Impaired fertility due to compromised ovarian function affects millions of American women today. The ovarian follicle (each contains one oocyte) is the fundamental functional tissue unit of the ovary. Therefore, isolation and cryopreservation of ovarian follicles for in vitro culture to obtain healthy fertilizable oocytes have been regarded as a promising strategy for restoring and preserving female fertility. However, none of the methods used today for follicle culture recapitulates the mechanical heterogeneity experienced by both the primary and pre-antral follicles in the ovary. Using a microfluidic device, we have recently fabricated a biomimetic ovarian microtissue that consists of a more rigid alginate hydrogel shell and a softer collagen core to mimic the harder ovarian cortex and softer ovarian medulla, respectively. The follicle is partially embedded both in the core and shell, which recapitulates the mechanical heterogeneity experienced by the follicles in vivo. With this biomimetic ovarian microtissue, we revealed that the mechanical heterogeneity is crucial for developing early pre-antral follicles to the antral stage and stimulating antral follicles to ovulate and release oocytes. We hypothesize that the design of the biomimetic ovarian microtissue system can be further developed for in vitro culture of both primary and early pre-antral follicles. For follicle cryopreservation, contemporary approaches require either a highly toxic concentration (up to ~8 M) of membrane-penetrating cryoprotectants (CPAs) to vitrify (i.e., cooling to cryogenic temperature without ice formation) the follicles, or slowly freezing the follicles to form extracellular ice and dehydrate them. The latter is associated with inevitable physicochemical damage to cells due to ice formation. Our recent studies revealed that alginate hydrogel microencapsulation is exceptional in suppressing ice formation and growth, which allows vitrification of a variety of stem cells at a low CPA concentration (1.5-2 M) with high viability and intact function post cryopreservation. This low-CPA vitrification approach combines all the advantages of the conventional approaches for cell cryopreservation while avoiding their shortcomings. We hypothesize that this low-CPA vitrification approach can be developed to cryopreserve primary and early pre-antral follicles encapsulated in the biomimetic ovarian microtissue, due to the presence of an alginate hydrogel shell in the microtissue. The objective of this project is to test the aforementioned two hypotheses by developing 1), a microfluidic device with the novel and important capability of on-chip label-free selective extraction of the follicle-laden microcapsules with high efficiency (> 90%); 2), the biomimetic ovarian microtissue system with novel design to facilitate follicle development and ovulation; and 3), a new method for cryopreserving follicles in the biomimetic ovarian microtissue using the QMC-based low-CPA vitrification technology. The novel microfluidic device, innovative biomimetic ovarian microtissue system, and new low-CPA vitrification technology are invaluable for studying follicle biology, screening pharmaceutical drugs, and restoring and preserving the fertility of women.
-Relevance to Public Health We propose to develop a novel microfluidic device and biomimetic ovarian microtissue system for 3D culture of primary and early pre-antral follicles and for their cryopreservation. This project will have a significant impact on the restoration and preservation of the future fertility of professional women who may want to delay childbearing, and women who may have impaired fertility as a result of ovarian disorder that is either genetic or acquired due to exposure to environmental/occupational hazards or aggressive medical treatments such as extirpative surgery, radiation, and chemotherapy. This project will also provide a valuable in vitro model of the ovarian tissue for both studying follicle biology and screening drugs in terms of gonadotoxicity.
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