Journal article
Production and Use of Customizable Agarose Molds for Scaffold-Free Mouse Ovarian Follicle Culture
Journal of visualized experiments, Vol.224(224), e68871
10/24/2025
DOI: 10.3791/68871
PMCID: PMC12688028
PMID: 41212809
Abstract
The ovarian follicle is the functional unit of the ovary that produces hormones and gametes needed to sustain female reproductive function and health. The ability to recapitulate folliculogenesis, ovulation, and luteinization in vitro has broad basic, translational, and clinical utility. The most advanced in vitro follicle growth systems maintain the follicle's three-dimensional (3D) architecture, which is crucial for the development of meiotically competent metaphase II oocytes in humans. Recently, a scaffold-free method for in vitro follicle growth of mouse multilayer secondary follicles was developed and validated. For this, custom 3D printed molds were used to micropattern agarose with microwells that accommodate the volumetric expansion of follicles. Follicles grown in this scaffold-free environment showed comparable hormone production and viability relative to well-established alginate-based encapsulated in vitro follicle growth (eIVFG) systems. Importantly, agarose microwells are a scalable method, less technically demanding, and show improved follicle growth and ovulation rates relative to eIVFG. This methodology produces customizable molds that are biocompatible with the oocyte, a cell highly sensitive to material-specific leachates and other environmental contaminants. Further, follicles in this system are cultured in the same focal plane, enabling real-time timelapse imaging and analysis. To increase the accessibility of this new approach, this article details the methods needed to design and 3D-print master molds, create silicone molds for 24- or 96-well plates, and culture isolated multilayer secondary ovarian follicles in the agarose molds. This setup can also be integrated with a cost-effective time-lapse imaging system, enabling morphokinetic analysis. In addition, molds can be paraffin-embedded for downstream histological analyses. Overall, this user-friendly method is a versatile tool for follicle culture and can be customized further to promote the differentiation and maturation of germ cells within the context of the follicle to sustain complete in vitro gametogenesis.
Details
- Title: Subtitle
- Production and Use of Customizable Agarose Molds for Scaffold-Free Mouse Ovarian Follicle Culture
- Creators
- Hannes CampoPrianka H. HashimEmily J. Zaniker-GomezSamuel GauthierZihang YanHao F ZhangJames A. AnkrumFrancesca E. Duncan
- Resource Type
- Journal article
- Publication Details
- Journal of visualized experiments, Vol.224(224), e68871
- DOI
- 10.3791/68871
- PMID
- 41212809
- PMCID
- PMC12688028
- NLM abbreviation
- J Vis Exp
- ISSN
- 1940-087X
- eISSN
- 1940-087X
- Publisher
- JOURNAL OF VISUALIZED EXPERIMENTS; CAMBRIDGE
- Grant note
- National Institutes of Child Health and Human Development: R01HD105752, T32HD094699 NIH Common Fund's SenNet program: U54AG075932, UH3CA268105 Bill & Melinda Gates Foundation: INV-003385
This work was funded by the National Institutes of Child Health and Human Development (R01HD105752 to F.E.D and T32HD094699 to E.J.Z) and the NIH Common Fund's SenNet program (U54AG075932 and UH3CA268105 to F.E.D.) and the Bill & Melinda Gates Foundation Grant (INV-003385 to F.E.D.) . Under the grant conditions of the Bill & Melinda Gates Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission.
- Language
- English
- Date published
- 10/24/2025
- Academic Unit
- Roy J. Carver Department of Biomedical Engineering; Fraternal Order of Eagles Diabetes Research Center
- Record Identifier
- 9985019150302771
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