Determining Optimal Inhibitory Tumor Treating Field Frequency for Cancer Cells: A Non-invasive In Vitro Method to Assess the Effect of TTFields on Cancer Cell Viability

Overview

This video demonstrates the effect of TTField application on the growth of ovarian cancer cell lines in vitro. This assay can help determine the optimal frequency of the electric field that can lead to a maximum reduction in the number of cancer cells.

Protocol

1. TTFields In Vitro Application System - Base Plate and Dish Maintenance

1. Rinse dishes and dish covers under running tap water. Then, rinse with deionized water and air-dry face down.
2. If a chemotherapeutic agent/drug has been added to dishes in a prior experiment, fill each dish with a 5% light detergent solution and leave overnight.
   1. Rinse the dishes thoroughly under running tap water to avoid any traces of detergent inside the dish. Rinse the dishes and dish covers with deionized water and air-dry face down.
3. Insert the clean and dry dishes with their covers into sterilization bags. Seal and place the bags in an autoclave, with the dishes face down. Autoclave for 30 min at no higher than 121 °C.
4. Set the autoclave on a dry program, partly open the autoclave door, and dry the dishes for 30 min.
5. Wipe the base plate with a cloth lightly moistened with 70% ethanol.

2. Experiment Setup

NOTE: All equipment and materials used in this protocol are described in the Materials List. All steps below should be performed inside a laminar flow cabinet while sterile conditions are maintained.

1. Prepare clean and sterile TTFields dishes and covers, as described in section 1. Wipe the base plate with a cloth lightly moistened with 70% ethanol.
2. Install the TTFields dishes with the covers onto the base plate by pressing down gently on the dish and rotating clockwise by about 5 mm until the rim of the dish locks onto the three pins on the base plate. Place a sterile 22-mm coverslip (treated plastic or glass) on the bottom of each dish.
3. Prepare the cell suspension in complete growth medium (Dulbecco's modified Eagle's medium for U-87 MG and F98; RPMI for A2780 and OVCAR-3; supplemented as described in Materials List).
   NOTE: The cell concentrations depend on the cell types and experiment duration; 80%-90% confluent cell growth should not be exceeded by the end of the experiment.
   CAUTION: Human cell lines may represent a biological hazard and should be handled with the appropriate safety measures.
4. Place 200 µL of cell suspension (usually 5,000-20,000 cells in 200 µL for a 72-h experiment, depending on the doubling time) as a drop in the center of each coverslip and cover with the dish lids.
5. Incubate at 37 °C in a CO₂ incubator until the cells adhere; an overnight incubation is possible at this stage.
6. Aspirate the fluids from the drop using a 200- or 1,000-µL pipette.
7. Gently pipet 2 mL of complete growth medium to fill each dish. Use a sterile tip to pipet and softly tap on the coverslip edges to release the air bubbles occasionally caught under the slide.
8. Cover the dishes with their lids and place them inside a CO₂ incubator at 37 °C until the start of the TTFields treatment.

3. TTFields Application

1. Transfer the base plates with the attached TTFields dishes to a refrigerated CO₂ incubator.
   NOTE: In vitro TTFields application will generate heat; therefore, a refrigerated incubator is required to compensate for the warming of the dishes. Higher TTFields intensities will produce more heat and will require lower incubator ambient temperatures (see Table 1). Clinically relevant TTFields intensities are achieved when the incubator temperature for TTFields application is in the range of 18-30 °C.
2. Connect the flat cable female connector to the base plate. Switch the TTFields generator on.
3. Start the TTFields in vitro application system software and select the experiment settings.
   1. Define a new experiment. Type the name of the experiment and the experiment owner in the software user interface on the computer screen. Adjust the frequency and target temperature for each dish or base plate.
4. Start the TTFields application using the software.
5. Verify that all dishes are connected properly and appear light blue on the monitor. If a dish is circled with a red frame (meaning that it is not properly connected), press it down gently and rotate slowly back and forth until the contacts are restored and the dish appears light blue.
6. Leave the TTFields in vitro application system running for up to 24 h.
7. If the experiment reaches its endpoint, stop the experiment by clicking on the END EXPERIMENT button in the software and proceed to step 5. If not, click on PAUSE experiment.
8. Disconnect the flat cable connector from the base plate. Remove the base plate with the dishes from the incubator into a laminar flow cabinet.
9. Replace the medium in all dishes every 24 h and return the base plate to the refrigerated incubator. Connect the base plate to the generator using the flat cable. Continue the experiment by clicking on the CONTINUE button.

Incubator ambient temperature	Expected TTFields intensities
(°C)	(V/cm RMS)
18	1.62
19	1.55
20	1.48
21	1.41
22	1.33
23	1.26
24	1.19
25	1.12
26	1.04
27	0.97
28	0.9
29	0.83
30	0.76

Table 1. Incubator ambient temperature and expected TTFields intensities inside the TTFields dish.

4. Control Samples

Note: Grow control cells in similar conditions to TTFields-treated cells, excluding TTFields application.

1. Plate control samples with the same suspension and on a similar surface used for plating TTFields-treated samples.
2. Place control dishes in a CO₂ incubator at 37 °C.
3. Replace the medium in the dishes every 24 h to match the TTFields-treated dish medium conditions.

5. Experiment End

1. After clicking on END EXPERIMENT, wait for the software to retrieve all records from the system and to save the records to the computer.
2. Use the REPORTS button to review the temperature, current, and resistance log files of each base plate to verify that all dishes were treated according to the experimental plan.
   NOTE: All reports and logs are saved to the computer after the end of the experiment and can be reviewed at any time.
3. Switch off the TTFields generator.
4. Disconnect the flat cable from the base plate and remove it from the incubator.
5. To remove a ceramic dish, press down and turn the dish counterclockwise to unlock it from the base plate.
6. Take the dishes into a laminar flow cabinet and aseptically remove the coverslips. Transfer them to sterile Petri dishes containing fresh medium or phosphate-buffered saline (PBS) for further inspection and evaluation.

Materials

Name	Company	Catalog Number	Comments
Invitro system and software	Novocure	ITG1000 and IBP1000	Each unit contains 1 TTFields generator, 1 base plate, 8 TTFields dishes with covers and 1 flat cable.
Sterilization bags	Westfield medica	24882
Plastic cover slides	Thermo Scientific (NUNC)	174977	Pre treated and sterilized
Glass cover slides	Thermo Scientific (Menzel-Gläser)	CB00220RA1	Sterilize if necessary
Dulbecco’s modified Eagle’s medium	Biological Industries (Israel)	01-055-1A	Warm in 37 °C water bath before use
RPMI 1640	Gibco	21875-034	Warm in 37 °C water bath before use
Fetal Bovine Serum (FBS)	Biological Industries (Israel)	04-007-1A	Warm in 37 °C water bath before use
L-Glutamine 200 mM (100x)	Gibco	25030-029
Pen/Strep (10,000 U/mL Penicillin, 10,000 µg/mL Streptomycin)	Gibco	15140-122
Sodium Pyruvate solution 100 mM	Biological Industries (Israel)	03-042-1B
Hepes buffer 1 M	Biological Industries (Israel)	03-025-1B
Insulin solution from bovine pancreas	Sigma-Aldrich	10516-5ML
0.25% Trypsin/EDTA	Biological Industries (Israel)	03-050-1B	Warm in 37 °C water bath before use
Methanol	Merck	1.06009.2511	Cool to -20 °C in the freezer before use
PBS	Biological Industries (Israel)	02-023-1A	Without calcium and magnesium
A2780	ECACC	93112519	Grow in RPMI 1640 supplemented with FBS (10%), pen/strep (100 U/ mL / 100 µg/mL), sodium pyruvate (1 mM) and Hepes buffer (12 mM).
F98	ATCC	CRL-2397	Grow in Dulbecco’s modified Eagle’s medium supplemented with FBS (10%), pen/strep (100 U/mL / 100 µg/mL), sodium pyruvate (1 mM) and glutamine (2 mM).
Ovcar-3	ATCC	HTB-161	Grow in RPMI 1640 supplemented with FBS (20%), pen/strep (100 U/ mL / 100 µg/mL), sodium pyruvate (1 mM), Hepes buffer (12 mM) and insulin (10 µg/mL).
U-87 MG	ATCC	HTB-14	Grow in Dulbecco’s modified Eagle’s medium supplemented with FBS (10%), pen/strep (100 U/mL / 100 µg/mL), sodium pyruvate (1 mM) and glutamine (2 mM).
Refrigerated CO2 incubator	CARON	7404-10-3
Laminar flow cabinet	ADS Laminair	Bio12 and VSM12