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Encyclopedia of Experiments

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

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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.

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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

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