Construction and Performance Evaluation of an Astaxanthin–Chitosan/Chitooligosaccharide Hydrogel System for Ex Vivo Culture of Murine Spermatogonial Stem Cells

Spermatogonial stem cells (SSCs) are the only adult male germline stem cells capable of lifelong self-renewal and differentiation into spermatozoa. Scalable ex vivo survival is essential for endangered species germplasm banking, genetic resource conservation, and male infertility therapy. Here, chitosan (CO) or chitosan oligosaccharide (COS) was cross-linked into injectable, biodegradable 3D hydrogels loaded with the natural astaxanthin (AST). CCK-8 optimization identified 0.3% CO + 0.2% AST (CHAG) and 0.2% COS + 0.2% AST (COAG) as superior formulations. After 7 or 14 d of 3D culture, CHAG yielded significantly more colonies than controls (<i>p</i> < 0.01), with elevated EdU incorporation, alkaline phosphatase activity, and positive OCT4 and PLZF staining, confirming preserved stemness. Caspase-3 expression was markedly reduced, indicating the AST-mediated suppression of oxidative apoptosis. RNA-seq showed distinct transcriptome pathways (<i>p</i> < 0.01): CHAG up-regulated adhesion and ECM-receptor and cell cycle pathways, whereas COAG enriched immune-modulatory and signaling modules, enabling context-specific use. AST-loaded CO/COS hydrogels are inexpensive, cytocompatible, and scalable, doubling as a biomimetic niche that accelerates SSC proliferation while delaying senescence. The platform provides a robust, controllable 3D system for SSC expansion and establishes a pre-clinical basis for translating CO/COS/AST composites to reproductive stem cell biotechnology.