ABSTRACT This is a multi-PI, multi-institutional grant to develop physiomimetic systems that mimic human testis development and spermatogenic lineage development, ex vivo. Chemotherapy and radiation treatments for cancer or gender affirming treatments for transgender patients can cause infertility. Adult patients have the option to cryopreserve eggs or sperm prior to treatment that can be thawed in the future and used to achieve pregnancy with standard assisted reproductive technologies, including IVF. Those options are not available to all adult patients or to prepubertal patients who are not able to produce mature eggs or sperm. Although prepubertal boys or transgender girls who are on gender affirming treatments are not producing sperm, they do have spermatogonial stem cells SSCs in their testes that have the potential to produce sperm. The MPIs in Chicago and Pittsburgh have been cryopreserving immature testicular tissues for young patients for more than a decade with anticipation that those tissues can be thawed in the future and matured to produce sperm. Each patient donates a portion of their tissue to research to develop next generation technologies that will allow them to use their tissues for reproduction. We will use those tissues to gain fundamental insights into human testis development from newborn to adult stages of life. This will establish benchmarks to evaluate our progress engineering three novel testis physiomimetic systems that support spermatogenesis, ex vivo. Aim 1 will replicate mouse testicular tissue organotypic culture at the air/liquid interface using a microfluidics device and a static PDMS roof-transwell system before translating the approach to human testis tissues. Aim 2 will establish mouse and human testicular organoids, which will then be induced to fuse into elongated seminiferous tubule-like structures. Aim 3 will print seminiferous tubule like scaffolds using human testis extracellular matrix (htECM) and pig testis ECM (ptECM) hydrogels as bioink and then seed the scaffolds with mouse and human testicular somatic cells and germ cells. These testicular physiomimetic systems will provide fundamental insights into human testicular somatic cell and germ cell development and establish experimental platforms to test toxicologic or pharmaceutical compounds or drugs that promote or prevent sperm production. The overarching objective is to mature human testicular tissues in one or more of these physiomimetic systems to produce fertilization competent sperm, ex vivo.

PROJECT NARATIVE Immature testicular tissues have been cryopreserved for thousands of patients worldwide with anticipation that they can be thawed in the future and matured to produce sperm. Autologous transplantation of frozen/thawed testicular tissues/cells will not be appropriate or safe for all patients. In this proposal, we test three different physiomimetic systems for maturing immature testicular tissues or cells and producing sperm outside the body. In addition to clinical applications, these systems can be used to gain fundamental insights into human testis development or used as platforms to screen of toxicologic or pharmaceutical compounds or develop drugs that promote or prevent sperm production.