A Three-Dimensional Gelatin Methacryloyl (GelMA) Hydrogel Platform for In Vitro Bovine Ovarian Tissue Culture and Primordial Follicle Activation

Conventional two-dimensional (2D) culture systems fail to preserve the three-dimensional (3D) follicular architecture, permitting granulosa cell migration away from the oocyte and disrupting paracrine signaling essential for folliculogenesis. Hydrogel-based 3D culture platforms have emerged as a promising alternative for maintaining tissue integrity during extended in vitro culture. This protocol describes a 3D ovarian tissue culture method using 4% (w/v) gelatin methacryloyl (GelMA) hydrogel scaffolds with 0.1% (w/v) lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as the photoinitiator. Bovine ovarian cortical fragments (1 mm × 1 mm × 1 mm) embedded in photocrosslinked GelMA scaffolds are cultured for 6 days under standard conditions (38.5 °C, 5% CO₂). Compared with conventional 2D culture and Matrigel-based systems, this method enhances primordial follicle activation and progression to the secondary stage, as confirmed by follicle stage distribution analysis and diameter measurements. Key outcome measures include baseline follicle viability assessed by neutral red staining prior to culture, histological analysis with hematoxylin and eosin (H&E) staining, scanning electron microscopy (SEM) of scaffold architecture, and quantitative assessment of follicle diameter and developmental stage distribution. The protocol is reproducible, relies on commercially available materials, and provides a physiologically relevant platform for investigating primordial follicle activation and early folliculogenesis in large animal models.