Production and Use of Customizable Agarose Molds for Scaffold-Free Mouse Ovarian Follicle Culture

Hannes Campo; Prianka H. Hashim; Emily J. Zaniker-Gomez; Samuel Gauthier; Zihang Yan; Hao F. Zhang; James A. Ankrum; Francesca E. Duncan

doi:10.3791/68871

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Method Article

Production and Use of Customizable Agarose Molds for Scaffold-Free Mouse Ovarian Follicle Culture

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DOI:

10.3791/68871

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October 24th, 2025

Hannes Campo^*¹ , Prianka H. Hashim^*¹ , Emily J. Zaniker-Gomez¹ , Samuel Gauthier¹ , Zihang Yan² , Hao F. Zhang² , James A. Ankrum³ , Francesca E. Duncan¹

¹Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, ²Department of Biomedical Engineering, Northwestern University, ³Roy J. Carver Department of Biomedical Engineering, Pappajohn Biomedical Institute, University of Iowa

^* These authors contributed equally

Summary

We describe methods to culture ovarian follicles in a novel scaffold-free agarose mold that complement in vitro gametogenesis.

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.

Introduction

The oocyte, or female gamete, contributes the bulk of the cytoplasm and half of the genetic material to the embryo at fertilization, which is essential for the development of the next generation. Oogenesis starts during embryonic development when primordial germ cells enter meiosis and arrest in prophase I. These oocytes reside within primordial follicles and constitute the ovarian reserve, a finite and nonrenewable pool from which they are progressively recruited. Follicles develop in a process known as folliculogenesis, with the goal of ultimately producing a mature gamete capable of fertilization¹^,²^,