Modeling Ovarian Cancer Metastasis in Peritoneal Cavity Lining: A Method To Establish an In Vitro 3D Model of the Peritoneal Lining

1. Plating the Organotypic Culture

1. Release NOF (normal omental fibroblasts ) from a 75 cm² culture flask by rinsing with 10 ml of PBS followed by 3 ml of 0.25% trypsin/25 mM EDTA for no more than 5 min. Neutralize trypsin with at least 3 times the volume of full growth media.
2. Transfer trypsinized cells into a 50 ml conical tube and spin down at 0.5 g x 3 min. Remove supernatant and bring cells back up in 5 ml of full growth media. Use a cell counter to count the cells recovered from the culture flask.
3. Dilute the cells in the appropriate volume of full growth media to plate 2,000-4,000 NOF per 100 µl onto a black, clear-bottomed, 96-well plate. Add 0.5 µg/100 µl of rat-tail collagen I to the cell mixture before plating. Let the cells sit at 37 °C in 5% CO₂ in a humidified environment for at least 4 h, or until the cells adhere to the plate surface.
4. Release HPMC (primary human mesothelial cells) from the culture flask using the same methods described in steps 1.1 and 1.2. Dilute the cells in the appropriate amount of full growth media to plate 10,000-20,000 HPMC per 50 µl on top of the NOF already plated with collagen I in 96-well plates. Let the cells sit at 37 °C in 5% CO₂ in a humidified environment for at least 18 h before beginning experiments.
5. Culture green fluorescent protein (GFP)-expressing HeyA8 ovarian cancer cells in full growth media. HeyA8 ovarian cancer cells are fluorescently labeled by expression of a lentiviral vector expressing copepod cGFP (pCDH-CMV-MCS-EF1). HeyA8 ovarian cancer cells should be 80-90% confluent on the day of use (logarithmic growth phase). Release cells from the culture plate and count using the same methods described in steps 1.1-1.2 for primary cells.
6. Allow ovarian cancer cells to recover in full growth media for 15-20 min at 37°C in a conical tube with the lid loosely sealed.