Human Primary Ovarian Surface Epithelial Cell Isolation for Obtaining Organoids for In Vitro Tissue Studies

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 240G 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 240G for five minutes. Cryo-preserve the human ovarian surface epithelial cell pellet for future culture use.