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

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

To begin, install TTFields dishes with covers onto a base plate and prepare an additional 8 dishes that will be used for growing control cells. Place a sterile 2-millimeter coverslip on the bottom of each dish. Next, suspend 5 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 microlitres 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 pipette. Then, carefully pipet 2 milliliters of complete growth medium onto each dish. Gently tap with a sterile pipette 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 the 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 TTField&#39;s treatment in the software and check if all dishes appear light blue on the monitor, which confirms their proper connection. To re-establish 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 degree 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.

determining-optimal-inhibitory-tumor-treating-field-frequency-for

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Cancer Research

Gynecologic Cancer

Assays & techniques

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1. TTFields In Vitro Application System -&#160;Base Plate and Dish Maintenance
<ol>
	<li>Rinse dishes and dish covers under running tap water. Then, rinse with deionized water and air-dry face down.</li>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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 &#176;C.</li>
	<li>Set the autoclave on a dry program, partly open the autoclave door, and dry the dishes for 30 min.</li>
	<li>Wipe the base plate with a cloth lightly moistened with 70% ethanol.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Prepare the cell suspension in complete growth medium (Dulbecco&#39;s modified Eagle&#39;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.</li>
	<li>Place 200 &#181;L of cell suspension (usually 5,000-20,000 cells in 200 &#181;L 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.</li>
	<li>Incubate at 37 &#176;C in a CO2 incubator until the cells adhere; an overnight incubation is possible at this stage.</li>
	<li>Aspirate the fluids from the drop using a 200- or 1,000-&#181;L pipette.</li>
	<li>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.</li>
	<li>Cover the dishes with their lids and place them inside a CO2 incubator at 37 &#176;C until the start of the TTFields treatment.</li>
</ol>
3. TTFields Application
<ol>
	<li>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 &#176;C.</li>
	<li>Connect the flat cable female connector to the base plate. Switch the TTFields generator on.</li>
	<li>Start the TTFields in vitro application system software and select the experiment settings.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Start the TTFields application using the software.</li>
	<li>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.</li>
	<li>Leave the TTFields in vitro application system running for up to 24 h.</li>
	<li>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.</li>
	<li>Disconnect the flat cable connector from the base plate. Remove the base plate with the dishes from the incubator into a laminar flow cabinet.</li>
	<li>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.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Incubator ambient temperature&#160;</td>
			<td>Expected TTFields intensities&#160;</td>
		</tr>
		<tr>
			<td>(&#176;C)</td>
			<td>(V/cm RMS)</td>
		</tr>
		<tr>
			<td>18</td>
			<td>1.62</td>
		</tr>
		<tr>
			<td>19</td>
			<td>1.55</td>
		</tr>
		<tr>
			<td>20</td>
			<td>1.48</td>
		</tr>
		<tr>
			<td>21</td>
			<td>1.41</td>
		</tr>
		<tr>
			<td>22</td>
			<td>1.33</td>
		</tr>
		<tr>
			<td>23</td>
			<td>1.26</td>
		</tr>
		<tr>
			<td>24</td>
			<td>1.19</td>
		</tr>
		<tr>
			<td>25</td>
			<td>1.12</td>
		</tr>
		<tr>
			<td>26</td>
			<td>1.04</td>
		</tr>
		<tr>
			<td>27</td>
			<td>0.97</td>
		</tr>
		<tr>
			<td>28</td>
			<td>0.9</td>
		</tr>
		<tr>
			<td>29</td>
			<td>0.83</td>
		</tr>
		<tr>
			<td>30</td>
			<td>0.76</td>
		</tr>
	</tbody>
</table>
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.
<ol>
	<li>Plate control samples with the same suspension and on a similar surface used for plating TTFields-treated samples.</li>
	<li>Place control dishes in a CO2 incubator at 37 &#176;C.</li>
	<li>Replace the medium in the dishes every 24 h to match the TTFields-treated dish medium conditions.</li>
</ol>
5. Experiment End
<ol>
	<li>After clicking on END EXPERIMENT, wait for the software to retrieve all records from the system and to save the records to the computer.</li>
	<li>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.</li>
	<li>Switch off the TTFields generator.</li>
	<li>Disconnect the flat cable from the base plate and remove it from the incubator.</li>
	<li>To remove a ceramic dish, press down and turn the dish counterclockwise to unlock it from the base plate.</li>
	<li>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.</li>
</ol>

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

Source: Porat, Y. et al. <a href="https://jove.unicartagenaproxy.elogim.com/t/55820">Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields (TTFields).</a> J. Vis. Exp. (2017)
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.

Source: Porat, Y. et al. Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields (TTFields). J. Vis. Exp. (2017)
This ...

Tumor Treating Fields, or TTFields, use alternating electric fields to disrupt cancer cell proliferation. To study the effect of TTFields on cancerous cells in vitro, begin by assembling TTFields compatible dishes over a base plate. A pair of electrodes positioned on the dish walls helps deliver electric fields internally.
Place a glass coverslip inside the assembled dish. Seed a drop of cancer cell suspension onto the coverslip. Incubate to allow the cells to settle and adhere to the coverslip surface. Discard the spent media. Supplement it with a fresh growth media to support the growth and expansion of cancer cells.
Transfer the assembly to a refrigerated environment to compensate for dish heating during electric field application. Next, connect the assembly to a power supply. Subject the cells to an appropriate electric field for the desired duration.
Within rapidly dividing cells, the applied field causes charged molecules like tubulin dimers and septins to forcefully align in its direction. This mechanism interferes with key mitotic events like microtubule polymerization and septin fiber localization. The resulting mitotic arrest triggers apoptosis leading to tumor cell death.

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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

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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