3D Culture of Human Ovarian Tissue on Conductive Hybrid Scaffolds with and without Electrical Stimulation

Background The survival, development, and hormonal activity of ovarian follicles depend on the ovarian tissue microenvironment, making its preservation critical for effective fertility preservation. To maintain the structural and functional integrity of human ovarian cortical tissue during in vitro culture, we developed a conductive scaffold supplemented with hormones and electrical stimulation. Methods Conductive hybrid scaffolds were fabricated from alginate (Alg), carboxymethyl cellulose (CMC), and polypyrrole (PPy) using in situ polymerization of pyrrole monomer followed by ionic cross-linking. These scaffolds supported three-dimensional culture of human ovarian cortical tissue. Three formulations (PPy to Alg-CMC ratios of 2:1, 5:1, and 10:1) were evaluated for structural stability. Human ovarian cortical fragments (1 mm × 1 mm × 500 μm) were cultured on the scaffolds for 15 days; one group received daily electrical stimulation (100 mV for 1 h), while the control group did not. Stromal and follicular responses were assessed via immunohistochemical staining for α-smooth muscle actin (α-SMA), Ki-67, and p53 to evaluate ECM remodeling, proliferation, and stress/apoptosis. Results The 2:1 PPy/Alg-CMC scaffold exhibited superior performance, with biocompatibility, conductivity, and no cytotoxicity. Under electrical stimulation, stromal, granulosa, and endothelial cells displayed strong activation, including sustained α-SMA expression (indicating a myofibroblast-like phenotype and vascular endothelial growth), elevated Ki-67 levels (indicating enhanced proliferation), and absent p53 expression (confirming no cellular stress or apoptosis). Conclusion This study demonstrates that a conductive hybrid scaffold (2:1 PPy to Alg-CMC) combined with daily electrical stimulation promotes stromal proliferation, preserves stromal integrity and tissue structure, and supports primordial follicle progression to the preantral and antral stage, suggesting effective maintenance of the follicular niche.