The overall goal of this procedure is to establish proliferative tetraploid cells, free of a diploid population, from normal human fibroblasts. This method can help answer key questions in the field of carcinogenesis research, such as how aneuploid cells develop in the early stages of carcinogenesis. The main advantage of this technique is that proliferative tetraploid cells, free of a diploid population, can be established by relatively simple procedures.
While some strains become completely tetraploid by simple treatment with spindle poison, demecolcine, most need to be handled with the shake-off method during that treatment to emulate a diploid population. To begin the induction of tetraploidy without the shake-off method, passage approximately five times 10 to the 1/5 cultured cells into a 100-millimeter dish. Incubate the dish in a 5%carbon dioxide atmosphere at 37 degrees Celsius for 24 hours.
After this, treat the exponentially-growing cells with medium containing 0.1 micrograms per milliliter DC.Incubate in a 5%carbon dioxide atmosphere at 37 degrees Celsius for four days. Then replace the DC-containing medium with drug-free medium. Incubate in 5%carbon dioxide at 37 degrees Celsius until proliferation resumes.
To induce tetraploidy utilizing the shake-off method, passage the cultured cells into T-75 flasks, making sure that each flask contains at least one times 10 to the 1/6 cells. Incubate the flasks in a 5%carbon dioxide atmosphere at 37 degrees Celsius for 24 hours. The next day, treat the cells with medium containing DC for 16 to 18 hours to arrest them in mitosis as outlined in the text protocol.
After incubation, check the cells in a flask under the inverted phase contrast microscope. And then gently shake each flask to begin the collection of loosely-adhered mitotic cells. Careless handling, such as violent shaking or touching the surface of the flask by the pipette tip should be avoided.
Do not contaminate the mitotic cells with adhered interface cells. Gently wash the flasks with warm medium, and transfer the cells to centrifuge tubes. Utilize an atomic cell counter to count the cell number.
After cell counting, centrifuge at 300 times g for five minutes. Reseed the collected mitotic cells into 16-millimeter culture dishes. Then treat the plated cells with medium containing 0.1 micrograms per milliliter DC.Incubate in 5%carbon dioxide at 37 degrees Celsius for three days.
After this, replace the DC-containing medium with drug-free medium. Incubate in a 5%carbon dioxide atmosphere at 37 degrees Celsius until proliferation resumes. For analysis of DNA ploidy, wash the cells with PBS.
Then add a solution containing 0.05%trypsin and 0.02%EDTA to the cells. Incubate for 10 minutes at 37 degrees Celsius. After this, transfer the cell suspension to a centrifuge tube.
And neutralize the trypsin by adding medium containing 10%fetal bovine serum. Centrifuge the cells in medium at 300 times g for five minutes. Resuspend the cells in five milliliters of PBS.
Then centrifuge again at 300 times g for five minutes. Next, resuspend cells in one milliliter of PBS. Add four milliliters of 100%ethanol drop-wise with mixing.
Let the suspension rest at room temperature for 30 minutes to fix the cells. After this, centrifuge the cells in fixative solution at 300 times g for five minutes. And resuspend cells in five milliliters of PBS.
Centrifuge the suspension at 300 times g for five minutes. Then treat the cells with 475 microliters of 0.5 milligrams per milliliter RNA solution. Add 25 microliters of one milligram per milliliter propidium iodine solution to stain the cells.
And then mix. Allow the suspension to rest at room temperature for 30 minutes. Next, use a flow cytometer with a 488 nanometer laser and red channel emission filter to analyze the DNA content of the cells.
Analyze a reference sample prepared from genuine diploid cells as well to assess ploidy of target cells. To begin, treat exponentially-growing cells with medium containing 0.1 micrograms per milliliter DC for four hours to arrest the cells in metaphase. After this, harvest at least five times 10 to the 1/5 cells by trypsinization.
Suspend the harvested cells in medium containing FBS. Centrifuge the suspension at 300 times g for five minutes. Then add five milliliters of a 0.075 molar potassium chloride solution.
Incubate at 37 degrees Celsius for 10 minutes. After incubation is complete, centrifuge the suspension at 300 times g for five minutes. Resuspend the cells in five milliliters of Carnoy's fixative.
And then centrifuge the suspension at 300 times g for five minutes. Then aspirate the supernatant and resuspend cells in 0.5 milliliters of Carnoy's fixative. Drop 30 microliters of the cell suspension onto a glass slide.
Finally, stain and analyze the cells via either chromosome counts or karyotype analysis. In this study, proliferative tetraploid cells, free of a diploid population, are cultured from normal human fibroblasts. Although some cell strains can become completely tetraploid by continuous treatment with DC, most cells need to be treated with the shake-off method during the course of DC treatment to eliminate a diploid population.
The representative results for the induction of tetraploidy in three fibroblast strains are shown here. The strain TIG-1 becomes almost entirely tetraploid through simple continuous treatment with DC.However, this treatment causes the BJ and TIG-hT strains to become a mixture of diploid and tetrapod populations. Therefore, isolation of mitotic cells by the shake-off method is necessary during the treatment process.
A chromosome count is performed in the established tetraploid cells. As can be seen, the modal chromosome number is 92 for both TIG-1 and BJ cells, and 91 for TIG-hT cells. Once mastered, this technique can be done in two weeks if it is performed properly.
While attempting this procedure, it's important to remember to gently shake the flasks in the shake-off step, not to cause contamination of mitotic cells by adherent interface cells.
