A Modified Co-Culture System for Understanding Granulosa-Theca Cell Interactions in the Bovine Ovary

The co-culture of bovine theca and granulosa cells serves as a reliable foundation to analyze paracrine signaling and substrate transport between the steroidogenic cells of the follicle. The model enables the compartmentalization of theca and granulosa cells similar to the in vivo environment, and the usage of commercially available inserts allows a reproducible and standardized cell culture. We could create a physiological relevant environment to investigate granulosa cell interaction in more detail, like substrate exchange or follicular dynamics during the process of follicular genesis.

To begin, obtain bovine ovaries. Wash the bovine ovaries three times in PBS supplemented with antibiotics to remove blood and residues from the surface. Place the ovaries in a tumbler.

Fill it with PBS just enough to cover the ovaries and discard the solution. Then add more PBS. Place one ovary in a glass dish.

Use a ruler to measure and select follicles that are between five to 11 millimeters in diameter for dissection. Now aspirate the follicular fluid by puncturing a selected follicle with an 18 gauge needle attached to a three milliliter syringe. Discard the follicular fluid immediately.

Transfer the follicles under a binocular microscope. With a pair of scissors, cut the follicle open at the puncture site. Using tweezers, grasp the theca internal layer from the inner surface of the opened follicle.

Peel off the theca interna gently from the inner follicular wall. Transfer the theca cell layer into a dish containing PBS supplemented with antibiotics to wash off remaining granulosa cells. Then use a scalpel to gently scrape the granulosa cells off the membrane surface.

Place the membrane into a new dish with fresh PBS for washing. Rinse the membrane by swirling it gently in the buffer to remove any residual cells. Transfer the theca interna into a prepared digestion solution within a well of a 12-well plate.

Use a scalpel to cut it into one to three millimeter sized pieces. Then transfer the tissue pieces into a prepared 1.5 milliliter reaction tube. Incubate the tubes in a thermos-shaking incubator.

After 30 minutes of incubation, vortex the tubes for three to five seconds before returning them to the incubator. Repeat this vortexing step three more times over the remaining incubation period. When incubation is complete, use a pipette to resuspend the digested cells.

Pass the solution through a 100 micrometer cell strainer placed on a 50 milliliter tube to remove any undigested tissue. Prepare the inoculation chamber for culturing theca cells. Place the coated insert upside down into a larger plate, such as a 12-well plate.

Place the cut and autoclave tube on top of the inverted insert, ensuring it fits securely to prevent media leakage. Thaw the cryo-preserved theca cells quickly in a 37 degrees Celsius water bath for three to five minutes. Immediately transfer the thawed cell suspension into pre-warmed media.

Centrifuge the theca cell suspension at 500 G for three minutes at approximately 20 degrees Celsius. Discard the supernatant. Then resuspend the pellet in supplemented alpha minimum essential medium.

Seed 200 microliters of the cell suspension into the inoculation chamber. To close the cell culture dish, increase the distance between the plate and the lid using cut and autoclaved caps from 1.5 milliliter reaction tubes placed in each corner of the dish. Close the well plate carefully without displacing the inserts.

Then gently transfer the closed plate into a humidified incubator. Fill a 24-well plate with 500 microliters of media in each desired well. Gently remove the chamber from the insert using tweezers.

Place the insert containing attached theca cells into the 24-well plate with the theca facing side down. For seeding granulosa cells, pipette 250 microliters of the cell suspension inside each insert. Incubate the co-culture at 37 degrees Celsius with 5%carbon dioxide for six days.

Perform a media exchange every 48 hours after granulosa cell seeding by replacing 2/3rds of the media volume. Theca cells expressed CYP17A1 exclusively, while granulosa cells predominantly expressed CYP19A1. After three days in culture, theca cells exhibited a flattened and elongated morphology on the collagen-coated membrane, indicating successful attachment.

When cultured alone for nine days, theca cells proliferated and reached confluence by day nine. Granulosa cells cultured alone for six days developed a fibroblast-like morphology and formed clusters characteristic of in vitro behavior. In the co-culture system, no morphological differences were observed in either cell type compared to their respective mono-cultures.

Estradiol levels were similar between granulosa cell mono-cultures and co-cultures. While theca cell mono-cultures produced only negligible amounts. CYP17A1 expression remained confined to theca cells under both mono and co-culture conditions.

CYP19A1 expression remained confined to granulosa cells under both mono and co-culture conditions.