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

This research has been focused on developing a 3D culture system for in vitro follicle growth and oocyte maturation. Creating such a system using components of a cell's native extracellular matrix will enhance follicle development. The biggest challenge is applying the knowledge from animal models to human follicle culture.

Access to human ovarian tissue is limited. And human follicles require a significantly longer time in culture to achieve oocyte maturation. The 3D culture model developed in this study may be especially useful for culturing human ovarian follicles, allowing retention of 3D architecture during the prolonged time needed in culture for oocyte maturation.

To begin, obtain ovaries from 10 to 14 day old B6D2F1 mouse pups for the procedure. Pipette 6 milliliters of pre-equilibrated follicle culture medium, or FCM, into two 60 millimeter dishes and overlay with mineral oil, and incubate. Pipette 1 milliliter of collagenase to the center well of the dish, and 3 milliliters of ovarian tissue, or OT media, in the outer well.

Move the ovaries to the collagenase solution. At the end of the collagenase incubation, move the collagenase treated ovaries to the outer well to rinse. Change micropipettes and transfer the ovaries to a fresh dish filled with OT medium.

To harvest the follicles, repeatedly aspirate and expel enzyme treated ovarian tissue through pipette tips cut to different sizes. Mechanically tease tissue using 27 gauge needles to eight release of follicles. Examine under a dissecting microscope at 40x magnification and pick secondary preantral follicles with a centrally located oocyte enclosed within an intact basement membrane to a fresh OT dish.

Place the FCM2 dish in the incubator for 60 minutes before starting the embedding process. Make two 25 microliter drops of HA gel without hydrogen peroxide on the lid for rinsing. Move follicles or follicle clusters to be embedded from the FCM dish to a drop of the hyaluronan, or HA gel, to rinse free of culture medium.

Place the FCM dish back under the bubbler to gas. For embedding follicles, place 1 microliter of 0.03%hydrogen peroxide drop onto the lid of a 60 millimeter dish. Now, add 25 microliters of 3 milligrams per milliliter HA gel and mix.

Pipette approximately eight to 10 microliters of the HA hydrogen peroxide mixture into two separate wells of the eight well culture dish. And quickly transfer the follicles, follicle clusters, or ovarian tissue fragments into the center of each drop. Ensure there are no bubbles within the drop.

After three minutes, add 100 microliters of preequilibrated follicle culture medium to each well. Overlay with warm preequilibrated mineral oil and incubate the dish. Prepare the equipment and the solutions required for the vitrification process.

Position the RI carrier into the slot in the cryo box containing liquid nitrogen. Place two drops of prewarmed vitrification solution 1, or VS1, side by side on the lid of a 60 millimeter dish near the top. Now, place two follicles in the first drop, rinse, and quickly move them to the second drop of VS1 for a five minute incubation.

While the follicles incubate, make three VS2 drops just below the VS1 drops. Then quickly move the follicles sequentially through the three VS2 drops within 60 seconds. For vitrification, pick up the follicles from VS2.

View the RI carrier under the dissecting scope. And adjust the focus to visualize the tiny hole in the plastic stick. Deposit follicles with minimal fluid into the hole.

Drop the plastic stick into the precooled outer straw. And use an ultrasonic sealer to close the straw. Place the straw in a goblet attached to the cryo cane.

Cover the cane with a plastic protective sleeve and plunge it into liquid nitrogen. For open carrier vitrification, prepare the carrier by inserting the metal CL stem into the magnetized vial cap, ensuring a firm hold. Place a rack into the cryo box to hold cryo vials, ensuring liquid nitrogen is below the top of the vials.

Fill a magnetized invented cryo vial with liquid nitrogen and place in the rack. Grasp the CL open carrier by its attached magnetic cap using a magnetic wand. Dip the CL carrier in a separate drop of VS2 to create a cryoprotectant film.

Pick up all follicles or follicle clusters and place them on the film with minimal fluid. Immediately immerse the CL into the cryo vial filled with liquid nitrogen to vitify the sample. Place the cryo vial on the cryo cane, and then cover with a plastic sleeve.

Plunge the cane into a liquid nitrogen storage tank. Prepare a center well dish with 3 milliliters of preequilibrated FCM in the outer well and 1 milliliter in the center well. After overlaying the medium with oil, place the dish under the bubbler to gas.

Place 0.5 milliliters of warming solutions WS1 and WS2 preequilibrated to 37 degrees Celsius into two labeled center well dishes. Move the cryo canes with samples from the storage tank into a cryo box filled with liquid nitrogen. First, remove the plastic cane cover.

Then keeping the straw immersed, remove it from the goblet and slide it into the holding slot in the cryo box. Cut the outer straw just above the black dot. Lift the inner plastic stick out of the outer straw and quickly immerse the stick into WS1, gently swirling to unload the follicles within 10 seconds.

After two minutes in WS1, use a micro pipette to transfer follicles or follicle clusters to WS2 for a three minute incubation. Then transfer them to an FCM dish and incubate the follicles for one to two hours in the center well before embedding, For open carrier CL warming, repeat the FCM and WS dish preparation. Transfer the cryo cane into a cryo box and remove the plastic cane cover.

Use a magnetic wand to lift the cryo vial cap until the metal stem of the CL is visible. Grasp the cap with the attached CL carrier using the magnetic wand. And quickly immerse the CL into WS1, swirling to unload follicles within 10 seconds.

Preantral follicle growth was maintained in the three-dimensional hyaluronan gel culture, preserving native follicle architecture with an antrum visible by day 12. Encapsulated follicles in hyaluronan gel exhibited radial expansion due to granulosis cell proliferation with follicle diameters increasing from around 139.8 micrometers to 385.6 micrometers over the culture period. Ovulated metaphase two oocytes were found near the follicle after human chorionic gonadotropin trigger.

The oocytes measured approximately 84.8 micrometers in diameter. With fresh ovaries, both individual follicles and follicle clusters exhibited good maturation rates. Follicle clusters from cryopreserved ovaries exhibited lower maturation rates leading to premature oocyte extrusion.

Isolated follicle cryopreservation was more effective than whole ovary preservation. Follicle vitrification on both carriers achieved high maturation rates, regardless of cooling rate differences. The CL open carrier system allowed for efficient cryo-preservation of multiple follicles, reducing processing time.