Name: Production and Use of Customizable Agarose Molds for Scaffold-Free Mouse Ovarian Follicle Culture (Video) | JoVE Journal
Uploaded: 2025-10-24T13:10:00.000+00:00
Duration: 9 min 50 s
Description: Learn scaffold-free follicle culture using customizable agarose molds for mouse ovarian follicles. This method improves growth, ovulation rates, and hormone production while enabling time-lapse imaging.

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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&#39;s SenNet program (U54AG075932 and UH3CA268105 to F.E.D.) and the Bill &#38; Melinda Gates Foundation Grant (INV-003385 to F.E.D.). Under the grant conditions of the Bill &#38; 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.

The authors have no conflicts of interest to disclose.

In vitro gametogenesis and maturation are key technologies for fundamental understanding of oocyte biology, with potential applications in assisted reproductive technology, fertility preservation, and species conservation. The methods to support IVG often rely on co-culture with somatic cells to create reconstructed ovaries30. The importance of these support cells is well-known in vivo, where the follicular somatic microenvironment is critical to healthy oocyte growth and develop...

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 fertilization1,2,3,4,5. Follicle development begins with an early, gonadotropin-independent phase where paracrine signaling is essential for the survival and growth of the follicle. As follicles mature to the secondary stage, they become reliant on gonadotropins, marking a shift to endocrine signaling. A key characteristic of secondary follicles is an oocyte surrounded by a minimum of two complete layers of cuboidal granulosa cells, along with the newly developed theca layer. Theca cells form the follicle&#39;s outermost layer, providing androgens and structural support6,7. Finally, a fluid-filled antral cavity forms and ruptures in response to a luteinizing hormone (LH) surge during ovulation, releasing a fertilization-competent gamete arrested at Meiosis II2,8,9.
The oocyte achieves developmental competence for fertilization and the ability to support early preimplantation embryo development, referred to as nuclear and cytoplasmic maturation, during oogenesis and folliculogenesis10,11,12. Cytoplasmic maturation is apparent morphologically, as oocytes expand throughout folliculogenesis. As a result of somatic cell proliferation, including theca, granulosa, and cumulus cells, follicles themselves experience significant volumetric expansion. In mice, the diameter of the follicle increases fivefold from early secondary to pre-ovulatory follicles. Bi-directional communication between the oocyte and surrounding somatic cells is essential for oogenesis. In primary follicles, the zona pellucida forms from proteins secreted by the oocyte, and communication between these follicle compartments is partly mediated by transzonal projections, which provide physical contact between these two follicle compartments13,14. Hence, the follicular microenvironment is vital to correctly orchestrate the growth as well as nuclear and cytoplasmic maturation of the oocyte2,5. To better understand the discrete mechanisms that support these processes, there is a need for advanced and physiologically relevant in vitro culture techniques. The ability to culture follicles in vitro has broad applications, including assisted reproductive technologies and fertility preservation15,16,17,18, species conservation19,20, drug development21, and toxicology screening22,23.
In vitro follicle culture systems are also an essential intermediary step for the ability to generate gametes from pluripotent stem cells, also known as in vitro gametogenesis (IVG)24. Researchers have utilized embryonic stem cells, mesenchymal stem cells, and induced pluripotent stem cells to develop gametes in a variety of species, often relying on somatic cells to recreate the native follicle microenvironment25,26,27,28,29,30. While major proof-of-concept milestones have been achieved through these methods, there is still a challenge of low-quality oocytes, reflected in low embryo development and live offspring rates following in vitro fertilization in mice31,32. No live births have been achieved yet in other species, underscoring the need for continued refinement of protocols to fully support oogenesis and folliculogenesis.
Currently, several strategies are utilized for follicle culture. The technically least demanding and highest throughput approaches are scaffold-free methods, where follicles are placed in a plastic dish or membrane insert33,34. Here, granulosa cells adhere to the culture surface, losing contact with the oocyte33,34. The breakdown of the follicular architecture limits the usability and biological relevance of these techniques. However, scaffold-free culture in low attachment plates or inverted drops allows the follicle to maintain structural integrity in several mammalian species35,36,37. Unfortunately, with these techniques, it is difficult to control the interaction between follicles and the amount of mechanical support they receive. Hence, to better recapitulate in vivo physiology, culture methods have been developed where follicles are encapsulated in hydrogels with defined, tunable chemical compositions and/or mechanical properties33,34,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53. For example, the alginate-encapsulated in vitro follicle growth system can recapitulate key transcriptomic signatures observed in vivo21.
Despite the success and value of encapsulated culture protocols, there are several disadvantages. They can be technically challenging, laborious, low-throughput, and are not compatible with automatic imaging methods. Further, encapsulated culture methods need multi-step approaches for larger mammalian species due to the higher volumetric expansion of the follicle37. Moreover, encapsulated techniques exert uniform support in all directions, and follicles need to be released from the hydrogel prior to ovulation. This does not occur in vivo, where the follicle interacts with the surrounding ovarian microenvironment, which provides dynamic biomechanical support54. For example, the stiff ovarian cortex maintains quiescence of primordial follicles, while large growing follicles are typically found in the softer medullary compartment55,56. Once a follicle with a competent oocyte has reached its terminal size, follicle wall and ovarian surface epithelium modifications allow for ovulation. Therefore, culture techniques where follicles are allowed to grow in one single direction with little resistance could better mimic in vivo physiology.
In a novel approach, a reproducible, scalable, technically less demanding, and cost-effective culture method for mouse multilayer secondary follicles was developed57. Here, ultra-low adhesion agarose microwells with precisely controllable geometry (shape, dimensions) and spatial arrangement (number, proximity) are manufactured using affordable methods. Follicles in the agarose molds experience mechanical support in all but one direction and are cultured in the same focal plane, enabling timelapse imaging for advanced morphokinetic analysis. Stereolithography (SLA) 3D printing was utilized as an approachable method to manufacture a master mold, followed by molding steps that create leachate-free silicone molds to cast the agarose. Improved follicle growth and ovulation outcomes were achieved using the new system compared to traditional eIVFG, without compromising overall follicle survival, maturation, or luteinization57. This protocol paper describes the steps necessary to customize and manufacture agarose molds, how to seed and culture multilayer secondary follicles, process, and finally perform timelapse analysis (Figure 1).

<table><tbody><tr><td>&lt;strong&gt;Materials and Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;50 mL Polystyrene Centrifuge Tubes</td><td>Globe Scientific</td><td>941-11017-CS</td><td></td></tr><tr><td>1.5 mL Microcentrifuge Tubes</td><td>Fisher Scientific</td><td>MCT-150</td><td>To snap freeze and store media</td></tr><tr><td>100 mm Dish, Non-Treated</td><td>Fisher Scientific</td><td>08-757-100D&amp;nbsp;</td><td>To make and sstore agarose micromolds</td></tr><tr><td>100% Ethanol</td><td>Mercedes Scientific</td><td>1200</td><td>Tissue Processing</td></tr><tr><td>1000 &amp;micro;L&amp;nbsp; Pipette Tips, Low Retention</td><td>MIDSCI</td><td>PR-1000BK</td><td>Culture tips&amp;nbsp;</td></tr><tr><td>200 &amp;micro;L&amp;nbsp; Pipette Tips, Low Retention</td><td>MIDSCI</td><td>PR-200BK</td><td>Culture tips&amp;nbsp;</td></tr><tr><td>24-well plate</td><td>Corning</td><td>353047</td><td>Culture</td></tr><tr><td>35 mm dishes</td><td>Falcon</td><td>351008</td><td>To isolate ovaries and incubate in enyzmatic media</td></tr><tr><td>95% Ethanol</td><td>Mercedes Scientific</td><td>1210</td><td>Tissue Processing</td></tr><tr><td>Agarose</td><td>Hoefer</td><td>GR140</td><td>Micromolds</td></tr><tr><td>BioXtra Mineral Oil, Light Oil, Suitable for mouse embryo cell culture</td><td>Sigma-Aldrich</td><td>M5310-500ML</td><td>Used as a biocompatible demolding agent</td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;</td><td>MP Biomedicals</td><td>103700</td><td>Growth Media</td></tr><tr><td>Cleaning Dusters, 10 Oz., Pack Of 6</td><td>Office Depot&amp;nbsp;</td><td>110284</td><td>Compressed air</td></tr><tr><td>Clear V4 Resin 1 L</td><td>Formlabs, Inc</td><td>RS-F2-GPCL-04</td><td>3D printing resin</td></tr><tr><td>Corning Reusable Plastic Low Form 100 mL Beaker, Polypropylene, Graduated</td><td>Corning</td><td>1000P-100</td><td>Beaker for mixing silicone components&amp;nbsp;</td></tr><tr><td>DNAse I</td><td>Qiagen</td><td>79254</td><td>Enzymatic Media</td></tr><tr><td>Ecoflex 00-45 Near Clear</td><td>Smooth-On, Inc</td><td>B09M8Y9PTV</td><td>Silicone material</td></tr><tr><td>F-12 + GlutaMAX</td><td>Gibco</td><td>31765-035</td><td>Growth Media</td></tr><tr><td>Fetal Bovine Fetuin</td><td>Sigma</td><td>F-3385</td><td>Growth Media</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Peak Serum</td><td>PS-FB2</td><td>Dissection Media, Enzymatic Media,&amp;nbsp; Maintenance Media &amp;amp; Maturation Media</td></tr><tr><td>Fisherbrand&amp;nbsp;Sterilization Pouches</td><td>Fisher Scientific</td><td>01-812-50&amp;nbsp;</td><td>Sterilization Pouches</td></tr><tr><td>Follicle stimulating hormone (FSH)</td><td>EMD Serono, Inc.</td><td>Gonal-F Rx only&amp;nbsp;</td><td>Growth Media &amp;amp; Maturation Media</td></tr><tr><td>Gibco&amp;nbsp;DPBS, calcium, magnesium</td><td>Fisher Scientific</td><td>14-040-182&amp;nbsp;</td><td>Dulbecco&#039;s phosphate-buffered saline</td></tr><tr><td>Human chorionic gonadotropin (hCG)</td><td>Sigma Aldrich</td><td>C1063-1VL</td><td>Maturation Media</td></tr><tr><td>Insulin-transferrin-selenium (ITS)</td><td>ThermoFisher</td><td>41400045</td><td>Growth Media</td></tr><tr><td>Invitrogen Molecular Probes Rhodamine Phalloidin</td><td>Fisher Scientific</td><td>R415</td><td>Egg Cytoskeleton Stain</td></tr><tr><td>IVF Dishes</td><td>Thermo Scientific</td><td>150260</td><td>To isolate, select for, and pre-equilibrate follicles</td></tr><tr><td>Leibovit&#039;z 15 (L15)</td><td>Gibco</td><td>11415-064</td><td>Dissection Media &amp;amp; Enzymatic Media</td></tr><tr><td>Liberase</td><td>Sigma Aldrich</td><td>5401119001</td><td>Enzymatic Media</td></tr><tr><td>MEM? + GlutaMAX</td><td>Gibco</td><td>32561-037</td><td>Maintenance Media,&amp;nbsp; Growth Media &amp;amp; Maturation Media</td></tr><tr><td>Mouse epidermal growth factor (EGF)</td><td>BD Biosciences</td><td>354010</td><td>Maturation Media</td></tr><tr><td>Nalgene Transparent Polycarbonate Classic Design Desiccator</td><td>Thermo Scientific</td><td>5311-0250</td><td>Vacuum Desiccator&amp;nbsp;</td></tr><tr><td>Paraffin</td><td>Fisher Scientific</td><td>83-30</td><td>Tissue Processing &amp;amp; Embedding</td></tr><tr><td>Penicillin-streptomycin</td><td>Gibco</td><td>15140-122</td><td>Dissection Media, Enzymatic Media &amp;amp; Maintenance Media</td></tr><tr><td>Stripper tips</td><td>Origio</td><td>MXL3-200</td><td>Culture</td></tr><tr><td>VECTASHIELD PLUS Antifade Mounting Medium with DAPI</td><td>Vector Laboratories</td><td>H-2000</td><td>Egg nucleus stain</td></tr><tr><td>Xylene</td><td>Mercedes Scientific</td><td>9840</td><td>Tissue Processing</td></tr><tr><td>&amp;alpha;-Tubulin (11H10) Rabbit mAb (Alexa Fluor 488 Conjugate)</td><td>Cell Signaling Technology</td><td>5063S</td><td>Egg Tubulin stain</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Build Platform 2</td><td>Formlabs, Inc</td><td>BP-F3-02-01&amp;nbsp;</td><td>3D printer component</td></tr><tr><td>Dino-Lite Edge AF4915ZTL, USB 2.0</td><td>Dino-Lite</td><td>AF4915ZTL</td><td>Imager for timelapse setup</td></tr><tr><td>Form 3 Resin Tank V2.1&amp;nbsp;</td><td>Formlabs, Inc</td><td>RT-F3-02-01</td><td>3D printer component</td></tr><tr><td>Form 3B+ 3D Printer</td><td>Formlabs, Inc</td><td>F3B-P-PRINTER</td><td>Formlabs&#039; stereolithography (SLA) 3D printer</td></tr><tr><td>Form cure&amp;nbsp;</td><td>Formlabs, Inc</td><td>FH-CU-01</td><td>Curing platform&amp;nbsp;</td></tr><tr><td>Form wash</td><td>Formlabs, Inc</td><td>FH-WA-01</td><td>Washing platform</td></tr><tr><td>Spray bottle w/ pump vaporizer</td><td>VWR</td><td>0309-3005</td><td>To apply demolding agent</td></tr><tr><td>Light focuser/concentrator cap for Edge series (excluding T5, T8 models)</td><td>Dino-Lite</td><td>N3C-R</td><td>Imager component for timelapse setup</td></tr><tr><td>Mount Holder bundle combining the RK-10 rack and RK-10-EX Arm Extension</td><td>Dino-Lite</td><td>RK-10A</td><td>Imager component for timelapse setup</td></tr><tr><td>Tissue Embedder</td><td>Leica</td><td>HistoCore Arcadia</td><td>To embed micromolds</td></tr><tr><td>Tissue Processor</td><td>Leica</td><td>TP1020</td><td>To process micromolds</td></tr><tr><td>211DS Shaking Incubator (Orbital Shaker)</td><td>Labnet International</td><td>I-5211-DS</td><td>To incubate ovaries in enzymatic media for release of follicles</td></tr><tr><td>&lt;strong&gt;Software&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Autodesk Fusion</td><td>Autodesk, Inc</td><td></td><td>Computer-aided design (CAD) software. Autodesk offers free educational access through the Autodesk Education program.</td></tr><tr><td>DinoCapture 3.0 version 1.1.1.3&amp;nbsp;</td><td>Dino-Lite</td><td></td><td></td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td></td><td>To analyze OCT images and measure follicles</td></tr><tr><td>PreForm</td><td>Formlabs, Inc</td><td></td><td>Free print preparation software designed to work with Formlabs&#039; stereolithography (SLA) printers.&amp;nbsp;</td></tr></tbody></table>

production and use of customizable agarose molds for scaffold-free mouse ovarian follicle culture

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

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.

To determine whether follicle survival and growth were supported using this novel technology as described in protocol section 7, follicles from the same mice were isolated and cultured in parallel in agarose micromolds or encapsulated in 0.5% alginate. Representative images of follicles from every other day of culture demonstrated normal development, growth, and formation of an antral cavity throughout the culture period (Figure 6A). Previously published results demonstrated that follicle su...

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

The ovarian follicle is the functional unit of the ovary that produces hormones and gametes needed to sustain female reproductive function and health. The ...

Our research aims to develop a customizable, physiomimetic, and scaffold-free follicle culture methods to determine if it can improve the quality of in vitro-grown follicles and the oocytes they develop. Currently, the most advanced culture method uses a hydrogel-encapsulated, in vitro follicle growth system, which allows follicles to maintain their three-dimensional architecture during folliculogenesis. Hydrogen encapsulation is technically challenging, laborious, low-throughput and is unfortunately not compatible with automatic imaging methods.Additionally, 3D encapsulation provides uniform support, which does not mimic in vivo physiology. We created 3D-printed, biocompatible molds that support oocytes'highly sensitive cells. Follicles grown in the scaffold-free environment showed improved growth and ovulation without compromising hormone production compared to established techniques.We're addressing the need for a user-friendly and customizable culture system that could better mimic the follicles'natural environment. Time-lapse imaging also allows live tracking and analysis of follicle performance. To begin, launch the CAD software on a computer system.Open the 24-well master mold base design step file on the software. Select the internal surface area of the object. Then navigate to the Solid tab in the design workspace, and select Create Sketch to insert the desired micromold design.Now, introduce the desired x and y dimensions and number of the microwells. Then select Finish Sketch on the toolbar. Create the 800-micrometer-deep microwells using the Extrude function by sequentially selecting Solid, Create, and Extrude.Now, choose a 0.1-millimeter radius fillet for the top of the microwells by clicking on Design, Solid, Modify, and Fillet. Add a 0.25-millimeter radius fillet to create a round-bottom microwell. Save the new master mold design and export a copy in step format by selecting File and pressing Export.Open the 24-well silicone-cast container one step file. Then insert the new master mold design as an external component by right-clicking the saved file and selecting Insert Into Current Design. Next, right-click on the inserted component and select Break Link to remove the reference to the master mold design.Center the new master mold design within the 24-well silicone-cast container one surface, ensuring it faces inward. Select OK to align the objects. Select the Cut operation, using the container as the target body and the master mold as the tool body to create the silicone molding cavity.Save and export the new 24-well silicon mold one as stl and step files. Open the stl files of the new 24-well silicone mold one and 24-well silicone-cast container two using 3D print preparation software. Orient the print with micropillars facing upwards, and use the Drill Hole function to create a one-millimeter-wide opening to the side of the print.Print both container designs at a 25-micrometer layer thickness. Wash the finished prints according to the manufacturer's instructions. Then spray the micropillar section extensively with 95%isopropanol.Remove any remaining ethanol with compressed air. After drying and curing, inspect each print using a stereo microscope. Carefully ensure that all micropillars are separated and uniform in size and appearance.After curing, cover the outside of the print with Parafilm if the drill channel is not filled, and store until further use. Next, place the prepared silicone in a vacuum desiccator for five minutes to remove air bubbles. Pour the silicone mixture with a uniform flow into the 3D-printed mold, ensuring the level matches or is slightly below the mold's surface for flatness.Remove trapped air with a P200 pipette tip and degas again if needed. Cure the silicone at room temperature in a desiccator for at least five hours or preferably overnight. After curing, remove the silicone from the mold, and inspect the micromolds under a stereo microscope.Remove any excess material from the drill channel, and discard molds with bridging or damaged wells. Next, place the silicone mold into silicone mold container two for the second molding step, ensuring the side opening is aligned. Spray the silicone mold lightly with embryo-safe mineral oil.Remove excess oils from the microwells using compressed air. Repeat the silicone-mixing procedure and pour the new mixture into the second container, maintaining the level at or below the mold's surface. The next day, separate the silicone from the 3D-printed mold and the silicone molds one and two from each other.After inspecting them microscopically, discard the molds with compromised micropillars or excess oil in the wells. Now, wash the silicone mold with 70%ethanol. Let it air dry for 30 minutes inside a laminar flow hood.Once dry, place the mold into a sterilization pouch before autoclaving. Bend the stripper tip slightly to enhance precision during follicle transfer. Transfer the follicles quickly to prevent pH or temperature fluctuations.Under a microscope, use a 200-micrometer stripper tip to transfer 10 multilayer secondary follicles per micromold, placing them in adjacent microwells. Confirm that all 10 follicles are similarly sized before finalizing the setup. Next, place a light focuser cap on a handheld microscope.Insert the microscope with mount into an incubator, and connect to a laptop with the software. Position and align the culture well under the microscope and adjust height and focus so all follicles are visible. Use auto white balance, AWB, and LED control.Turn off auto exposure, AE, and select optimal exposure time. Start time-lapse imaging with a duration of eight days and an interval of 30 minutes. Select photo and turn off LED when not capturing images.Follicles cultured in agarose micromolds showed continuous growth and antral cavity formation. After induction of ovulation, follicles cultured in agarose micromolds yielded more ovulated eggs compared to those in alginate. Further demonstrating biocompatibility, the spindle morphology of M2 eggs did not differ significantly between groups.Alginate-encapsulated follicles did not maintain their position during culture and are incompatible with time-lapse imaging. During time-lapse imaging, individual follicle morphology, including Feret's diameter, circularity, and aspect ratio, could be tracked and measured instantly throughout the culture period. Optical coherence tomography enabled 3D visualization of internal follicle structures and also captured dynamic follicle rupture events during ovulation.Agarose micromolds enabled histological analysis of multiple follicles in the same plane after paraffin embedding and sectioning, allowing identification of tissue presence or absence and supporting H&E staining. To demonstrate the customizability of this method, a high-throughput, 96-well version was created featured 10 microwells per mold, and was successfully fabricated. As with the 24-well design, it was compatible with time-lapse imaging of follicles.

production-use-customizable-agarose-molds-for-scaffold-free-mouse

Research

JoVE Journal

Developmental Biology

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Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

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Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

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Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

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Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

This protocol describes a novel three-dimensional (3-D) culture model using a tyramine-linked hyaluronan hydrogel to encapsulate and culture preantral follicles from the mouse ovary. We also detail two approaches to ovarian follicle cryopreservation by vitrification.

Application of a Novel Hyaluronan Hydrogel for Three-Dimensional Follicle Culture and Methodology for Mouse Ovarian Follicle Cryopreservation

This study presents a cost-effective and efficient methodology for generating 3D cell structures using a stamp-based system to create microwells in agarose molds. The system promotes the formation of uniform spheroids/organoids, thus improving cell interactions. This approach reduces experimental variability and supports applications in drug testing and tissue engineering.

Generation of Three-Dimensional Spheroids/Organoids from Two-Dimensional Cell Cultures Using a Novel Stamp Device

We describe methods for isolating mouse ovarian somatic cells from a stroma-enriched fraction of the ovary and for generating mouse ovarian somatic organoids using a scaffold-free approach.

Probing the Ovarian Microenvironment: Methods for Isolation, 2D Culture, and Organoid Culture of Mouse Ovarian Somatic Cells

Learn scaffold-free follicle culture using customizable agarose molds for mouse ovarian follicles. This method improves growth, ovulation rates, and hormone production while enabling time-lapse imaging.

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

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