3D Co-culture with Direct Interaction: Co-culturing Cancer Cells with Monocytes to Study Their Interaction

To begin, add DQ collagen IV, a fluorescent dye quenched protein substrate into an extracellular matrix extract or ECME. Pour this extract into each well of a two well slide. Label the first well as co-culture, second as control. Add an appropriate amount of fluorescently labeled cancer cell suspension into wells, and incubate the slide at 37 degrees Celsius for the desired time to promote cell attachment.

Add a required amount of monocytes cell suspension to the first well. Let the ECME solidify at 37 degrees Celsius. Pour equal ratios of cancer cell and monocyte culture media into each well and incubate the slide at 37 degrees Celsius for the desired duration in a carbon dioxide environment. In the co-culture well, cancer cells secrete proteins such as monocytes chemoattractant protein to attract monocytes.

In contrast, monocytes produce matrix metalloproteinase or MMP, to degrade the collagen, promoting cancer cells invasion. Observe the slide under a confocal microscope. The DQ collagen degradation emits fluorescence which can be seen more in the co-culture cells indicating cell interaction, while less in control cells. In the following protocol, we will co-culture breast cancer cells with monocytes to study their interaction.

To label BRC cells and monocytes with commercially available coumarin and rhodamine before cell culture, prepare a monolayer of two times 10 to the six BRC cells of interest and replaced the standard medium with sufficient prewarmed working solution of the fluorescent dye. Incubate the cells at 37 degrees Celsius in a humidified 5% carbon dioxide environment for 30 minutes. Then aspirate the dye solution and use sufficient 1x PBS to gently rinse the cells.

Aspirate the PBS and add the standard medium. To trypsinize the cells add two milliliters of 0.05% trypsin and 0.48 millimolar EDTA to the flask, and incubate the cells at 37 degrees Celsius for 5 to 10 minutes. Add 200 microliters of FBS to stop the trypsinization and seven milliliters of the corresponding medium without supplements.

Resuspend the cells by pipetting, then transfer 10 microliters of the cells to a Neubauer chamber and count them. Next, pellet the cells at 430 x g at room temperature for five minutes, then discard the supernatant and use milliliters of prewarmed fluorescent dye working solution to suspend the cells.

Incubate the cells suspension at 37 degrees Celsius in a humidified 5% carbon dioxide environment for 30 minutes. After pelleting the cells again, discard the dye working solution and use 5 to 7 milliliters of 1x PBS to gently resuspended the cells. Then after pelleting the cells once more and discarding the PBS, resuspended the cells in 5 to 7 milliliters of standard medium.

Count 10 microliters of the cell suspension. Set up a co-culture by spreading enough ECME at the bottom of the well to form an even layer in each well of a four-well chamber slide system. Plate 20 microliters of a single cell suspension containing 5 times 10 to the fifth labeled BRC cells per well. After 15 to 20 minutes at 37 degrees Celsius, add a suspension of 2.5 times 10 to the fifth labeled monocytes in 80 microliters of assay medium, supplemented with 60% ECME.

Allow the ECME to solidify a 37 degrees Celsius for 15 to 20 minutes. Now, add 1 milliliter of a 1:1 mixture of the BRC cells and monocyte culture medium. Incubate the co-cultures at 37 degrees Celsius in a humidified 5% carbon dioxide environment for 24 hours, 48 hours, or five days to track changes at different time points.

To degrade the ECM proteins and recover the cells from the cultures, aspirate and discard the medium then add 0.5 milliliters of 1x PBS with 0.1% trypsin and 0.25% EDTA to the culture. Incubate the cells at 37 degrees Celsius for three hours. Following the incubation, add 0.5 milliliters of 1x PBS with 10% FBS to neutralize the trypsin and resuspend the cells by vigorously pipetting to obtain a single cell suspension.

After pelleting the cells, discard the supernatant and resuspended the cells in five milliliters of 1x PBS with 10% FBS. Repeat the step once. Subject the cell suspension to FACS with the appropriate instrument. Ensure that the final populations are at least 95% pure.