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Chitosan Hydrogel Supports Integrity of Ovarian Follicles during In Vitro Culture: A Preliminary of A Novel Biomaterial for Three Dimensional Culture of Ovarian Follicles.

ABSTRACT: Objective:Testing novel biomaterials for the three dimensional (3D) culture of ovarian follicles may ultimately lead to a culture model which can support the integrity of follicles during in vitro culture (IVC). The present study reports the first application of a chitosan (CS) hydrogel in culturing mouse preantral follicles. Materials and Methods:In this interventional experiment study, CS hydrogels with the concentrations of 0.5, 1, and 1.5% were first tested for fourier transform infrared spectroscopy (FT-IR), Compressive Strength, viscosity, degradation, swelling ratio, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity and live/dead assay. Thereafter, mouse ovarian follicles were encapsulated in optimum concentration of CS (1%) and compared with those in alginate hydrogel. The follicular morphology, quality of matured oocyte and steroid secretion in both CS and alginate were assessed by enzyme-linked immunosorbent assay (ELISA). The expression of folliculogenesis, endocrine, and apoptotic related genes was also evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and compared with day that in 0. Results:The rates of survival, and diameter of the follicles, secretion of estradiol, normal appearance of meiotic spindle and chromosome alignment were all higher in CS group compared with those in alginate group (P?0.05). The expression of Cyp19a1 and Lhcgr in CS group was significantly higher than that of the alginate group (P?0.05). Conclusion:The results showed that CS is a permissive hydrogel and has a beneficial effect on encapsulation of ovarian follicle and its further development during 3D culture.

SUBMITTER: Hassani F

PROVIDER: S-EPMC6722450 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Chitosan Hydrogel Supports Integrity of Ovarian Follicles during In Vitro Culture: A Preliminary of A Novel Biomaterial for Three Dimensional Culture of Ovarian Follicles.

Hassani Fatemeh F Ebrahimi Bita B Moini Ashraf A Ghiaseddin Ali A Bazrafkan Mahshid M Hassanzadeh G Holamreza GH Valojerdi Mojtaba Rezazadeh MR

Cell journal 20190729 4

<h4>Objective</h4>Testing novel biomaterials for the three dimensional (3D) culture of ovarian follicles may ultimately lead to a culture model which can support the integrity of follicles during <i>in vitro</i> culture (IVC). The present study reports the first application of a chitosan (CS) hydrogel in culturing mouse preantral follicles.<h4>Materials and methods</h4>In this interventional experiment study, CS hydrogels with the concentrations of 0.5, 1, and 1.5% were first tested for fourier ...[more]

PMID: 31376330

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Project description: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.

Dataset Information

Chitosan Hydrogel Supports Integrity of Ovarian Follicles during In Vitro Culture: A Preliminary of A Novel Biomaterial for Three Dimensional Culture of Ovarian Follicles.

Publications

Chitosan Hydrogel Supports Integrity of Ovarian Follicles during In Vitro Culture: A Preliminary of A Novel Biomaterial for Three Dimensional Culture of Ovarian Follicles.

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