Name: 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
Uploaded: 2023-04-30T13:26:44
Description: Source: Krishnan, V. et al. In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose. ...

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

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

Grow and prepare fluorescently tagged cells and resuspend at a concentration of 2 million cells per milliliter. Apply approximately 6 microliters of the tissue adhesive to the membrane of the culture insert and allow it to air dry. Then, wash the membrane twice with sterile water to remove any excessive adhesive before air drying the membranes under a laminar hood.
Carefully excise the omenta and attach it to the adhesive-coated membrane using sterile forceps. After allowing the tissue to adhere to the membrane for one minute, add 500 microliters of the cell suspension on top of each omentum in each culture insert.
Then, fill the area around the transwell chamber with 2.5 milliliters of DME/F-12 media. Incubate the omenta with cell suspension for six hours at 37 degrees Celsius in a 5% CO2 environment. Carefully remove and wash the omenta with about 10 milliliters of PBS. Finally, visualize fluorescent cancer cell foci using an appropriate fluorescent imaging system.

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Cancer Research

Gynecologic Cancer

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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
<ol>
	<li>Establishment of basic ex vivo omental organ culture conditions
	<ol>
		<li>Place each omentum into a single culture plate insert and place within one well of a 12-well tissue culture plate containing 500 &#181;l of DME/F12 media containing 20% FCS. Maintain organ cultures at 37 &#176;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).</li>
		<li>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&#38;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.</li>
		<li>To measure tissue function, place omenta (n=3) in separate wells of a 24-well plate containing 500 &#181;l of DME/F12 media with 20% FCS. Allow omenta to condition the media at 37 &#176;C in a 5% CO2 environment for 24 hr and then remove 250 &#181;l for use in an IL-6 ELISA assay.</li>
	</ol>
	</li>
	<li>Ex vivo co-culture of the mouse omentum with SKOV3ip.1-GFP cells
	<ol>
		<li>Grow and prepare fluorescently tagged cells by pipetting 1x106 &#160;thawed cells in 10 ml of media in a T-75 flask. Allow the cells to grow at 37 &#176;C in a 5% CO2 environment until they reach 70% confluence. Trypsinize and resuspend at a concentration of 2 x 106&#160;cells per ml.</li>
		<li>Apply ~6 &#181;l 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.</li>
		<li>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 &#181;l 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 .</li>
		<li>Incubate omenta with cell suspension for 6 hr at 37 &#176;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.</li>
	</ol>
	</li>
</ol>

Source: Krishnan, V. et al. <a href="https://www.jove.com/t/52721">In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose.</a> J. Vis. Exp. (2015)
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.

Source: Krishnan, V. et al. In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose. ...

The omentum, an adipose-rich tissue within the peritoneal cavity, consists of fat depots that provide a suitable microenvironment for metastatic cancer cells to colonize and form secondary tumors. To study this colonization ex vivo, begin by positioning a permeable membranous cell culture insert inside a culture plate.
Apply a tissue adhesive over the inner region of the insert membrane. Place a mouse omental explant on the adhesive to immobilize the tissue. Gently seed a suspension of fluorescently-labeled ovarian cancer cells on top of the omentum.
Next, fill the sides of the well with a suitable medium to supplement cell growth and incubate for the desired time. The omental tissue contains specialized structures called milky spots, which are clusters of immune cells packed around networks of blood vessels. These immune cells secrete certain chemical factors that attract cancer cells and promote their implantation. The colonized cancer cells proliferate, simulating metastatic conditions.
Finally, discard the spent media. Observe the culture using a fluorescent microscope to assess fluorescent cancer cell colonies within the omental tissue.

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Video: 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 - Experiment

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

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