Isolation, Expansion, Molecular Characterization, and Synergistic Antioxidant Protection of Rat Spermatogonial Stem Cells Using Mangiferin and Sodium Selenite

Gonadal toxicity from cancer treatments highlights the need for efficient spermatogonial stem cell (SSC) isolation and expansion protocols for fertility preservation. This study aimed to isolate and culture rat SSCs and evaluate the cytoprotective and antioxidant effects of mangiferin (M) and sodium selenite (S), individually and in combination (M+S). Rat testicular tissue was isolated through a two-step enzymatic digestion protocol, and baseline passage 1 (P1) cells were characterized using RT-qPCR and immunocytochemistry. Cultured cells were allocated into four experimental groups: control, 100 μg/mL mangiferin alone, 2 ng/mL sodium selenite alone, and combined co-treatment. Supplementation with sodium selenite alone reduced viability to 62.00%, whereas mangiferin alone demonstrated high biocompatibility (81.00% viability). The combined M+S group partially mitigated selenite-induced toxicity, restoring viability to 75.50%. Notably, co-supplementation exerted a potent combined cryoprotective action, elevating post-thaw cellular viability to 92.67% compared to 71.33% in the untreated control. Molecular and immunocytochemical profiling confirmed the enrichment of a functional undifferentiated germline population expressing stemness markers (Gfra1, Zbtb16, Vimentin) without somatic lineage markers (CD18, CD31, AE1/AE3). In conclusion, the co-supplementation of mangiferin and sodium selenite effectively counteracts in vitro hyperoxic and cryogenic oxidative insults, establishing a robust framework for germ cell cryobanking and male fertility restoration.