A Novel Cultivation System for Germ Cell Proliferation and Sustaining Whole Testicular Niche

Abstract Childhood cancer treatments often impair male fertility due to gonadotoxic effects. While in vitro culture of prepubertal testis tissue offers a potential solution to preserve fertility, producing sufficient sperm remains a major barrier. To address this, we developed a hydrogel microneedle‐based culture system, which is used to culture mouse testes from 5 days postpartum (d pp ) and establish a model of ‘whole testicular spermatogonia pool’ (WTSP). This system promotes over fourfold expansion of undifferentiated spermatogonia, compared to declining numbers during in vivo development. Transplantation of WTSP into nude mice doubled spermatids count per tubule compared to conventional whole testes transplantation. Furthermore, in vitro meiosis induction of WTSP significantly enhanced spermatid proportion, thus generating fertile offspring. Using this system, we also cultured the gonads harvested from aborted human male fetuses and observes significant proliferation of spermatogonia. Lastly, it is shown that the cellular states of the WTSP closely resemble those of 5 d pp mouse testes, and the role of EPHA2 in promoting spermatogonia proliferation by activating the PI3K‐AKT‐mTOR pathway. In conclusion, the WTSP offers a promising method for preserving fertility in prepubertal male cancer patients by maintaining and expanding spermatogonia extracted before treatment.