This method can help answer key questions in the bone metastasis research field, such as what changes are induced in the bone marrow adipocyte growth and their development in a cancer microenvironment. The big advantages of this technique are that it is easy to perform and that the candidate molecules can be quickly obtained. This method can provide insights into the crosstalk between bone marrow adipocytes and the melanomal cells.
It can also be applied to other cell types, such as osteoblasts. Begin by seeding two times 10 to the fifth B16F10 melanoma cells in a 100-millimeter dish with 10 milliliters of DMEM supplemented with 10%FBS, for a 24-hour incubation at 37 degrees Celsius, and 5%CO2. The next day, wash the about 80%confluent cell culture two times with five milliliters of PBS per wash, and starve the cells with 10 milliliters of serum-free culture medium in the cell culture incubator for 18 to 24 hours.
The next day, use a pipette to transfer the conditioned supernatant into a 50-milliliter conical tube for centrifugation, and load the medium into a syringe equipped with a 0.45 micron-pore filter. Then, strain the solution into a sterile container to remove any remaining cell debris, and store the conditioned medium at minus 20 degrees Celsius for up to one month. To induce bone marrow adipocytes, culture mouse F14 1.1 bone marrow stromal cells in a 60-millimeter dish with complete DMEM to an 80%confluency.
At the appropriate stage of culture, harvest the cells with 1.5 milliliters of a 0.25%trypsin solution for one to two minutes at 37 degrees Celsius. When the cells have detached, arrest the enzymatic reaction with five milliliters of medium supplemented with 10%FBS, and adjust the cells to a one to two times 10 to the fifth cells-per-milliliter concentration with fresh serum-supplemented culture medium. Seed five times 10 to the fourth cells in 0.5 milliliters of adipogenic induction medium per well in a 24-well plate, and return the cells to the cell culture incubator for another two days, until they reach about 90%confluency.
Then, replace the supernatant with one milliliter of adipogenic maintenance medium per well, and continue to culture the cells until adipocyte maturation. When the cells look like small to large soap bubbles under light microscopy, wash these mature adipocytes two times with PBS, followed by fixation in 0.2 milliliters of 3.7%formaldehyde for one hour. Rinse the fixed cells in 60%isopropanol, and allow the wells to dry completely.
Add 0.2 milliliters of Oil Red O working solution to the cells for a 30 to 120-minute incubation at room temperature, and wash the cells with water to remove any unbound dye for imaging of the adipocytes by light microscopy. Alternatively, upon their maturation, add 0.5 milliliters of ready-to-use RNA isolation reagent to each adipocyte culture well, and use the appropriate primers to confirm the adipocyte-specific gene signature of the cells. Then, run the reaction in a thermal cycler using the indicated settings.
To set up a bone marrow-derived adipocyte melanoma cell co-culture, first add 150 microliters of serum-free DMEM to the inner chambers of 0.4 micron-pore size membrane inserts, and place each insert into one well of a 24-well plate containing 500 microliters of serum-free medium per well. Dilute B16F10 melanoma cells from an 80%cell culture to an experimentally-appropriate concentration in fresh serum-free medium, and replace the medium in the inner chamber of each insert with 150 microliters of cells. Next, replace the adipogenic maintenance medium supernatant in each well of a 90%confluent mature bone marrow adipocyte culture with 600 microliters of complete DMEM, and transfer the melanoma cell-containing inserts to each well of the mature adipocyte culture plate.
When all of the inserts have been transferred, place the plate in the cell culture incubator. After two days, remove the inserts and wash the bottom of each well two times with 500 microliters of PBS per wash. Then, stain the adipocytes with the Oil Red O working solution, or analyze the adipocyte gene expression by quantitative PCR, as demonstrated.
Mature adipocytes differentiated in tumor-conditioned medium contain lipid droplets, allowing the differentiated adipocytes to be easily identified by Oil Red O staining. The adipocyte-specific gene signature of these cells can also be confirmed by quantitative PCR. A reduction in lipid storage is observed in adipocytes co-cultured in the presence of high numbers of melanoma cells, as well as a de-differentiation in adipocyte phenotype, as evidenced by a decreased adipocyte-specific gene expression in these formerly mature cells.
After watching this video, you should have a good understanding of how to design and set up a 2D co-culture system for studying the interactions between two different cell types.
