MON-149 - Open Channel Culture Systems Generate Murine Testicular Assembloids With Resilient Germ Cell Populations and Enclosed Seminiferous Tubule-like Structures

Gonadotoxic cancer therapies can leave surviving pediatric cancer patients unable to have children, which they often cite as an important quality of life metric. Male patients do not produce mature sperm and thus can only preserve fertility by saving immature gametes via testicular tissue cryopreservation. However, there are no technologies that have successfully restored fertility using this tissue in humans. In vitro spermatogenesis within testicular organoids is one technology that is currently being investigated. Our goal was to create a microenvironment to support in vitro spermatogenesis in organoids generated from prepubertal primary murine testis cells. Specifically, we aimed to improve upon previously published testicular organoid models by establishing organoids with native-similar testicular architecture, enclosed seminiferous tubule-like structures (TLSs), and a mature spermatogonial stem cell (SSC) niche. We engineered 3D-printed open channel culture systems (OCCSs) of varying widths and oxygen tensions that facilitated the merging of organoids into larger assembloids. OCCS design was found to regulate TLS density, diameter, and lumen. Assembloids cultured within submerged OCCSs of 400- and 600-micron widths more consistently contained enclosed TLSs, germ cells localized adjacent to Sertoli cells, and Leydig cells localized outside TLSs than assembloids cultured at the air-liquid interface of OCCSs of identical widths. More specifically, the 400 and 600-micron submerged OCCSs produced assembloids containing the largest percentage of DDX4-expressing germ cells (1.0% and 1.5% of total cells, respectively) and nearly 100% were localized adjacent to Sertoli cells. Furthermore, our OCCSs were able to support proliferating spermatogonia, including SSCs, over 13 days of culture. Whole mount imaging of assembloids cultured in submerged OCCSs indicates that TLSs are largely enclosed structures. Quantitative measurements of 29 tubules within a 400-micron submerged assembloid were taken, with the minor diameters ranging from approximately 50-150 microns and the major diameters ranging from approximately 75-225 microns. The significant difference in major and minor diameters for each TLS indicates that the TLSs within assembloids are elongated and elliptical, despite not forming long cord structures as in the native testes. Preliminary data indicates higher expression of androgen receptor than AMH within assembloids at the end of the culture period, suggesting the stem cell niche is maturing. Ongoing work aims to characterize the hormonal output of assembloids throughout culture, test the effect of vascularization on TLS formation, improve TLS elongation, and promote the formation of a fully mature stem cell niche. These advancements will inform future studies performed with human tissues, bringing us one step closer to in vitro spermatogenesis.

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