The overall goal of this simple in vitro coculture model is to mimic the characteristics of an in vivo tumor microenvironment, enrich individual cell populations, and investigate various modes of intercellular communication. This method can help answer key questions in the cancer and radiobiology fields, specifically regarding the factors underlying the generation of cancer-associated fibroblasts and the responses to therapeutic agents. The main advantage of this technique is that it allows the generation of individual cell populations from a mixed cell culture without stress-inducing fluorescence tagging or cell sorting.
Begin by washing the cell culture of interest two times with five milliliters of PBS. After the second wash, detach the cells with one milliliter of room temperature trypsin-EDTA for two minutes at room temperature. Then quench the reaction with nine milliliters of complete growth medium, gently pipetting the medium over the surface of the flask 10 times to dissociate the cells.
After counting, dilute the cells to a 2.5 times 10 to the fifth cells per milliliter concentration in fresh medium, and transfer the cells to a sterile 15 milliliter centrifuge tube. Spin down the cells by centrifugation and resuspend the pellet in fresh growth medium supplemented with 50%fetal bovine serum at 2.5 times 10 to the fifth cells per 70 microliters of medium concentration. Next, transfer inserts of the appropriate experimental pore size from their packaging into individual wells of a multi-well dish and cover the dish.
Then holding the dish with both hands, gently invert the dish until the insert bottoms are facing upwards. Remove the bottom of the dish. Using sterile forceps in one hand, hold one insert in place and use the other hand to slowly aspirate 70 microliters of cells into a micropipette tip.
Then slowly dispense the cells across the surface of what is now the topside of the insert. After seeding each of the inserts, carefully replace the dish bottom and incubate the inverted dish at 37 degrees Celsius and 5%carbon dioxide with humidity for 30 to 45 minutes. At the end of the incubation, in a laminar flow biological safety cabinet carefully reinvert the dish such that the bottoms of the inserts now face down.
Then slowly and carefully immerse the bottom of each insert in two milliliters of pre-warmed complete medium, and place the dish back into the humidified incubator. After 48 hours, replace the medium in the bottom of each well with two milliliters of fresh growth medium. When all of the medium has been refreshed, seed 2.5 times 10 to the fifth cells of the second population of interest onto the top of the inserts in one milliliter of fresh medium, and return the dish to the incubator.
24, 48, and 96 hours later, replace the medium in the top of each insert with one milliliter of fresh complete medium. And the medium at the bottom of each well with two milliliters of fresh complete medium. After 120 hours of coculture, transfer one insert at a time into individual 35-millimeter cell culture dishes containing one milliliter of PBS.
And wash the bottoms and topsides of the inserts with one milliliter of PBS. To collect the cells grown on the bottom of the insert, place the inserts bottom side down into 200 microliters of room temperature trypsin-EDTA. After two minutes at room temperature, stop the reaction with 800 microliters of complete growth medium.
Then holding the insert at a slight angle, gently pipette the supernatant over the surface of the cells 10 times, collecting the cells in the dish. When all of the inserts have been stripped, resuspend the cells at a concentration of two times 10 to the fifth cells per milliliter of growth medium. And add 250 microliters of cells onto sterile individual glass coverslips.
Place the coverslips into the humidified incubator for one hour. At the end of the incubation, in a laminar flow biological safety cabinet, carefully add two milliliters of complete growth medium to the dish and incubate at 37 degrees Celsius and 5%percent carbon dioxide with humidity for 48 hours. Then wash the cells three times with PBS.
After the third wash, fix the cells in 4%formaldehyde in PBS for ten minutes at room temperature, followed by five washes with Tris-buffered saline. After the last wash, permeabilize the cells with 0.25%Triton X-100 supplemented with 0.1 saponin for five minutes at room temperature, followed by incubation in blocking solution for one hour at room temperature. Next, label the samples with the primary antibody of interest in blocking solution at four degrees Celsius overnight.
The next morning, remove the unbound antibody with three minute washes in wash solution, followed by an one hour room temperature incubation in the appropriate secondary antibody in blocking solution. At the end of the incubation, wash the unbound secondary antibody as just demonstrated and mount the coverslips onto individual slides with antifade mounting medium containing DAPI. Seal the coverslip edges with clear nail polish.
Then image the cells under 63x oil magnification on an inverted microscope equipped with an external light source for fluorescence excitation. This system allows two different cell populations to be grown on either side of the porous membranes of cell culture inserts for at least 120 hours, maintaining a greater than 99%purity in the cell populations on either side of the membrane, when inserts with 0.4 or one micron pores are used. Inserts with three micron pores however, are large enough to allow the cells to migrate across the membrane as observed in this experiment using a GFP-positive human breast cancer cell line.
0.4 micron pore inserts also limit the formation of functional gap junctions between the cell cultures on both sides of the insert, constraining the communication to secreted factors. Inserts with one and three micron pores however, allow the functional coupling of cells through the gap junctions as indicated by the transfer of the fluorescent label across the membrane in these cocultures. Importantly, this system can be used to effectively generate cancer-associated fibroblasts from normal human diploid fibroblasts following their coculture with breast cancer cells, as evidenced by the reduced expression of caveolin-1 on the fibroblasts cocultured with the human breast cancer cells.
While attempting this procedure, it's important to select inserts with a pore size adequate for the question being explored. And to see the first cell population on the bottom side of the insert in medium that facilitates their attachment. Following this procedure, other methods like immunoblotting, in-situ immunofluorescence, or most other cell-based assays can be performed to answer additional questions about protein expression alterations, changes in invasion and migration or differences in responses to therapeutic agents.
After its development, this technique paved the way for researchers in the field of cancer biology and radiation biology to explore the factors that contribute to the development, the evolution of the tumor microenvironment, and in our case to the spread of the harmful effects of ionizing radiation from targeted cells with radiation to the bystander cells in the vicinity. After watching this video, you should have a good understanding of how to prepare a mixed cell coculture, to maintain the coculture, and to harvest high purity enriched cell populations from the coculture for further analysis. Don't forget that working with human cell strains or cell lines can be hazardous and that the appropriate proper personal protection equipment should be worn and the proper biosafety regulations should be adhered to while performing this procedure.
Thanks for watching and good luck with your experiments.
