Optimized culture system to maximize ovarian cell growth and functionality in vitro.

Ovaries are the primary physiological source of female sex hormones, which play a crucial role in maintaining ovarian cycle, determining secondary sexual characteristics and preparing the endometrium for implantation. In vitro follicle engineering has been used to investigate follicle development, including ovarian hormone production and gamete maturation. To engineer functional follicles, culture and expansion of the primary ovarian cells are essential. However, the phenotypic and functional characteristics of primary ovarian cells are often lost during culture. The objective of this study is to develop an optimized culture system for maintaining ovarian cell growth and functionality. Granulosa cells (GCs) and theca cells (TCs) were isolated from female rats. The addition of follicle-stimulating hormone (FSH) or luteinizing hormone (LH) to the basal culture media significantly enhanced the secretion of estradiol from GCs and androstenedione from TCs. Serum concentrations of 5% and 10% had a similar role in promoting ovarian cell expansion and secretion of estradiol and androstenedione hormones from both types of cells. Growth differentiation factor 9 (GDF9), bone morphogenic protein 15 (BMP15), BMP7 and basic fibroblast growth factor (bFGF) enhanced GC proliferation and estradiol production, respectively. Among them, the effect of bFGF was most significant. bFGF also enhanced TC proliferation. When GCs and TCs were cultured in 5% serum, gonadotropin and bFGF-containing medium, they proliferated exponentially throughout the culture period of up to 40 days while maintaining their functional characteristics. Taken together, these results indicate that our medium formula is optimal for maximizing proliferation of functionally differentiated ovarian cells.