Novel ex vivo culture of neonatal mouse testicular organoid maintained in a hanging droplet with retinoic acid

Abstract

To propose a method to sustain germ cell characteristics of neonatal germinal epithelial cells in the form of self-organized organoids and initiate meiotic differentiation under the exposure of retinoic acid (RA). We tested the feasibility of utilizing an ex vivo three-dimensional culture system maintained in a hanging droplet (HD) to sustain and induce maturation of murine neonatal testicular cells. After trypsinization of 5-day-old newborn mouse testicular tissue, isolated cells were cultured in medium designed for spermatogonial stem cells (SSCs) composed of DMEM/F12 with GDNF, FGF2, 2-mercaptoethanol, L-glutamine, and B27 supplement in a gelatin-treated well for 3 days. Cell culture was then trypsinized and washed in SSC medium. The resulting cell pellet was resuspended in SSC medium void (control) or with 1 μM RA (HDRA). Cell suspensions were then adjusted to 40,000 cells/ml, and approximately 1,000 testicular cells were placed in each 25-μl HD. Cell characterization was performed every 3 days by germ cell stage–specific markers on an H&E-stained background. After culturing neonatal testicular cells in HDs, initial aggregation was observed 48 hours after HD culture. The earliest complete self-formation of spheroidal organoids was observed at day 3 for both control and HDRA. In the control group, continuing and consistent expression of OCT4 (>70%) and nuclear DAZL (>75%) throughout the experiment until day 21 determined that the SSCs retain stemness. In HDRA, a downregulated expression of OCT4 was recorded as early as day 3 in approximately 50% of the cells. A shift from nuclear to perinuclear positivity of DAZL in 16% of the cells in the HDRA group at day 21 confirmed differentiation into spermatocytes. Cytoplasmic VASA expression in the HDRA group confirmed meiotic/post-meiotic differentiation of the germ cells. Positive vimentin staining in 25% of the cells indicated the presence of nurturing pre-Sertoli cells in both groups. The attempt to maintain germ cell characteristics of neonatal testicular cells in the form of self-organized organoids appears to be an effective strategy for studying ex vivo spermatogenesis in the long-term. With the essential supplement of RA, germinal epithelial maturation was achieved. Once the ability to induce late-stage gametogenesis is confirmed, this technique may benefit cancer survivors who underwent gonadotoxic therapy in prepubertal age with irreversible damage of the germinal epithelium.