Optimization of Conditions for Cryopreservation of Enriched Spermatogonial Stem Cells in Olive Flounder (Paralichthys olivaceus)

Spermatogonial stem cells (SSCs) are pivotal in surrogate broodstock technology. However, species-specific protocols for the efficient enrichment and long-term preservation of SSCs in olive flounder (Paralichthys olivaceus) are not yet fully established. In this study, we evaluated and optimized methods for the isolation and cryopreservation of P. olivaceus SSCs. First, we compared two enrichment methods, including Percoll density gradient centrifugation (PDGC) and differential plating (DP). Although SSCs enriched by both methods showed increased expression of SSC-specific marker genes, PDGC resulted in significantly greater enrichment than DP. A combination of PDGC and DP did not further improve enrichment efficiency, suggesting that PDGC alone is sufficient in P. olivaceus. Second, we optimized cryopreservation conditions according to various cryoprotectants. Among the conditions, SSCs cryopreservation using 1.3 M propylene glycol (PG) as a permeating agent and 0.2 M raffinose (Raf) as a non-permeating cryoprotectant provided the highest cell viability (56.1%), demonstrating a synergistic protective effect. Finally, preliminary in vivo migration and localization ability of the cryopreserved SSCs was confirmed through xenotransplantation into zebrafish (Danio rerio) larvae. PKH26-labeled donor cells exhibited successful initial localization and short-term persistence within the presumptive gonadal ridge of the recipients at 5 days post-transplantation. These findings provide an optimized protocol for the handling and preservation of P. olivaceus germline resources, contributing to the technical advancement of surrogate reproduction strategies in this species.