Formation of testicular organoids for male reproductive research

The decrease of sperm counts observed over the last decades represents a social and biological crisis associated with male infertility, higher disease burden and healthcare costs. However, current knowledge in reproductive biology and male reproductive health is not complete, hindering research on male infertility, its diagnosis and treatment. Because they offer the possibility to manipulate cells in time and space through genetic modifications, or though inclusion and exclusion experiments, TOs allow in-depth studies on testis development, function and disease. The TO concept refers to the reconstitution of the 3D testicular architecture from dissociated cells and complex interactions in vitro from dissociated cells to support spermatogenesis.
Following the identification of extrinsic factors that drive testicular morphogenesis from testicular cell suspensions in 3D cultures (Chapter 3), we established our own murine TO cultures using 3D-printed alginate(-RGD)based macroporous scaffolds (Chapter 4) and agarose 3DPDs (Chapter 5). The 3D organoid cultures were primarily designed to delimitate the area in which testicular cells can grow and scaled to an anatomical unit of the testis; a single tubule-like structure devoid of core degeneration, and with surrounding interstitium. In Chapter 4, the 3D printed-TO culture platform was free of exogeneous ECM-support, which is important to reduce variability to the 3D cell culture conditions. We demonstrated that prepubertal murine testicular cells have an intrinsic property to reestablish the compartmentalized structure of the testis, relying entirely on their own morphogenic properties and matrix production. However, a significant decrease of germ cells and meiotic arrest were observed.
In Chapter 5, using a biphasic culture approach, including a reorganization and a differentiation phase with dedicated sequential media, we formed TOs resembling the anatomical unit of the testis and improved germ cell maintenance during long-term culture. Using specific starting cell densities and media, TOs had a formation efficiency of approximatively 85% in the 3DPD- TO culture platform and, most importantly, generated elongating spermatids. To our knowledge, this is the first study to adopt this approach in the TO field, and to achieve all benchmarks of TOs. The limitations identified in earlier reports were addressed and improved in current TOs. Our observations show that the testosterone secretion pattern of the TOs can still be optimized to further enhance IVS. Moreover, stress responses related to the culture procedure itself cannot be excluded and may possibly disturb spermatogenesis by means of inflammatory reactions, known to have unfavorable effects on testicular morphogenesis and spermatogenesis. In vitro study models that allow reproducible mechanistic studies, such as the 3DPD-TO platform, are necessary to gain insight and put clinical attention on andrology, male reproductive biology and health research.