Establishment of a 3D Co-Culture System Using OFT Cell Line and Effect of Hormone Supplementation on Germline Gene Expression in Olive Flounder (Paralichthys olivaceus).

In vitro 3D cell culture systems provide a physiologically relevant microenvironment that mimics native tissue architecture. However, establishing functional 3D testicular aggregates in teleosts remains challenging. In this study, we established a novel 3D co-culture aggregate system for olive flounder (Paralichthys olivaceus) primary testicular cells by incorporating the established olive flounder testicular cell (OFT) line. Primary testicular cells alone failed to form 3D aggregates within the first 24 h in ultra-low attachment plates. However, co-culturing with OFT cells rapidly initiated 3D aggregate assembly within 24 h. Furthermore, supplementation with a cocktail of sex hormones and growth factors maintained high cell viability without cytotoxic effects. Notably, quantitative real-time PCR analysis revealed that hormone and growth factor supplementation significantly upregulated the transcript levels of both plzf (2.15-fold) and scp3 (2.34-fold). These results demonstrate that the OFT cell line is essential for facilitating the assembly and incorporation of primary testicular cells into 3D aggregates, while sex hormones and growth factors promote spermatogonial stem cell maintenance and meiotic progression within the aggregates. Overall, this 3D co-culture platform serves as a valuable in vitro tool for fish spermatogonial stem cell research and reproductive biotechnology in aquaculture.