Hydrogel Based 3-Dimensional (3D) System for Toxicity and High-Throughput (HTP) Analysis for Cultured Murine Ovarian Follicles

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.

Vitamin C and sodium bicarbonate enhance the antioxidant ability of H9C2 cells and induce HSPs to relieve heat stress

“…The protective functions of drugs are generally dosedependent and time-dependent, and this holds true for vitamin C (Jaiswal et al 2017;Padayatty and Levine 2016). An important difference between drugs and toxicants is this dose-dependence (Bae et al 2008;Zhou et al 2015). The physiology of vitamin C is closely related to its dose (Levine Fig.…”

Ovarian extracellular matrix‐based hydrogel for human ovarian follicle survival in vivo: A pilot work

“…8 To circumvent this problem, previous studies with mouse follicles aimed to combine alginate with other natural polymers. [9][10][11][12] Despite their promising results, alginate-based hydrogels still lack many bioactive factors present in the ovarian extracellular matrix (ECM). 13 To develop a 3D scaffold as similar as possible to the native organ, hydrogels from decellularized ECM have been proposed.…”

Biomaterial strategies to replicate gynecological tissue

“…80 When mouse follicles were encapsulated in a fibrin alginate mixture, the hydrogels provided excellent architecture for cell attachment, proliferation, differentiation, and cell-cell signaling. 114 In vitro models of the ovary have also used alginate 58,90 or PEG 113,115 as supporting materials. These biomaterials also promote the survival, maturation, and autonomous function of follicles.…”