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 recapitulate the mechanical heterogeneity experienced by both the primary and pre-antral follicles in the ovary. Using a non-planar 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 ovulation to release oocytes. We hypothesize that 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 used to cryopreserve the primary and early pre-antral follicles encapsulated in the biomimetic 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 with three specific aims: 1), to develop a computational model for understanding the complex multi-phase flow occurred during microfluidic encapsulation of follicles in the biomimetic ovarian microtissue; 2), to microencapsulate both primary and early pre-antral follicles for biomimetic 3D culture in vitro; and 3), to cryopreserve primary and early pre-antral follicles encapsulated in the biomimetic ovarian microtissue by low-CPA vitrification. The novel non-planar microfluidic device, biomimetic ovarian microtissue system, and low-CPA vitrification technology are valuable for studying follicle biology, screening pharmaceutical drugs, and restoring/preserving the fertility of women.

Public Health Relevance

-Relevance to Public Health We propose to develop a novel non-planar microfluidic device and biomimetic ovarian microtissue system for miniaturized 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 studying follicle biology and for drug screening in terms of gonadotoxicity.

National Institute of Health (NIH)
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Rampulla, David
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University of Maryland College Park
Biomedical Engineering
Biomed Engr/Col Engr/Engr Sta
College Park
United States
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Liu, Xiaoli; Zhao, Gang; Chen, Zhongrong et al. (2018) Dual Suppression Effect of Magnetic Induction Heating and Microencapsulation on Ice Crystallization Enables Low-Cryoprotectant Vitrification of Stem Cell-Alginate Hydrogel Constructs. ACS Appl Mater Interfaces 10:16822-16835
Sun, Mingrui; Durkin, Patrick; Li, Jianrong et al. (2018) Label-Free On-Chip Selective Extraction of Cell-Aggregate-Laden Microcapsules from Oil into Aqueous Solution with Optical Sensor and Dielectrophoresis. ACS Sens 3:410-417
Stewart, Samantha; He, Xiaoming (2018) Intracellular Delivery of Trehalose for Cell Banking. Langmuir :
He, Xiaoming (2017) Microscale Biomaterials with Bioinspired Complexity of Early Embryo Development and in the Ovary for Tissue Engineering and Regenerative Medicine. ACS Biomater Sci Eng 3:2692-2701
Zhao, Gang; Zhang, Zhiguo; Zhang, Yuntian et al. (2017) A microfluidic perfusion approach for on-chip characterization of the transport properties of human oocytes. Lab Chip 17:1297-1305
Huang, Haishui; Zhao, Gang; Zhang, Yuntian et al. (2017) Predehydration and Ice Seeding in the Presence of Trehalose Enable Cell Cryopreservation. ACS Biomater Sci Eng 3:1758-1768
Huang, Haishui; Yu, Yin; Hu, Yong et al. (2017) Generation and manipulation of hydrogel microcapsules by droplet-based microfluidics for mammalian cell culture. Lab Chip 17:1913-1932
He, Xiaoming (2017) Microfluidic Encapsulation of Ovarian Follicles for 3D Culture. Ann Biomed Eng 45:1676-1684
Wang, Hai; Agarwal, Pranay; Xiao, Yichao et al. (2017) A Nano-In-Micro System for Enhanced Stem Cell Therapy of Ischemic Diseases. ACS Cent Sci 3:875-885
He, Xiaoming; Toth, Thomas L (2017) In vitro culture of ovarian follicles from Peromyscus. Semin Cell Dev Biol 61:140-149

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