Modeling Ovarian Cancer Cell Colonization of Omentum: An Ex Vivo Method to Establish Milky Spot Colonization of Fluorescently Labeled Ovarian Cancer Cells in Murine Omentum Explants

Overview

In this video, we establish an ex vivo omental organ culture overlaid with GFP-expressing ovarian cancer cells to study ovarian cancer colonization within the omental explant. This protocol analyzes immune cell structures within the omentum known as milky spots that promote metastases.

Protocol

All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Milky Spot Colonization Ex Vivo

1. Establishment of basic ex vivo omental organ culture conditions
   1. Place each omentum into a single culture plate insert and place within one well of a 12-well tissue culture plate containing 500 µl of DME/F12 media containing 20% FCS. Maintain organ cultures at 37 °C in a 5% CO₂ environment for 24 to 48 hr and/or additional time points. Use three independent omenta (triplicate samples) for each condition (e.g., media type/time point).
   2. To confirm the integrity of tissues at the experimental endpoints, fix and process samples as described in Step 1.1.3. Counting healthy versus necrotic adipocytes on H&E sections can assess tissue integrity. A minimum of ~120 cells from five biological samples is required to formulate a percentage of live tissue present.
   3. To measure tissue function, place omenta (n=3) in separate wells of a 24-well plate containing 500 µl of DME/F12 media with 20% FCS. Allow omenta to condition the media at 37 °C in a 5% CO₂ environment for 24 hr and then remove 250 µl for use in an IL-6 ELISA assay.
2. Ex vivo co-culture of the mouse omentum with SKOV3ip.1-GFP cells
   1. Grow and prepare fluorescently tagged cells by pipetting 1x10⁶  thawed cells in 10 ml of media in a T-75 flask. Allow the cells to grow at 37 °C in a 5% CO₂ environment until they reach 70% confluence. Trypsinize and resuspend at a concentration of 2 x 10⁶ cells per ml.
   2. Apply ~6 µl of the tissue adhesive to the membrane of the culture insert and allow it to air dry. Wash the membrane twice with sterile water to remove any excessive adhesive. Air dry the membranes under a laminar hood.
   3. Carefully excise the omenta and attach it to the adhesive-coated membrane using sterile forceps. Allow the tissue to adhere to the membrane for 1 min prior to adding media. Add 500 µl of the cell suspension on top of each omentum in each culture insert. Fill the area around the transwell chamber with 2.5 ml of DME/F-12 .
   4. Incubate omenta with cell suspension for 6 hr at 37 °C in a 5% CO₂ environment. Carefully remove and wash the omenta with ~10 ml of PBS. Visualize fluorescent cancer cell foci using an appropriate fluorescent imaging system.

Materials

Name	Company	Catalog Number	Comments
Dissection Tools	Fine Science Tools	NA
Giemsa	Fluka/Sigma Aldrich	48900
5% Formalin	Sigma	HT501320
DMEM	Corning	10-013-CV
PBS	Corning	21-040-CV	Without calcium and Magnesium
Collagenase	Worthington	LS004196
Millicell culture plate insert	Millipore	PICM01250
Cell-Tak	Corning	354240