Genovese, Vincenzo (2024) Advanced in vitro culture of bovine and human ovarian tissue using a perifusion bioreactor: sustaining tissue viability and boosting follicular growth. [Tesi di dottorato]

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Abstract

This PhD thesis investigates the development and optimization of dynamic culture systems for in vitro folliculogenesis in ovarian tissue. Given the limitations of static ovarian tissue culture methods, this work explores the use of continuous perfusion dynamic culture systems (PB) to enhance follicle activation, growth, and survival. The dynamic perifusion system offers a more controlled microenvironment, mimicking the in vivo conditions necessary for successful folliculogenesis. The research focuses on optimizing culture conditions and examining the impact of different bioreactor materials on tissue viability. Chapter 1 introduces the concept of dynamic culture and its advantages over static culture systems. This chapter describes the development and the optimization of a continuous perfusion dynamic culture system as an experimental model for culturing ovarian tissue strips in vitro. The effectiveness of this system is analyzed and validated by comparing it with static in vitro culture in both conventional dish (CD) and a permeable dish (PD), the latter representing the current gold standard. Bovine ovarian tissue is used as an experimental model to establish the efficacy of the dynamic culture system, which subsequently applied to human tissue. Building on this validation, Chapter 2 focuses on identifying the most suitable biocompatible materials and the evaluation of its compatibility for producing of the bioreactor device. Cytotoxicity assays are conducted to select the optimal medical-grade materials that ensure compatibility with ovarian tissue (both bovine and human) without compromising tissue integrity. Although dynamic culture in bioreactors has been shown to improve follicular progression, quality, and viability compared to static culture, the role of extracellular matrix (ECM) remodeling remains underexplored. The ECM in ovarian tissue is a structural scaffold for follicles and stromal cells. The mechanical properties of ECM, including the stiffness or softness of the microenvironment surrounding ovarian follicles, play a critical role in mechanotransduction, affecting follicle activation and growth. In this direction, Chapter 3 examines ECM remodeling in ovarian tissue cultured under dynamic conditions. ECM stiffness essential for mechanotransduction, is shown to influence follicle activation and growth. This chapter investigates how dynamic culture fosters a more favorable ECM environment for follicular development, compared to static culture systems. Recent research highlights the importance of anti-Müllerian hormone (AMH), a glycoprotein belonging to the transforming growth factor-beta (TGF-β) superfamily, produced by granulosa cells in growing follicles, particularly preantral and antral secondary follicles. AMH is a recognized biomarker, with serum levels strongly correlating with ovarian reserve indicators. The concentration of AMH in serum is a reliable marker of antral follicle count, reflecting the remaining primordial follicles population. Thus, Chapter 4 explores the role of AMH as a marker 12 of follicular progression in long-term cultures. Monitoring AMH levels at various time points provides insights into the developmental stages of the follicles during in vitro culture. Another key consideration is the cryopreservation of ovarian tissue prior to gonadotoxic treatment which is currently the only recommended option for fertility preservation in prepubertal girls. Due to the technical complexity of this procedure, only a limited number of centers worldwide offer it. As a result, harvested ovarian tissue must be maintained at hypothermic temperatures (4°C) for extended periods during transportation. The time between tissue collection and cryopreservation may affect cell functionality during fertility restoration. Chapter 5 presents a study on the effects of prolonged pre-freezing preservation of follicular viability. Using prepubertal bovine ovarian tissue as a model, this study assesses the impact of extended hypothermic storage (4°C) on tissue quality offering recommendations for handling and transporting ovarian tissue prior to cryopreservation. This dissertation aims to provide valuable insights into the optimization of in vitro folliculogenesis techniques, opening new avenues for fertility preservation.

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