In vitro reconstitution of the hormone-responsive testicular organoids from murine primary testicular cells

Increasing rates of male infertility require more experimental models to understand the mechanisms underlying male infertility. In vitro organoids hold unprecedented promise for this purpose; however, the development of organoids with tissue architecture similar to that of the testis in vivo remains a challenge. Here, we generated testicular organoids derived from testicular cells by combining hanging drop culture and rotation a culture system. Our results indicated that testicular cells could self-assemble into spheroid organoids with tubule-like structures in hanging drop culture. The organoids can subsequently be cultured and maintained in a rotation culture systems. These established organoids have gene expression profiles similar to those of adult testis tissue, produce testosterone with preserved gonadotropin responsiveness, and exhibit sensitivity to reproductive toxicants. More importantly, each testicular Organoid can be generated from only 2000 cells, and they maintain their proliferative ability after freezing and thawing. These features make it possible to obtain fresh primary testis cells from a testicular biopsies taken from patients or endangered wild species, and to build individual-specific biobanks. These findings will help enable the exploration of self-organization process of testicular cells and provide an experimental model for reproductive biology research, pharmacotoxicology testing, and regenerative medicine.

hanging drop; rotation culture; testicular organoid.