MP70-05 DEVELOPMENT OF A NOVEL 3 DIMENSIONAL (3D) TESTICULAR ORGANOID MODEL FOR THE STUDY OF HUMAN SPERMATOGENESIS AND GENOTOXICITY IN VITRO

Abstract

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology1 Apr 2016MP70-05 DEVELOPMENT OF A NOVEL 3 DIMENSIONAL (3D) TESTICULAR ORGANOID MODEL FOR THE STUDY OF HUMAN SPERMATOGENESIS AND GENOTOXICITY IN VITRO Samuel Pendergraft, Hooman Sadri-Ardekani, Tanya Reid, Anthony Atala, and Colin Bishop Samuel PendergraftSamuel Pendergraft More articles by this author , Hooman Sadri-ArdekaniHooman Sadri-Ardekani More articles by this author , Tanya ReidTanya Reid More articles by this author , Anthony AtalaAnthony Atala More articles by this author , and Colin BishopColin Bishop More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1430AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Mammalian spermatogenesis is regulated through paracrine-endocrine activity, cell signaling, and local control mechanisms. These highly specific signaling interactions are effectively absent upon placing testicular cells into two-dimensional (2D) primary culture. 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 3D testis organoid in vitro by combining stem cell and tissue engineering approaches. This model can be utilized as a means to evaluate gonadotoxic agents, and to address critical deficiencies in our understanding of basic human spermatogenesis Human testes tissues for this study were obtained through the National Disease Research Interchange (NDRI). METHODS Development of this model system consisted of (1) Identification and analysis of specific cellular components necessary for use in a 3D culture method, (2) establishment of basic design parameters, culture conditions, and characterization of human testicular organoids using live cell imaging, immunofluorescence, immunohistochemistry, gene expression, and viability assays, and (3) assess the potential to demonstrate testicular organoid function and as a tool for screening for testicular drug toxicity. RESULTS Human Spermatogonial stem cells (SSCs), Sertoli, and Leydig cells were isolated from human adult testes tissues, characterized, and expanded. These cell types were integrated successfully into 3D organoids and maintained viability as determined by ATP and Live/Dead assays for over 3 weeks in culture. These organoids were responsive to hCG and able to produce testosterone in vitro. Changes in gene expression within these multicellular human testis organoids was measured over time for cell and stage-specific markers including UCHL1, ITGA6, DAZL, SYCP3, PRM1, ACROSIN, FSHr, CYP19a1, HSD3B1, and CYP11a1. Dose response curves were generated using a luminescent viability assay in response to exposure to 3 commonly used genotoxic chemotherapeutic drugs and dose-response was compared to corresponding cells cultured in 2D. Calculated IC50 values were determined to be significantly higher for 3D organoid cultures. CONCLUSIONS Functional human 3D testicular organoids were successfully generated. Future directions include optimizing the spermatogenic capacity of the organoids and continuing to evaluate their use as a novel testicular toxicity model © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e907 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Samuel Pendergraft More articles by this author Hooman Sadri-Ardekani More articles by this author Tanya Reid More articles by this author Anthony Atala More articles by this author Colin Bishop More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...