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 Trypan 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 240 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 pre-warmed 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 medium, add 100 microliters of ice cold advanced DMEM F12 to each well. Scrape the bottom of the wells with a P1000 pipette tip to detach the gel droplets and transfer each floating gel droplet to a 1.5 milliliter tube. Centrifuge the tube at 240 G for five minutes.
After discarding the supernatant, resuspend the pellet in 300 microliters of cell dissociation buffer and place the tubes in a pre-warmed bead bath at 37 degrees Celsius for 5 to 10 minutes. Next, add 300 microliters of human OSE organoid basic medium, and centrifuge at 240 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-lumenized cell clusters.
