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

Overview

In this video, we demonstrate the development of  an in vitro  three-dimensional model of the lining of the peritoneal cavity. This organotypic culture can help to investigate ovarian cancer cell adhesion, invasion, and proliferation.

Protocol

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.

Materials

Name	Company	Catalog Number	Comments
PBS	Fisher Scientific	SH3001304
15 cm culture dishes	BD Biosciences	353025
DMEM with L-Glutamine	Corning	10-013-CV
MEM Vitamins	Corning	25-020-Cl
MEM Nonessential amino acids	Corning	25-025-CI
Penicillin-Streptomycin	Corning	30-002-CI
Shaker	Thermo-Fisher	MaxQ 4450
Centrifuge	Eppendorf	5702
Incubator	Thermo-Fisher	Forma Series II Water Jacketed CO2 Incubator Model 3100
Trypsin EDTA, 1x (0.25%)	Corning	25-053-CI
T-75 Flasks	BD Biosciences	353136
T-175 Flasks	BD Biosciences	353112
Pipet tips	Rainin	P2, P10, P20, P200 and P1000
Pipet tips	Corning	Filtered tips P2, P10, P20, P200 and P1000
Cell Counter	Invitrogen	Countess
Countess Cell Counting Chamber Slides	Invitrogen	C10313
Trypan Blue Stain (0.4%)	Gibco	15250-061
Collagen Type I (Rat Tail)	BD Biosciences	354236
96 well plate, clear bottom, black	BD Biosciences	353219