Current studies use cold culture systems comprised of isolated adipocytes and ovarian cancer cells. These adipocytes are differentiated in vitro from pre-adipocytes. These cells are then separated through a Transwell insert and therefore lack direct cell-to-cell contact.
This ex vivo culture method offers a model to study the direct interaction of cancer cells with the adipose microenvironment. Our model preserves the structure of the human omentum and therefore maintains the various cell types that are typically in this organ. In addition to adipocytes, these cells include supporting fibroblasts as well as immune cells.
This model also maintains these cells in their inherent matrix. This model provides an excellent platform for the development and evaluation of novel therapeutic approaches to target metastatic cancer cells in this niche. To begin, remove the freshly excised human omentum from the collection container inside a laminar flow hood.
Immerse the omentum in PBS to gently wash away any residual blood or debris. Using a scalpel and forceps, cut the tissue into pieces. Place the omentum pieces into individual wells of a 24-well culture plate.
Then fill the wells with 500 microliters of omentum culture media. Add 10 milliliters of sterile PBS to a culture flask containing 75%confluent human mCherry positive ovarian cancer cells. Gently rock the flask to wash the cells.
Remove the PBS and add three milliliters of 0.05%trypsin-EDTA. Gently rock the culture flask again to evenly distribute trypsin-EDTA on the attached cells. Then, place the flask in an incubator for five minutes at 37 degrees Celsius under 5%carbon dioxide.
After that, remove the flask from the incubator. Now, tap the culture flask to completely detach the cells, then add three milliliters of omentum culture media to neutralize the trypsin. Pipette the cell suspension multiple times to mix it well.
Transfer the suspension into a 15-milliliter conical tube. Centrifuge the suspension at 1, 200 G for five minutes at 24 degrees Celsius. Now, pipette out the supernatant and re-suspend the cell pellet in six milliliters of fresh omentum culture media.
Take 10 microliters of the cell suspension to count the cells using a cell counter. Dilute the cell suspension with fresh culture media to the required density. With a one-milliliter syringe, draw up the cancer cell suspension.
Attach a 26 gauge needle to the syringe. Now, use a pair of small surgical forceps to pick up a piece of the cut omentum and place the omentum piece in a separate working sterile dish. Then, inject about 100 microliters of the cancer cell suspension into the tissue.
Alternatively, mix equal volumes of thawed basement membrane matrix with omentum culture. Place the mixture on ice until injection. After immersing the omentum pieces into the matrix mix in the wells of the culture plate, incubate the plate for 20 minutes.
After that, remove the plate from the incubator. Once the mixture has solidified, inject the omentum pieces with the cancer cells. Confirm the successful injection of the cancer cells using fluorescent imaging.
A streak of fluorescent signal can be seen at the injection sites as confirmation. To facilitate the co-culture of the omentum and cancer cells, add 500 to 200 microliters of fresh media every 48 to 72 hours. Finally, using a fluorescent microscope, monitor the growth of ovarian cancer tumors.
Tumor growth may be visible in two weeks. The ovarian cancer cells were successfully integrated into the omentum pieces by day 14. The cancer cells initially spread across the omentum surface in the first week.
Over time, the cells congregated to form tumors. The tumor burden was confirmed by the color change of the omentum media, which turned from red to yellow.
