Helium plasma-activated conditioned medium promotes spermatogonial stem cell recovery and redox homeostasis under bleomycin-induced testicular injury in SSCs-sertoli cell co culture.

Chemotherapy-induced testicular damage is a major cause of male infertility, primarily due to oxidative stress, apoptosis, and disruption of the spermatogonial niche. Spermatogonial stem cells (SSCs) as well as Sertoli cells are central regulators of spermatogenesis, and preserving their function is essential for maintaining male fertility. This study investigated the protective and regenerative potential of helium plasma-activated conditioned medium (PACM) against bleomycin-induced cytotoxicity in a neonatal rat SSCs-Sertoli cell co-culture model. Testicular cells were isolated and characterized using alkaline phosphatase assay and qRT-PCR for evaluating CD49f and Sox9 expression. Cells were treated with conditioned medium (CM), plasma-activated medium (PAM), and plasma-activated conditioned medium (PACM), either alone or in combination with bleomycin (Ble). Among all treatments, PACM exerted the strongest protective effects, significantly through enhancing cell viability and colony formation as well as reducing intracellular reactive oxygen species (ROS) and apoptosis. Based on findings, Ble markedly disrupted redox balance, apoptosis increment verified through Annexin-FITC externalization, suppressed Nrf2, c-KIT and CAT expression, attenuated catalase enzyme activity; however, co-treatment with PACM most effectively overwhelmed Ble insult, indicating activation of antioxidant defenses and spermatogenic signaling pathways. The superior efficacy of PACM appears to result from the synergistic interaction between plasma-generated reactive oxygen and nitrogen species (RONS) and the bioactive components of conditioned medium, including growth factors, cytokines, and exosomes. Collectively, these findings indicated that PACM restores the spermatogonial microenvironment, reinforces endogenous regenerative capacity, and offers a promising cell-free strategy to mitigate bleomycin-induced reproductive toxicity in vitro. It proposes that future studies are critical to identify the specific PACM components, the principal mediators and signaling pathways responsible for PACM effects. A graphical overview of the experimental workflow and outcomes is presented in Fig. 1.