I-5: Multicellular Human Testicular Organoid: A Novel 3D In Vitro Germ Cell and Testicular Toxicity Model

Background Background: Mammalian spermatogenesis is regulated through paracrine and endocrine activity, specific cell signaling, and local control mechanisms. These highly specific signaling interactions are effectively absent upon placing testicular cells into two-dimensional primary culture. The specific changes that occur between key cell types and involved spermatogenesis signaling pathways during primary culture remain to be elucidated. However, current protocols to produce mature germ cells in vitro are inefficient and are limited in supporting post-meiotic cells. In order to address these limitations we have developed a 3-dimensional testis organoid in vitro by combining stem cell and novel tissue engineering approaches. This model can be utilized as a means to evaluate potential gonadotoxic agents, and act as a means to address critical deficiencies in our understanding of basic human spermatogenesis. The overall goal of this study is to establish, characterize, and culture a multicellular, 3D, human testis organoid and to assess its functionality and spermatogenic capacity over time. Materials and Methods: Development of our model system consisted of (1) identification and analysis of specific cellular components necessary for use our 3-dimensional culture method, (2) establishment of basic design parameters, culture conditions, and (3) characterization of human testicular organoids using live cell imaging, immunofluorescence, immunohistochemistry, cell type and stage-specific gene expression, and viability assays. Results: Human spermatogonial stem cells (SSCs), Sertoli, and Leydig cells were isolated, characterized, and expanded from tissue obtained through the National Disease Research Interchange (Philadelphia, PA, USA). These cell types were integrated successfully into 3-dimensional organoids and maintained viability as determined by ATP and Live/Dead assays for over 4 weeks in culture. During extended culture, qPCR analysis revealed a significant upregulation of spermatogenic markers including DAZL, ACR, and PRM1, as well as an upregulation of the leydig cell functional marker HSD3B1 and sertoli cell functional marker FSHr. In addition, these organoids secrete androgens and are responsive to hCG stimulation in vitro. In order to establish enhanced feasibility of this system for high-throughput in vitro drug screening applications, we further demonstrated that these organoids can be successfully cryopreserved for future use without sacrificing proliferative capacity or functionality. Conclusion: Testicular in vitro oragnoids were successfully generated by using isolated human SSC, Sertoli, and Leydig cells and maintained long term. Future directions will include optimizing thec capacity of the organoids and evaluating their use as a novel testicular toxicity model. MaterialsAndMethods N;Results N;Conclusion N;