This study presents a method for modeling ovarian cancer metastasis using a 3D organotypic culture that mimics the peritoneal lining. The approach involves co-culturing fibroblasts and mesothelial cells, followed by the introduction of ovarian cancer cells to study their interactions.
The peritoneum, or the membrane that lines the abdominal cavity, constitutes peritoneal mesothelial cells supported over a layer of fibrovascular connective tissue. Metastasizing ovarian cancer cells adhere to this peritoneal lining and proliferate into cancerous growths.
To model ovarian cancer metastasis, begin by taking human fibroblasts in a conical tube and mix them with collagen - an extracellular matrix protein. Plate the suspension onto a culture dish. Incubate to facilitate collagen matrix solidification to embed the fibroblasts and mimic the connective tissue of the peritoneum.
Next, add a suspension of mesothelial cells over the fibroblast-collagen layer. Incubate the plate to allow the mesothelial cells to adhere and form a monolayer on the surface of the fibroblast-collagen layer. This 3D organotypic culture now mimics the composition and architecture of an in vivo peritoneal lining.
Finally, seed this organotypic culture with ovarian cancer cells and incubate for the desired duration. The ovarian cancer cells release certain signaling molecules that alter the mesothelial layer, enhancing cancer cell adhesion and invasion, mimicking in vivo metastatic conditions.
To release the normal omentum fibroblasts from culture, rinse the culture flask with 10 milliliters of PBS followed by 3 milliliters of trypsin for no more than 5 minutes. When the cells have detached, neutralize the trypsin with at least three times the volume of full growth medium and transfer the cells to a 50-milliliter conical tube. Spin down the cells and resuspend the pellet in 5 milliliters of full growth medium. Then, count the cells and dilute them to a 2 to 4 times 10 to the third fibroblast per 100 microliters of full growth medium concentration.
Next, add 0.5 micrograms per 100 microliters of rat-tail collagen I to the cells and plate 100 microliters of cells per well into a black, clear bottom 96-well plate. Then, transfer the plate to a cell culture incubator for at least 4 hours or until the cells adhere to the plate surface. While the normal omentum fibroblasts are settling, detach the HPMC from their culture flasks and dilute these cells to 1 to 2 times 10 to the fourth HPMC per 50 microliters of full growth medium.
Then, add 50 microliters of HPMC per well to the attached normal omentum fibroblasts and return the plate to the cell culture incubator for at least 18 hours before experimental use. The next day, seed the 3D organotypic co-cultures with GFP expressing ovarian cancer cells from an 80% to 90% confluent culture at the appropriate dilution for the desired downstream assay.
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