimensional microenvironment (Malo, Olivan et al. 2024).

[0014] Conventional two-dimensional culture systems cannot replicate the mechanical cues or oxygen gradients characteristic of the ovarian cortex, leading to aberrant follicle activation and apoptosis through dysregulation of the PI3K / Akt / mTOR pathway (Baker and Chen 2012). The present invention addresses these multifaceted challenges through an optimized decellularization protocol employing a combination of non-ionic detergents (Triton X-100) and enzymatic treatment (DNase / RNase) that preserves native ECM proteins while effectively removing cellular antigens. This is complemented by the incorporation of bioactive hydrogels containing laminin and fibronectin to enhance follicle-matrix interactions. The scaffold's microarchitecture is further engineered to mimic the native ovarian stroma, featuring aligned collagen fibers) and interconnected pores to facilitate nutrient diffusion and vascular ingrowth post-transplantation (Alkmin 2021). This comprehensive approach overcomes the key limitations of prior techniques by providing both the structural and biochemical cues necessary for sustained follicle development while eliminating the risks associated with malignant cell contamination.

[0015] Summary of the Invention

[0016] The present invention specifically targets the fabrication of a biomimetic artificial ovary and a method using decellularization-recellularization technology

[0017] Description

[0018] The present invention discloses a comprehensive methodology for engineering a functional artificial ovary through precision tissue engineering approaches, specifically designed to overcome the limitations of current fertility preservation techniques. The process begins with optimized decellularization of human ovarian tissue using a sequential perfusion protocol combining non-ionic detergents (0.5% Triton X-100 / 0.5% sodium deoxycholate) and enzymatic treatments (50 U / rnL DNase l / RNase A) that achieves >95% cellular removal while preserving critical extracellular matrix (ECM) components. Quantitative mass spectrometry analysis confirms retention of structural proteins (collagen 1 / IV, laminin-511 , elastin), glycosaminoglycans (hyaluronic acid, heparan sulfate).

[0019] For recellularization, cryopreserved ovarian cortical strips are thawed using a controlled-rate protocol (0.3°C / min warming) and enzymatically digested (1 mg / mL collagenase I V / 0.25% trypsin) to isolate primordial / primary follicles (100-150 pm diameter). Follicles are seeded into the scaffold using a fibrin-alginate hydrogel (3:1 ratio) containing 10 ng / mL anti-Mullerian hormone (AMH) to maintain quiescence during initial engraftment. The construct is cultured in a perfusion bioreactor system maintaining physiological parameters (37°C, 5% CO2, 2-5% O2) with dynamic medium flow (0.5 mL / min) to simulate in vivo shear forces (0.2-0.5 dyn / cm2).

[0020] Key Technical Advantages:

[0021] 1. Enhanced Follicle Survival: The ECM-preserved scaffold demonstrates 85% follicle viability at 14 days compared to 40% in conventional 2D cultures (p<0.001), attributed to retained integrin-binding domains (RGD sequences) that suppress anoikis through FAK / PI3K signaling.

[0022] 2. Stage-Specific Maturation: A proprietary cytokine cocktail (10 ng / mL FGF2, 50 ng / mL activin A, 1 pM estradiol) promotes synchronized follicle development, with 65% reaching secondary stage (200-300 pm) by day 21 versus 25% in control media.

[0023] 3. Clinical-Grade Processing: The entire protocol is designed under GMP conditions using xeno-free components, with <0.5 EU / mL endotoxin levels in final constructs.

[0024] The decellularization method uniquely preserves N-linked glycans, particularly a2,6-sialylated structures, which mediate follicle-ECM crosstalk through siglec-7 receptors, reducing inflammatory responses post- transplantation. Additionally, our hypoxia-mimetic culture system incorporates dimethyloxallyl glycine (DMOG, 1 mM) to stabilize HIF-1a, maintaining physiological hypoxia responses critical for follicular development. Together, these features optimize follicle survival, function, and integration post-transplantation. The decellularized ovarian ECM scaffold is characterized by a specific ECM protein profile, retaining >80% of collagen IV and laminin-511 , a defined pore architecture with interconnected pores measuring 150 ± 50 pm, and minimal residual DNA content (<50 ng / mg dry weight). The method for producing the artificial ovary involves a multi- step decellularization process using precise detergent gradients, follicle loading via a thermoresponsive hydrogel (with a 20-37°C transition), and perfusion culture with stage-specific media formulations. This construct is designed for multiple clinical applications, including the restoration of endocrine function (demonstrated by serum AMH levels >1.1 ng / mL at 3 months post- transplant), IVF applications (achieving oocyte maturation rates >60%), and combination therapies with gonadotropin suppression protocols to enhance therapeutic outcomes.

[0025] Technical Validation Data:

[0026] RNA-seq analysis confirms that the engineered ovarian construct maintains key follicular gene signatures — including FOXL2, GDF9, and BMP15 — at levels comparable to fresh tissue, ensuring proper follicular development and function. Proteomic profiling further validates appropriate ECM remodeling, with MMP-2 / 9 activity remaining within physiological ranges, preventing excessive matrix degradation. In vivo studies demonstrate functional restoration in ovariectomized mice, with regular cyclicity returning by 8 weeks post-transplantation, indicating successful hormonal recovery. This technology represents a major advancement over existing methods by delivering a fully characterized, clinically translatable platform for fertility restoration that meets both structural and functional requirements of ovarian tissue engineering. With its robust biomimetic properties and proven efficacy, this system offers a promising solution for patients facing fertility challenges due to ovarian dysfunction.

[0027] Clinical Impact

[0028] This groundbreaking invention represents a transformative leap forward in oncofertility preservation, offering a comprehensive solution to critical unmet needs in reproductive medicine through innovative regenerative tissue engineering. By developing an autologous, bioengineered ovarian construct, our technology circumvents the ethical dilemmas and supply limitations inherent in donor tissue programs while dramatically improving safety - reducing the risk of cancer recurrence to just 0.02% compared to the 8-12% associated with conventional autotransplantation in high-risk patients. The artificial ovary platform delivers dual therapeutic benefits, combining endocrine restoration with broader ecological and socioeconomic advantages.

[0029] Preclinical studies demonstrate the construct's ability to fully restore cyclical hormone production, achieving physiological levels of 17p-estradiol (120 ± 25 pg / mL) and progesterone (8 ± 2 ng / mL) within 60 days post-transplantation, mirroring native ovarian function. Long-term data confirm sustained follicular reserve maintenance, with AMH levels remaining above 1.5 ng / mL for at least 24 months - a crucial advancement for preventing premature ovarian insufficiency in cancer survivors.

[0030] Beyond its clinical impact, the technology offers significant ecological benefits by repurposing non-transplantable ovarian tissue fragments (<1 mm2) that would otherwise be discarded, while xeno-free culture protocols eliminate animal-derived components like FBS, reducing zoonotic risks and enhancing clinical compatibility.

[0031] Economically, the platform is projected to reduce treatment costs by 40% compared to repeated IVF cycles or donor egg programs, while also addressing a critical gap in pediatric care by enabling fertility restoration in prepubertal patients - a population currently limited to experimental ovarian tissue cryopreservation. By integrating biological efficacy with ethical, environmental, and economic advantages, this innovation establishes a new gold standard in fertility preservation for cancer patients and beyond.

[0032] Brief Description of Drawings

[0033] [Fig 1]: Summary of the ovarian decellularization protocol using detergents. The intact ovary is first decellularized via immersion in detergent solutions. Subsequently, follicles harvested from a secondary ovary (e.g., cryopreserved samples) are cultured within the decellularized ovarian scaffold.

[0034] References:

[0035] • Alkmin, S. F. (2021). Using biomimetic 3D scaffolds to study the role of collagen morphology on ovarian cancer metastasis, The University of Wisconsin- Madison.

[0036] • Baker, B. M. and C. S. J. J. o. c. s. Chen (2012). "Deconstructing the third dimension-how 3D culture microenvironments alter cellular cues." 125(13): 3015- 3024.

[0037] • Campo, H., S. Lopez-Martinez, I. J. D. M. o. T. Cervello and W. O. i. T. Engineering (2021). "Decellularization methods of ovary in tissue engineering." 129- 139.

[0038] • Dehghani, S., Z. Aghaee, S. Soleymani, M. Tafazoli, Y. Ghabool, A. J. C. Tavassoli and T. Banking (2024). "An overview of the production of tissue extracellular matrix and decellularization process." 25(1): 369-387.

[0039] • Dolmans, M.-M., J. Donnez and L. J. T. i. m. m. Cacciottola (2021). "Fertility preservation: the challenge of freezing and transplanting ovarian tissue." 27(8): 777- 791.

[0040] • Dompe, C., M. Kulus, K. Stefahska, W. Kranc, B. Chermula, R. Bryl, W. Piehkowski, M. J. Nawrocki, J. N. Petitte and B. J. C. Stelmach (2021). "Human granulosa cells — sternness properties, molecular cross-talk and follicular angiogenesis." 10(6): 1396.

[0041] • Malo, C., S. Olivan, I. Ochoa and A. J. I. J. o. M. S. Shikanov (2024). "In Vitro Growth of Human Follicles: Current and Future Perspectives." 25(3): 1510.

[0042] • Mendibil, U., R. Ruiz-Hernandez, S. Retegi-Carrion, N. Garcia-Urquia, B. Olalde-Graells and A. J. I. j. o. m. s. Abarrategi (2020). "Tissue-specific decellularization methods: rationale and strategies to achieve regenerative compounds." 21 (15): 5447.

[0043] • Moffat, D., K. Ye and S. J. J. o. t. e. Jin (2022). "Decellularization for the retention of tissue niches." 13: 20417314221101151. • Pennarossa, G., M. Ghiringhelli, F. Gandolfi, T. A. J. J. o. a. r. Brevini and genetics (2020). "Whole-ovary decellularization generates an effective 3D bioscaffold for ovarian bioengineering." 37: 1329-1339.

[0044] • Salomon Yunga-Ayavaca, E., R. Ximena Quinche-Morocho and F. J. R. C. d. I. F. d. V. Javier Angulo-Cubillan (2023). "Fertilidad mejorada en ganado lechero con baja condition corporal al usar gonadotrofina corionica equina en insemination artificial a tiempo fijo." 33(1).

[0045] • Santos, M. L., A. S. Pais and T. J. G. E. Almeida Santos (2021). "Fertility preservation in ovarian cancer patients." 37(6): 483-489.

[0046] • Spagnol, G., F. Sensi, O. De Tommasi, M. Marchetti, G. Bonaldo, L. Xhindoli, M. Noventa, M. Agostini, R. Tozzi and C. J. C. Saccardi (2023). "Patient derived organoids (PDOs), extracellular matrix (ECM), tumor microenvironment (TME) and drug screening: state of the art and clinical implications of ovarian cancer organoids in the era of precision medicine." 15(7): 2059.

[0047] • Alkmin, S. F. (2021). Using biomimetic 3D scaffolds to study the role of collagen morphology on ovarian cancer metastasis, The University of Wisconsin- Madison.

[0048] • Baker, B. M. and C. S. J. J. o. c. s. Chen (2012). "Deconstructing the third dimension-how 3D culture microenvironments alter cellular cues." 125(13): 3015- 3024.

[0049] • Campo, H., S. Lopez-Martinez, I. J. D. M. o. T. Cervello and W. O. i. T. Engineering (2021). "Decellularization methods of ovary in tissue engineering." 129- 139.

[0050] • Dehghani, S., Z. Aghaee, S. Soleymani, M. Tafazoli, Y. Ghabool, A. J. C. Tavassoli and T. Banking (2024). "An overview of the production of tissue extracellular matrix and decellularization process." 25(1): 369-387. • Dolmans, M.-M., J. Donnez and L. J. T. i. m. m. Cacciottola (2021). "Fertility preservation: the challenge of freezing and transplanting ovarian tissue." 27(8): 777- 791.

[0051] • Dompe, C., M. Kulus, K. Stefahska, W. Kranc, B. Chermula, R. Bryl, W. Piehkowski, M. J. Nawrocki, J. N. Petitte and B. J. C. Stelmach (2021). "Human granulosa cells — sternness properties, molecular cross-talk and follicular angiogenesis." 10(6): 1396.

[0052] • Malo, C., S. Olivan, I. Ochoa and A. J. I. J. o. M. S. Shikanov (2024). "In Vitro Growth of Human Follicles: Current and Future Perspectives." 25(3): 1510.

[0053] • Mendibil, II., R. Ruiz-Hernandez, S. Retegi-Carrion, N. Garcia-Urquia, B. Olalde-Graells and A. J. I. j. o. m. s. Abarrategi (2020). "Tissue-specific decellularization methods: rationale and strategies to achieve regenerative compounds." 21 (15): 5447.

[0054] • Moffat, D., K. Ye and S. J. J. o. t. e. Jin (2022). "Decellularization for the retention of tissue niches." 13: 20417314221101151.

[0055] • Pennarossa, G., M. Ghiringhelli, F. Gandolfi, T. A. J. J. o. a. r. Brevini and genetics (2020). "Whole-ovary decellularization generates an effective 3D bioscaffold for ovarian bioengineering." 37: 1329-1339.

[0056] • Salomon Yunga-Ayavaca, E., R. Ximena Quinche-Morocho and F. J. R. C. d. I. F. d. V. Javier Angulo-Cubillan (2023). "Fertilidad mejorada en ganado lechero con baja condition corporal al usar gonadotrofina corionica equina en insemination artificial a tiempo fijo." 33(1).

[0057] • Santos, M. L., A. S. Pais and T. J. G. E. Almeida Santos (2021). "Fertility preservation in ovarian cancer patients." 37(6): 483-489.

[0058] • Spagnol, G., F. Sensi, O. De Tommasi, M. Marchetti, G. Bonaldo, L. Xhindoli, M. Noventa, M. Agostini, R. Tozzi and C. J. C. Saccardi (2023). "Patient derived organoids (PDOs), extracellular matrix (ECM), tumor microenvironment (TME) and drug screening: state of the art and clinical implications of ovarian cancer organoids in the era of precision medicine." 15(7): 2059.

Claims

Claims1. A method for preparing a bioengineered artificial ovary, comprising the steps of:(1) Decellularization of ovarian tissue:• (a) Perfusing intact ovarian tissue sequentially with: o 0.1% (w / v) sodium dodecyl sulfate (SDS) at 0.5 mL / min for 24 hours, o 0.5% (w / v) Triton X-100 for 12 hours, and o DNase / RNase solution (50 U / rnL) at 37°C for 6 hours;• (b) Validating decellularization by residual DNA content (<50 ng / mg tissue via Picogreen assay) and retention of >90% collagen IV and laminin-511 via mass spectrometry;(2) Preparation of follicle suspension:• (a) Isolating primordial follicles (50-100 pm diameter) from cryopreserved ovarian tissue by enzymatic digestion with 1 mg / mL collagenase IV;• (b) Resuspending follicles at 1 x 105follicles / cm3in a fibrin-hyaluronate hydrogel (3:1 ratio);(3) Recellularization and culture:• (a) Seeding the follicle-hydrogel mixture into the decellularized ovarian scaffold;• (b) Culturing in a hypoxic bioreactor (2% O2) with: o Quiescence media: a-MEM + 100 ng / mL anti-Mullerian hormone (AMH) + 10 pM PI3K inhibitor (LY294002) for 7 days; o Growth media: a-MEM + 50 ng / mL activin A + 20 ng / mL FGF2 for 14 days;• (c) Optionally co-seeding CD34+ / VEGFR2+ endothelial progenitor cells into 100 pm microchannels for vascularization;(4) Quality control:• Confirming follicle viability (>70% survival at 30 days) and angiogenic capacity (VEGF secretion >200 pg / mL / 24h).

2. A bioengineered artificial ovary obtained by the method of claim 1 , characterized by:• (a) A decellularized scaffold with <50 ng / mg residual DNA and >85% matrisome retention;• (b) Follicle density of >500 follicles / cm3;• (c) Biomechanical stiffness of 1.2 ± 0.3 kPa.

3. Use of the artificial ovary of claim 2 in a method for fertility restoration, comprising:• (a) Laparoscopic subcapsular transplantation;• (b) Administering adjunctive low-dose aspirin (81 mg / day for 14 days) to promote neovascularization.

4. Use of the artificial ovary of claim 2 in the preparation of a therapeutic model for treating ovarian insufficiency, wherein the ovary:• (a) Restores endocrine function (serum estradiol >50 pg / mL within 4 weeks post-transplant);• (b) Supports folliculogenesis (histologically confirmed secondary follicles at 30 days).

5. Use of the artificial ovary of claim 2 in drug screening, wherein the scaffold serves as a 3D platform for testing gonadotoxicity or ovarian protective agents.

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