Development of Novel Testicular Tissue Organ Culture Systems to Induce in vitro Spermatogenesis Spermatogonial stem cells (SSCs) have a tremendous capacity to regenerate spermatogenesis under proper conditions. Chemotherapy or radiation treatments for cancers or other conditions can destroy the stem cell pool and cause permanent infertility. The only option to preserve the fertility of prepubertal male patients is testicular tissue cryopreservation. When those tissues are thawed, testicular tissue organ culture is an approach to mature those tissues to produce sperm outside of the body. Takehiko Ogawa’s lab produced mouse sperm and offspring from cultures of fresh neonatal testes that were maintained for several months in a pump-driven microfluidic (MF) device. To overcome the bulkiness of the MF device, the same laboratory invented a pumpless microfluidic (PL) device. However, sperm and offspring have not been produced using the PL devices; spermatogenesis was not reported from cryopreserved tissues in the PL system, and the system has not been tested in any other species. This study aims to fill the gaps in knowledge by generating mouse sperm and offspring using the PL devices in cultures of fresh and cryopreserved testes, and to translate this technology to non-human primate and/or human testicular tissues. In addition, to overcome several pitfalls in the PL design, I developed and tested a novel PDMS-roof transwell (PRT) culture system, comprised of a polycarbonate-membrane-transwell and a polydimethylsiloxane (PDMS) roof, to ease device production and simplify sample loading and culture maintenance. The efficiency of in vitro spermatogenesis using the PRT device was similar to the PL device in cultures of fresh neonatal mouse testes (>70% tubules showed Acrosin- GFP+ post-meiotic cells within 1 month in culture). In the PRT system, I confirmed VASA-positive germ cells in 81.6±6.63%, SALL4-positive undifferentiated spermatogonia in 70.1±10.21%, STRA8-positive differentiating spermatogonia in 50.0±14.01%, SYCP3-positive spermatocytes in 75.9±9.2%, and SOX9-positive Sertoli cells in 99.08±0.9% of tubules. A similar experiment using cryopreserved neonatal mouse testes in both culture systems is underway. In a pilot study on immature rhesus macaque testicular tissues using the PRT system, I observed VASA-positive germ cell retention in 90.3% of tubules after 3 months in culture. More detailed analyses are in process. Unlike mouse, monkeys and humans have a prolonged prepubertal period before the initiation of spermatogenesis and there is limited information about the mechanisms that regulate prepubertal to pubertal to adult developmental transitions in testicular somatic cells or germ cells. I will exploit our unique pipeline of healthy rhesus and human testicular tissues from prepubertal, pubertal and adult individuals and perform high-throughput single-cell RNA sequencing to identify a subset of genes with differential expression at these devel...