# Microfluidic encapsulation of ovarian follicles for biomimetic 3D culture andcryopreservation

> **NIH NIH R01** · UNIV OF MARYLAND, COLLEGE PARK · 2020 · $416,976

## Abstract

Project Summary/Abstract
 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. ...

## Key facts

- **NIH application ID:** 9878847
- **Project number:** 5R01EB023632-04
- **Recipient organization:** UNIV OF MARYLAND, COLLEGE PARK
- **Principal Investigator:** Xiaoming He
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $416,976
- **Award type:** 5
- **Project period:** 2017-04-01 → 2022-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9878847

## Citation

> US National Institutes of Health, RePORTER application 9878847, Microfluidic encapsulation of ovarian follicles for biomimetic 3D culture andcryopreservation (5R01EB023632-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9878847. Licensed CC0.

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