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Updated: Apr 1, 2026
Application of a Novel Hyaluronan Hydrogel for Three-Dimensional Follicle Culture and Methodology for Mouse Ovarian Follicle Cryopreservation
Published on: May 9, 2025
Hydrogel Based 3-Dimensional (3D) System for Toxicity and High-Throughput (HTP) Analysis for Cultured Murine Ovarian
Hong Zhou1, Malika Amattullah Malik2, Aarthi Arab1
1Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, 48109, United States of America.
Abstract:
Various toxicants, drugs and their metabolites carry potential ovarian toxicity. Ovarian follicles, the functional unit of the ovary, are susceptible to this type of damage at all stages of their development. However, despite of the large scale of potential negative impacts, assays that study ovarian toxicity are limited. Exposure of cultured ovarian follicles to toxicants of interest served as an important tool for evaluation of toxic effects for decades. Mouse follicles cultured on the bottom of a culture dish continue to serve an important approach for mechanistic studies. In this paper, we demonstrated the usefulness of a hydrogel based 3-dimensional (3D) mouse ovarian follicle culture as a tool to study ovarian toxicity in a different setup. The 3D in vitro culture, based on fibrin alginate interpenetrating network (FA-IPN), preserves the architecture of the ovarian follicle and physiological structure-function relationship. We applied the novel 3D high-throughput (HTP) in vitro ovarian follicle culture system to study the ovotoxic effects of an anti-cancer drug, Doxorobucin (DXR). The fibrin component in the system is degraded by plasmin and appears as a clear circle around the encapsulated follicle. The degradation area of the follicle is strongly correlated with follicle survival and growth. To analyze fibrin degradation in a high throughput manner, we created a custom MATLAB® code that converts brightfield micrographs of follicles encapsulated in FA-IPN to binary images, followed by image analysis. We did not observe any significant difference between manually processed images to the automated MATLAB® method, thereby confirming that the automated program is suitable to measure fibrin degradation to evaluate follicle health. The cultured follicles were treated with DXR at concentrations ranging from 0.005 nM to 200 nM, corresponding to the therapeutic plasma levels of DXR in patients. Follicles treated with DXR demonstrated decreased survival rate in greater DXR concentrations. We observed partial follicle survival of 35% ± 3% (n = 80) in 0.01nM treatment and 48% ± 2% (n = 92) in 0.005nM, which we identified as the IC50 for secondary follicles. In summary, we established a 3D in vitro ovarian follicle culture system that could be used in an HTP approach to measure toxic effects on ovarian follicles.
Insights
A novel 3D ovarian follicle culture system using fibrin alginate interpenetrating network (FA-IPN) effectively assesses drug-induced ovarian toxicity. This high-throughput method accurately measures toxic effects, aiding in the evaluation of potential ovotoxicants.
Area of Science:
- Reproductive toxicology
- Biomaterials science
- High-throughput screening
Background:
- Ovarian toxicity from various toxicants and drugs poses a significant risk, yet assays for its study are limited.
- Current methods often rely on 2D cultures, which may not fully preserve ovarian follicle architecture and function.
- There is a need for advanced in vitro models to accurately assess ovarian toxicity.
Purpose of the Study:
- To develop and validate a novel 3-dimensional (3D) in vitro ovarian follicle culture system for high-throughput (HTP) toxicity assessment.
- To evaluate the utility of this 3D culture system in studying the ovotoxic effects of Doxorubicin (DXR).
Main Methods:
- A fibrin alginate interpenetrating network (FA-IPN) hydrogel was used to encapsulate mouse ovarian follicles, preserving their 3D structure.
- The system utilizes fibrin degradation as a biomarker for follicle health, quantified using automated image analysis with custom MATLAB® code.
- Cultured follicles were exposed to varying concentrations of Doxorubicin (DXR) to assess ovotoxicity.
Main Results:
- The 3D FA-IPN culture system successfully maintained follicle architecture and physiological structure-function relationships.
- Automated image analysis of fibrin degradation showed high correlation with follicle survival and growth, validating the method.
- DXR treatment resulted in decreased follicle survival rates, with an identified IC50 for secondary follicles at specific concentrations.
Conclusions:
- The established 3D HTP in vitro ovarian follicle culture system is a valuable tool for evaluating ovarian toxicity.
- This model offers a more physiologically relevant approach compared to traditional 2D methods for assessing ovotoxic effects of drugs and toxicants.
