A Novel Three-Dimensional Follicle Culture System Decreases Oxidative Stress and Promotes the Prolonged Culture of Human Granulosa Cells

Abstract

Tissue engineering advancements have made it possible to modify biomaterials to reconstruct a similar threedimensional structure of the extracellular matrix (ECM) for follicle development and to supply the required biological signals. We postulated that an artificial polysaccharide hydrogel modified with an ECM mimetic peptide may produce efficient irritation signals by binding to specific integrins providing a suitable environment for follicular development and influencing the behavior of human granulosa cells (hGCs). Laminin, an important component of the extracellular matrix, can modulate hGCs and oocyte growth. Specifically, follicles of mice were randomly divided into two-dimensional (2D) and three-dimensional (3D) culture systems established by a hydrogel modified with RGD or laminin mimetic peptides (IKVAV and YIGSR) and RGD (IYR). Our results showed that 3D cultured systems significantly improved follicle survival, growth, and viability. IYR peptides enhanced the oocyte meiosis competence. Additionally, we explored the effect of 3D culture on hGCs, which improved hGCs viability, increased the proportion of S-and G2/M-phase cells, and inhibited cell apoptosis of hGCs. On days 1 and 2, the secretion of progesterone was reduced in 3D-cultured hGCs. Notably, 3D-cultured hGCs exhibited delayed senescence, decreased oxidative stress, and elevated mitochondrial membrane potential. Moreover, the expression levels of cumulus expansion-related genes (COX2, HAS2, and PTX3) and integrin α6β1 were upregulated in 3D-cultured hGCs. In conclusion, a 3D culture utilizing hydrogels modified with Lamininmimetic peptides can provide a durable physical environment suitable for follicular development. The laminin-mimetic peptides may regulate the biological activity of hGCs by attaching to the integrin α6β1.

3D culture applied to reproduction in females: possibilities and perspectives

“…Natural components from the biological matrix can be also added to alginate hydrogel to improve the 3D environment ( Xu et al, 2013 ). Fibrin can be added to allow cellular proteases to degrade the matrix and create a dynamic mechanical environment throughout the culture, ( Jones and Shikanov, 2019 ; Zhao et al, 2023 ). Collagen and laminin, which are naturally present in the uterine extracellular matrix, can be added because of their elasticity that mimics the uterine environment, allowing the construction of very soft gels similar to the uterine tissue ( Lopez-Garcia et al, 2010 ).…”