This research aimed to compare the In Vitro growth, maturation, and gene expression in ovarian follicles collected from adult mice (6–8-week-old) between two-dimensional and three-dimensional cultures. First, we confirmed In Vitro follicle growth and maturation using
adult mice with outbred characteristics and analyzed the expression of genes related to follicular development. We found that the three-dimensional culture system utilizing a Matrigel drop to create an in vivo-like ovarian microenvironment was more efficient in terms of In Vitro
follicle growth, maturation, and gene expression than the two-dimensional system (non-physical environment). The in vivo-like three-dimensional culture of ovarian follicles provides new insights into the physiology and development of ovarian follicle in vivo, thereby contributing
to new strategies to improve female fertility.