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

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.