Human Ovarian Surface Epithelium Organoids as a Platform to Study Tissue Regeneration

Our research focuses on understanding the ovarian function in humans. Due to the still present knowledge gap in female reproductive health, as well as increasing need for new technology to treating fertility. We aim to study variant processes such as follicle regenesis or tissue regeneration in order to promote the development of new clinical protocols.

In recent years, we have witnessed great advances in 3D tissue culture protocols. These developments have enabled the formation of, for instance, artificial ovaries, which are follicle like structures, capable of supporting the maturation of an all site fully in vitro. Additionally, we are also seeing how organoids or tumoroids which are miniaturized versions of an organ or a tissue.

We are seeing how they are paving the way for further studies on ovarian biology and diseases. Access to variant samples coming from donors in reproductive age still represents a big challenge. On top of that, results obtain when using this material show a high inter donor variability, most likely due to factors such as differences in donor age, metabolic state, and endocrine function.

Altogether this heterogeneity makes it difficult to reproduce results and to translate the protocols into the clinic. Despite that, the ovaries stand out as one of the most dynamic and changing organs within the body its regenerative mechanisms are still very poorly understood and actually most of the knowledge obtained so far. It still comes from animal models.

With our OSC Organal protocol, we provide a platform to study the role of the human OSC layer in ovarian tissue regeneration and wound healing. Compared to others our protocol has been designed based on the use of non-pathological samples. This allow its application in developmental studies that are trying to find answers to fundamental mechanistic questions about ovarian function in humans.

To begin transport human ovaries to the lab in 0.9%sodium chloride or similar sterile saline solution on ice. Rinse ovaries with fresh cold saline solution in a Petri dish. Transfer each ovary to a 50 milliliter conical tube and add two to three milliliters of digestion medium to cover the ovary.

Place the tube in a bead bath, tempered at 37 degrees Celsius for 30 minutes. Then carefully transfer the ovary into a 60 millimeter Petri dish containing 10 milliliters of collection medium. Using a cell scraper scuff the ovarian surface containing the human ovarian surface epithelial cells gently.

Wash the scraper in the medium before scraping at least two more times. Transfer the human ovarian surface epithelial cells in the collection medium into a 15 milliliter tube. Spin down the cells at two 40 G for five minutes.

If the pellet shows a red color indicative of contamination with red blood cells, resuspend the pellet in one milliliter of red blood cell lysis buffer. Then add four milliliters of PBS with calcium and magnesium and centrifuge at two 40 G for five minutes. Cryopreserve the human ovarian surface epithelial cell pellet for future culture use.

To begin thaw cryopreserved human ovarian surface epithelial cells. Mix the cells with 10 milliliters of washing medium. After centrifuging the cells resuspend pellet in two milliliters of ovarian surface epithelium or OSE 2D medium and transfer it into two wells of a 12 Well plate.

Add one milliliter of extra OSE 2D medium into the well and culture at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide for 72 hours. Before the first media renewal, employ trippin blue staining to count freshly isolated or cryopreserved thawed live Human ovarian surface epithelial cells with an automated cell counter. Mix the desired number of cells in one milliliter of ice cold OSE organoid basic medium, and centrifuge the tube at two 40 G for five minutes.

Using a pipette resuspend the cell pellet in ice cold undiluted basement membrane extract solution. To obtain 10, 000 cells per 10 microliters, add 10 microliter droplets of OSE basement membrane extract solution per well in a prewarm multi-well chambered slide and place the plate upside down at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide for 15 minutes. After the gel solidifies, add 100 microliters of OSE 3D medium and culture at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide.

After removing the spent add 100 microliters of ice cold advanced DMEMF 12 To each well. Scrape the bottom of the wells with a P 1000 pipette tip to detach the gel droplets and transfer each floating gel droplet to a 1.5 milliliter tube Centrifuge the tube at two 40 G for five minutes. After discarding the supernatant, resuspend the pellet in 300 microliters of cell dissociation buffer and place the tubes in a prewarm bead bath at 37 degrees Celsius for five to 10 minutes.

Next, add 300 microliters of human OSE organoid basic medium, and centrifuge at two 40 G for five minutes. Finally, resuspend the pellet in the desired volume of undiluted basement membrane extract solution to reseed them at a suitable ratio for culturing. Primary human OSE cells exhibited cobblestone like morphology up to passage three, but showed signs of senescence thereafter.

Human OSE organoids from freshly isolated OSE cells grew larger than those from 2D expanded OSE cells after 14 days in culture, various morphologies were observed in 3D OSE organoids, including cystic monolayer, multilayered, and non aluminized cell clusters.