Application of a Novel Hyaluronan Hydrogel for Three-Dimensional Follicle Culture and Methodology for Mouse Ovarian Follicle Cryopreservation

The 3-D architecture of the ovarian follicle and the complex interactions between somatic cell components and the oocyte that are necessary for cytoplasmic and nuclear maturation are difficult to maintain in conventional two-dimensional (2-D) culture systems. We describe a novel 3-D culture model using a tyramine-linked hyaluronan hydrogel for encapsulation and culture of mouse ovarian follicles. The hyaluronan encapsulation technique allows 3-D growth of follicles and retention of trophic factors in close proximity to the developing follicles. This hydrogel is highly versatile and can be applied to isolated follicles as well as ovarian tissue fragments. The viscoelastic properties of the HA gel enable adjustment of rigidity as well as moldability based on gel concentration. Preantral follicles developing in this culture model are able to complete meiotic maturation within 10-12 days of culture and ovulate a metaphase II oocyte upon triggering with hCG. This paper also details two approaches to ovarian follicle cryopreservation by vitrification.