An in vitro follicle culture system could provide reproductive options to women facing infertility due to cancer treatment, as it would allow for the culture of cryopreserved ovarian tissue. The ovarian follicle is the reproductive unit of the ovary, consisting of a single centrally-located oocyte and its surrounding somatic cells. In our original culture system, the resulting follicles did not reach the size of follicles in vivo, nor do the cells differentiate similarly to those in the ovary. We therefore hypothesized that growth may be inhibited by the rigidity of the matrix, and decreasing the modulus of the alginate may allow for proper follicle development. Alginate modulus was varied using three methods: 1) gamma radiation (5 Mrad dose) was used to decrease the molecular weight of alginate, 2) sodium periodate was used to oxidize 1% of alginate monomers, thereby decreasing the molecular weight and rendering alginate susceptible to hydrolysis, and 3) the alginate percentage in the hydrogel was varied. Conditions were selected for subsequent follicle culture based upon the resulting alginate shear modulus (G). Selected alginate conditions consisted of: 1) a high modulus, high density condition (3% untreated alginate, G=2780 Pa), 2) a medium modulus, medium density condition (1.5% untreated alginate, G=1530 Pa), 3) a low modulus, low density condition (0.7% untreated alginate, G=226 Pa) 4) a low modulus, medium density condition (1.5% 50/50 blend of untreated and irradiated alginate, G=373 Pa), and 5) a chemically modified low modulus, medium density condition (1.5% oxidized alginate, G=532 Pa). Follicles were isolated at two stages of development (100-130 µm two-layered secondary follicles and 150-180 µm diameter multilayered secondary follicles) and encapsulated in alginate beads of each condition. Our findings indicate that decreased modulus of alginate results in larger follicles at the end of the culture period for both follicle stages (eg. for multilayered secondary follicles, a 15.9% size increase was observed in 3% alginate vs. a 131.5% size increase in 0.7% alginate).

In addition to increases in tissue growth, our results demonstrate that alginate rigidity, density, and chemistry regulate the types and morphology of tissue which develops in the encapsulated follicles. During follicle development in vivo, cells surrounding the follicle differentiate into theca cells and begin producing androgens. Decreased rigidity allowed for increased theca differentiation and development (eg. for two-layered secondary follicles, 21% develop a stratified theca layer in the 1.5% irradiated condition, while no follicles formed theca layers in unmodified alginate). In addition, a fluid-filled cavity (antrum) forms prior to ovulation from the ovary. Our findings indicate that decreasing the modulus of the matrix surrounding the follicle allows for increased incidence of antrum formation, and oxidation of alginate allows for maximal antrum formation (37.5% of follicles, vs 13.3% of follicles in untreated alginate). Together, these results demonstrate that the rigidity, density, and chemistry of the encapsulating matrix affect follicle growth as well as tissue differentiation, and are important considerations in developing an optimized culture environment.

Supported by NIH U54HD41857-01A1