Ovarian follicle growth and oocyte maturation require communications between the oocyte and its surrounding somatic cells. Three-dimensional structures provide cell culture conditions that increase interactions between the cellular components when compared to 2-dimensional structures. In this study, alginate matrices were employed to maintain a 3-dimensional architecture to examine its effects on in vitro culture outcomes using mouse ovarian follicles. Ovaries were obtained from the 14-day-old C57BL/6 mice, and preantral follicles were isolated and cultured in either a 2-dimensional culture system or 0.125/0.25% alginate matrices-based 3-dimensional culture systems. We successfully observed encapsulation within 0.125 and 0.25% alginate matrices, which maintained the follicular spherical architecture, with a centrally placed oocyte and layers of granulosa cells. All culture outcomes were comparable between the 2-dimensional control and 3-dimensional alginate group. However, 0.25% alginate hydrogels led the lower rates of follicle survival and antral formation in comparison to the 2-dimensional control and 0.125% alginate. These results demonstrate that a non-permissive stiffness from the high concentration of alginate could interfere with cell-to-cell communication of oocytes and follicles. The extracellular matrix interacts with the oocyte and granulosa cells, resulting in direct effects on their proliferation. The development of a 3-dimensional culture system can enhance follicle maturation and produce meiotically-competent oocytes to provide advanced reproductive options for IVF patients in the future.