The overall goal of the TTFields in vitro application system is to investigate their effect on cancer cells from different tumor types, to guide TTFields clinical administration and optimize treatment outcome. This method can help define the effect of different TTField parameters on cancer cells. The main advantage of this technique is that it allows for pre-clinical identification of the most effective TTField frequency that inhibits growth of a specific cancer cell line, which could be ultimately applied in humans.
By slight modification of this technique, it is possible to test the efficacy of the combined treatment of TTField and other treatment modalities, such as chemotherapy agents and radiation therapy. Demonstrating the procedure will be Yaara Porat, a researcher from our team. To begin, install TTFields dishes with covers onto a base plate and prepare an additional eight dishes that will be used for growing control cells.
Place a sterile two millimeter coverslip on the bottom of each dish. Next, suspend five times 10 to the fourth U-87 MG cells in one milliliter of DMEM medium. The appropriate number of cells plated in each dish depends on the properties of the cell line in use.
It is crucial to calibrate it prior to the experiment in order to avoid cell overgrowth. Pipet 200 microliters of the cell suspension onto each coverslip such that a drop is formed on its surface, and cover the dishes with their lids. Incubate all dishes at 37 degrees Celsius, 5%carbon dioxide overnight to allow the cells to adhere.
Once the cells are attached, aspirate the media from the coverslips using a 200 microliter pipet, then carefully pipet two milliliters of complete growth medium onto each dish. Gently tap with a sterile pipet tip on the coverslip edges to release any air bubbles caught under the slide. Afterward, incubate the cells at 37 degrees Celsius, 5%carbon dioxide until TTFields treatment begins.
To begin TTFields treatment, remove the base plate from the 37 degrees Celsius incubator and place it into a refrigerated carbon dioxide incubator. The refrigerated incubator temperature will determine electric field intensity. It is important to use the appropriate intensity, which is dependent on cell sensitivity to TTFields.
Then plug a flat cable female connector into the base plate and turn the TTFields generator on. Open the software dedicated for TTFields in vitro application system. After defining a new study by entering names of the experiment and experimenter, adjust the frequency and target temperature for each dish.
Click on the start button to initiate the TTFields treatment in the software, and check if all dishes appear light blue on the monitor, which confirms their proper connection. To reestablish the connection, gently press the dish down and rotate it slowly back and forth until the dish appears light blue on the screen. After 24 hours of the treatment, stop the experiment in the software by clicking pause.
Disconnect the flat cable connector from the base plate and then place the base plate in the laminar flow cabinet, then remove the base plate with the dishes and the control cells grown in standard conditions from their incubators. Replace the medium in all dishes with fresh complete growth medium, then place the control cells in a 37 degrees Celsius, 5%carbon dioxide incubator and return the base plate to the refrigerated incubator. Reconnect the base plate to the generator.
Continue the treatment by clicking the continue button. Once the treatment is completed, finish the experiment by clicking the end experiment button in the software, then save the data uploaded from the system. After turning the TTFields generator off, disconnect the flat cable from the base plate and remove the system from the incubator.
Press down and turn the ceramic dish counterclockwise to detach it from the base plate. Next, aseptically transfer the TTFields treated and control coverslips to sterile Petri dishes containing fresh medium for further evaluation. To evaluate the effect of TTFields treatment, remove the medium from each coverslip containing dish, then add 0.5 milliliters of 0.25%trypsin/EDTA, and incubate the dishes at 37 degrees Celsius, 5%carbon dioxide until the cells detach from the coverslip surface.
Neutralize the trypsin with 0.5 milliliters of complete growth medium, and re-suspend the cells by gently pipetting the suspension up and down. Finally, count the cells by flow cytometry. Presented here is the results of the frequency scan carried out in A2780 cells, revealing the frequency dependent influence of TTFields on the growth and clonogenic capacity of the studied cells.
The most prominent treatment efficacy was observed when the frequency of 200 kilohertz was applied. The efficacy of TTFields treatment carried out at two different intensities was investigated in OVCAR-3 cells. The study shows a clear relationship between the applied intensity and the treatment outcome, with the greater growth inhibitory activity of the higher field intensity.
Regardless of the applied field intensity, the strongest effect of the TTFields treatment was observed at the frequency of 200 kilohertz. Once mastered, this technique can be done in less than 30 minutes a day. While attempting this procedure, it's important to remember to avoid media spillage and to keep the dishes balanced when transferring them from the incubator to the hood, and back.
Following TTField application, other methods, like flow cytometry, alanine protein analysis or immunofluorescent microscopy can be performed in order to further investigate the treatment outcome and the mechanism underlying the observed activity. This technique is used to determine the optimal TTFields frequencies to be applied in the clinical settings for different types of cancers. After watching this video, you should have a good understanding of how to plan and execute TTFields in vitro experiments.
Do not forget that working with human cell lines can be hazardous, and precautions, such as protective gloves and clothes should always be taken while performing this procedure.
