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

Abstract

Source: Peters, P. N. et al. Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity. J. Vis. Exp. (2015)

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....

Materials

List of materials used in this article

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