This study investigates the role of the omentum in the colonization of metastatic ovarian cancer cells. By utilizing an ex vivo model, researchers can observe the interactions between cancer cells and the omental tissue.
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.
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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