Germ cell-containing testicular tubule-like structures form within murine assembloids using defined material constraints.

Testicular tissue cryopreservation (TTC) is currently the only option to preserve fertility in pre-pubertal individuals with testes. However, reimplantation of tissue collected during TTC has yet to result in a live human birth, despite recent progress in rhesus macaque models. Additionally, cell or tissue implantations are not options for individuals with metastatic disease, making it critical to develop alternative fertility restoration strategies using TTC specimens. Our goal was to generate primary cell-derived testicular assembloids with native-similar internal cell organization, enclosed seminiferous tubule-like structures (TLS), and surviving germ cells. Organoids derived from primary testicular cells harvested from 5-day old mice developed native-similar organization and enclosed TLSs when seeded at ~9,300 cells/microwell. We assessed the fusion and internal organization of merged organoids ("assembloids") in multiple open channel designs with differing physical constraints and access to media and oxygen. These conditions impacted assembloid ellipticity, cellular organization, TLS formation, TLS diameter, and germ cell survival. Open channels with 400- and 600-micron widths that were submerged within culture media most consistently produced assembloids with native-similar architecture. These data support a robust framework for the generation of murine testicular assembloids with germ cell-containing tubule-like structures and interstitial compartments. Optimization of this in vitro platform is an important step towards expanding fertility restoration options for prepubertal patients at increased risk for infertility.