RGD-modified dextran hydrogel promotes follicle growth in three-dimensional ovarian tissue culture in mice.

In vitro follicle growth is a promising technology to preserve fertility for cancer patients. We previously developed a three-dimensional (3-D) ovarian tissue culture system supported by mouse tumor cell-derived Matrigel. When murine ovarian tissues at 14 days old were cultured in Matrigel drops, antrum formation and oocyte competence were significantly enhanced compared with those cultured without Matrigel. In this study, we tested whether nonanimal-derived dextran hydrogels can support a 3-D ovarian tissue culture. We employed chemically defined dextran hydrogels consisting of dextran polymers crosslinked with polyethylene glycol (PEG)-based cell-degradable crosslinker. To determine the optimal gel elasticity for the 3-D tissue culture, we measured Young's modulus of dextran hydrogels at four concentrations (1.75, 2.25, 2.75, and 3.25 mmol/L), and cultured ovarian tissues in these gels for 7 days. As a result, 2.25 mmol/L dextran hydrogel with Young's modulus of 224 Pa was appropriate to provide physical support as well as to promote follicle expansion in the 3-D system. To mimic the natural extracellular matrix (ECM) environment, we modified the dextran hydrogels with two bioactive factors: ECM-derived Arg-Gly-Asp (RGD) peptides as a cell-adhesive factor, and activin A. The ovarian tissues were cultured in 2.25 mmol/L dextran hydrogels under four different conditions: Activin-/RGD- (A-R-), A + R-, A-R+, and A + R+. On Day 7 of culture, follicle and oocyte sizes were significantly increased in the RGD-modified conditions compared with those without RGD. The RGD-modified hydrogels also promoted mRNA levels of steroidogenic-related genes and estradiol production in the 3-D ovarian tissue culture. In vitro maturation and developmental competence of follicular oocytes were remarkably improved in the presence of RGD. In particular, blastocyst embryos were obtained only from A-R+ or A+R+ conditions after in vitro fertilization. We also determined synergistic effects of the RGD peptides and activin A on follicle growth and oocyte development in the 3-D tissue culture. In conclusion, our results suggest that RGD-modified dextran hydrogels provide an ECM-mimetic bioactive environment to support folliculogenesis in a 3-D ovarian tissue culture system.