We thank the Cleveland Clinic Department of Radiation Oncology for use of the linear accelerators. We thank Dr. Jeremy Rich for his generous gift of glioma stem-like cells. This research was supported by the Cleveland Clinic.

1. Cell preparation for suspension cell culture
<ol>
	<li>Culture glioma stem-like cells in stem cell culture media at approximately 5 x 106 cell/10 cm plates in a cell culture incubator with 5% CO2, 95% relative humidity at 37 &#176;C. 
	NOTE: The cell culture condition is the same throughout all the procedures. The media used in the protocol are complete media.</li>
	<li>Two days before scheduled irradiation, collect glioma stem-like cells from the culture plate with a sterile 5 mL pipette into a 15 mL centrifuge tube in a culture hood.</li>
	<li>Centrifuge the collected cells at 200 x g for 3 min in a countertop centrifuge.</li>
	<li>Discard the supernatant and digest the cell pellet (about 1 x 107 cells) with 1 mL of trypsin-EDTA at room temperature for 5 minutes to make a single cell suspension in trypsin-EDTA. Gently shake the bottom of centrifuge tube every 2 minutes during digestion to make sure the cells are digested thoroughly.</li>
	<li>Add 3 mL of stem cell culture media (see the Table of Materials) to quench trypsin. Centrifuge the collected cells at 200 x g for 3 min in a counter top centrifuge. Discard the supernatant and save the cell pellet.</li>
	<li>Resuspend the cells with 5 mL of cell culture media and count the cells with a hemocytometer.</li>
	<li>Plate 5 x 106 cells in two 10 cm plates containing 10 mL of cell culture media.</li>
	<li>Right before the scheduled irradiation, collect the cells and discard the supernatant after centrifugation as described in step 1.3. Re-suspend the cell pellet with 5 mL of cell culture media. Transfer 1 mL of cell suspension into a 35 mm plate containing 2 mL of cell culture media. 
	NOTE: The total volume of media is 3 mL in the 35 mm plate to make the liquid 1 cm in height in the plate.&#160;&#160;</li>
	<li>Transfer the plated cells in a secondary container, such as a plastic or foam box, to reduce the risk of contamination and bring the cells in the container to the irradiation facility on the utility cart.</li>
	<li>Irradiate the cells as described in step 4.</li>
</ol>
2. Cell preparation for attached cell culture
<ol>
	<li>One day before irradiation, in the cell culture hood, remove the DMEM media from the cell culture plate of the attached cell line, such as HEK-293 cells. The media can be suctioned using a Pasteur pipette connected to a vacuum.</li>
	<li>Wash cells with 5 mL of sterile PBS to the culture dish to rinse off residual media.</li>
	<li>Pipette 1 mL of trypsin-EDTA into the culture dish and gently tilt the culture plate to make sure that the entire dish is covered with trypsin-EDTA.</li>
	<li>Trypsinize cells at room temperature for 5 min. Quench the trypsin-EDTA reaction with 3 mL of DMEM media and collect cells with a 5 mL pipette into 15 mL centrifuge tube in the culture hood.</li>
	<li>Centrifuge the collected cells at 200 x g for 3 min in a counter top centrifuge. Discard the supernatant and save the cell pellet.</li>
	<li>Resuspend the cells with 5 mL of DMEM media and count the cells with a hemocytometer.</li>
	<li>Plate 2 x 105 cells in a 35 mm plate using 3 mL of cell culture media to a height of 1 cm of &#160;media.</li>
	<li>After 24 h of cell culture in the incubator at 37 &#176;C, transfer the plated cells in a secondary container (i.e., an insulated foam box) to the irradiation facility on a utility cart.</li>
	<li>Irradiate cells as described in step 4.</li>
</ol>
3. Cell preparation for immunostaining following irradiation
<ol>
	<li>Thaw commercial extracellular protein matrix on ice at 4 &#176;C overnight. Pre-chill 200 &#181;L pipette tips and 1.5 mL centrifuge tubes at 4 &#176;C overnight.</li>
	<li>Aliquot extracellular protein matrix with pre-chilled pipette tips and 1.5 mL centrifuge tubes at 200 &#181;L per tube.</li>
	<li>Dilute 200 &#181;L of extracellular protein matrix in pre-chilled 20 mL of cell culture media to make 1% extracellular protein matrix media.</li>
	<li>Place a sterilized coverslip (22 mm x 22 mm) in a 35 mm plate. Place 400 &#181;L of 1% extracellular protein matrix media on the coverslip.</li>
	<li>Place the 35 mm plate into the cell culture incubator &#160;at 37 &#176;C for 1 h to allow the extracellular protein matrix to polymerize on the coverslip.</li>
	<li>When using suspension cell culture, make a single cell suspension as described in steps 1.1 to 1.5.</li>
	<li>Plate 5 x 104 cells on an extracellular protein matrix-coated coverslip placed in a 35 mm plate. Return plated cells to the cell culture incubator overnight to ensure that cells are supported by the protein matrix. The total volume of the media in the plate should be 3 mL to make the height of media reach 1 cm in the culture dish.</li>
	<li>Observe the cells under a bright field microscope with 10x magnification objective lens. The cells should spread out on the coverslip instead of floating. Transfer the culture dish with plated cells into a secondary container such as a foam box to the irradiation facility on a utility cart and irradiate the cells as described in step 4.</li>
	<li>When using attached cell cultures (for example, HEK-293 cells), digest cells as described in steps 2.1 to 2.6. Place 5 x 104 cells on a sterile cover slip in a 35 mm plate one day prior to the irradiation to make sure cells are fully attached to the coverslip surface by observing them under a microscope as in step 3.9. Use 3 mL of DMEM media to make the liquid 1 cm in height in the plate.</li>
	<li>After growing cells on coverslips overnight or up to one day, transfer the culture dish with plated cells in a secondary container to the irradiation facility. A utility cart may be used to reduce the risk of spillage. Irradiate cells as described in step 4.</li>
</ol>
4. Irradiation with a linear accelerator (LINAC)
<ol>
	<li>Using the LINAC&#39;s console software, set the accelerator gantry and collimator to 0&#176;, open the X and Y jaws to a symmetrical 20 x 20 cm2&#160;field size and retract the multi-leaf collimators (MLCs) if present. 
	NOTE: LINACs may have a flattening filter free (FFF) mode, allowing very high dose rates. As the name suggests, this radiation is not uniform (flat), and the high dose rate is only achieved in the center of the beam. In this case a 7 x 7 cm2 field is used.</li>
	<li>Place at least 5 cm of water equivalent material on the treatment couch. Place the cell dish to be irradiated at 400 MU/min (standard dose rate) onto the water equivalent material and center it at the LINAC crosshairs.</li>
	<li>Place the cells to be irradiated at a depth of maximum dose in a 6 MV X-ray beam, around 1.5 cm. Place an additional 1 cm of water equivalent material on top of the dish. Combined with the 1 cm of medium throughout which the cells are suspended, this places the cells at an average depth of 1.5 cm.</li>
	<li>Affix the front pointer to the head of the LINAC. Extend the front pointer until it contacts the surface of the buildup material and note the distance. Adjust the table height until the distance from the source to the buildup surface is 100 cm. 
	NOTE: The distance can be confirmed with the optical distance indicator. Alternatively, the optical distance indicator may be used in lieu of the front pointer to set the source to buildup surface to 100 cm.</li>
	<li>Calculate the appropriate number of monitor units (MU) to deliver the desired dose of radiation to the cells and program the accelerator to deliver at 400 MU/min. 
	NOTE: For the source to surface distance (SSD) setup on a LINAC calibrated to deliver 1 cGy/MU at the depth of maximum dose, the required number of monitor units is calculated using MU = Dose(cGy) / (1 cGy/MU) / OF(20x20), where OF stands for output factor. This calculation will have to be altered for LINACs using alternate calibration setups.</li>
	<li>Leave the treatment vault and verify that all other individuals have exited. Vrify that there are no other cells in the room, or they will receive low doses of radiation. Close the vault door.</li>
	<li>Confirm the field size, MU and MU/min at the console and then enable the beam.</li>
	<li>Repeat steps 4.3-4.8 for the cells to be irradiated at higher or lower dose rates.
	<ol>
		<li>To achieve higher dose rates (e.g., 2100 MU/min or greater) with the accelerator, decrease the SSD in order to increase the effective dose rate to the cells according to the inverse square law: DoseRateEffective = Doserate * (100 cm / SSD_New)2.</li>
		<li>For low dose rates (e.g., 20 MU/min), increase the source to surface distance (SSD_New) to decrease the dose rate. For example, the cell culture dish may be placed on the floor of the treatment room.</li>
		<li>Recalculate the monitor unit setting on the accelerator if this setup is necessary, using MU = Dose(cGy) / (1 cGy/MU) / OF(20x20)/ (100 cm / SSD_New)2. For additional information on MU calculations, refer to reference by McDermott and Orton18.</li>
	</ol>
	</li>
	<li>Determine dose rate in Gy/minute by DoseRate(Gy/Min) = Dose(Gy)*(MU/min)/MU. e.g., 4 Gy delivered at 400 MU/min requires 380 MU, so 4*400/380 = 4.2 Gy/min. See Table 1.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59514/59514fig1.jpg" /> 
Figure 1: Set-up of the cell culture dish on linear accelerator. (A) A clinical linear accelerator is shown. (B) 5 cm of water equivalent material is placed on the treatment couch. (C) A cell culture dish is placed on the surface of the material. (D) The dish is centered using the accelerator crosshairs in the treatment field shown by the square light field. (E) 1 cm water equivalent material is placed on top of the cell culture dish. The source to surface distance (SSD) is checked using an optical distance indicator (F, G) or a front pointer (H, I). <a href="https://www.jove.com/files/ftp_upload/59514/59514fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Biological assays after irradiation
<ol>
	<li>After irradiation, return the cells to the cell culture incubator in the same manner as described above (step 1.9, 2.8 and 3.10).</li>
	<li>As needed, tailor a variety of biological assays to fit into the research project. 
	NOTE: Here, we show a representative cell cycle analysis16 as an example of a biological assay following irradiation.</li>
</ol>

<ol><li>Stupp, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Radiotherapy+plus+concomitant+and+adjuvant+temozolomide+for+glioblastoma%5BTitle%5D)+AND+%22The+New+England+Journal+of+Medicine%22%5BJournal%5D)">Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.</a> The New England Journal of Medicine. 352 (10), 987-996 (2005).</li>
<li>Stupp, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+radiotherapy+with+concomitant+and+adjuvant+temozolomide+versus+radiotherapy+alone+on+survival+in+glioblastoma+in+a+randomised+phase+III+study%3A+5-year+analysis+of+the+EORTC-NCIC+trial%5BTitle%5D)+AND+%22The+Lancet+Oncology%22%5BJournal%5D)">Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial.</a> The Lancet Oncology. 10 (5), 459-466 (2009).</li>
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<li>Hall, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Radiation+dose-rate%3A+a+factor+of+importance+in+radiobiology+and+radiotherapy%5BTitle%5D)+AND+%22The+British+Journal+of+Radiology%22%5BJournal%5D)">Radiation dose-rate: a factor of importance in radiobiology and radiotherapy.</a> The British Journal of Radiology. 45 (530), 81-97 (1972).</li>
<li>Steel, G. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+dose-rate+effect+in+human+tumour+cells%5BTitle%5D)+AND+%22Radiotherapy+%26+Oncology%22%5BJournal%5D)">The dose-rate effect in human tumour cells.</a> Radiotherapy & Oncology. 9 (4), 299-310 (1987).</li>
<li>Ling, C. C., Gerweck, L. E., Zaider, M., Yorke, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dose-rate+effects+in+external+beam+radiotherapy+redux%5BTitle%5D)+AND+%22Radiotherapy+%26+Oncology%22%5BJournal%5D)">Dose-rate effects in external beam radiotherapy redux.</a> Radiotherapy & Oncology. 95 (3), 261-268 (2010).</li>
<li>Castro, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amotosalen%2FUVA+treatment+inactivates+T+cells+more+effectively+than+the+recommended+gammadose+for+prevention+of+transfusion-associated+graft-versus-host+disease%5BTitle%5D)+AND+%22Transfusion%22%5BJournal%5D)">Amotosalen/UVA treatment inactivates T cells more effectively than the recommended gammadose for prevention of transfusion-associated graft-versus-host disease.</a> Transfusion. 58 (6), 1506-1515 (2018).</li>
<li>Gaddini, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exposing+primary+rat+retina+cell+cultures+to+%CE%B3-rays%3A+An+in+vitro+model+for+evaluating+radiation+responses%5BTitle%5D)+AND+%22Experimental+Eye+Research%22%5BJournal%5D)">Exposing primary rat retina cell cultures to γ-rays: An in vitro model for evaluating radiation responses.</a> Experimental Eye Research. 166, 21-28 (2018).</li>
<li>Simara, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+double-strand+breaks+in+human+induced+pluripotent+stem+cell+reprogramming+and+long-term+in+vitro+culturing%5BTitle%5D)+AND+%22Stem+Cell+Research+%26+Therapy%22%5BJournal%5D)">DNA double-strand breaks in human induced pluripotent stem cell reprogramming and long-term in vitro culturing.</a> Stem Cell Research & Therapy. 8 (1), 73(2017).</li>
<li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comparison+of+the+biological+effects+of+125I+seeds+continuous+low-dose-rate+radiation+and+60Co+high-dose-rate+gamma+radiation+on+non-small+cell+lung+cancer+cells%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">A comparison of the biological effects of 125I seeds continuous low-dose-rate radiation and 60Co high-dose-rate gamma radiation on non-small cell lung cancer cells.</a> PLoS One. 10 (8), 0133728(2015).</li>
<li>Lasio, G., Guerrero, M., Goetz, W., Lima, F., Baulch, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+varying+dose-per-pulse+and+average+dose+rate+in+X-ray+beam+irradiation+on+cultured+cell+survival%5BTitle%5D)+AND+%22Radiation+and+Environmental+Biophysics%22%5BJournal%5D)">Effect of varying dose-per-pulse and average dose rate in X-ray beam irradiation on cultured cell survival.</a> Radiation and Environmental Biophysics. 53 (4), 671-676 (2014).</li>
<li>Karan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Radiobiological+effects+of+altering+dose+rate+in+filter-free+photon+beams%5BTitle%5D)+AND+%22Physics+in+Medicine+and+Biology%22%5BJournal%5D)">Radiobiological effects of altering dose rate in filter-free photon beams.</a> Physics in Medicine and Biology. 58 (4), 1075-1082 (2013).</li>
<li>Sarojini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+combination+of+high+dose+rate+%2810X+FFF%2F2400+MU%2Fmin%2F10+MV+X-rays%29+and+total+low+dose+%280.5+Gy%29+induces+a+higher+rate+of+apoptosis+in+melanoma+cells+in+vitro+and+superior+preservation+of+normal+melanocytes%5BTitle%5D)+AND+%22Melanoma+Research%22%5BJournal%5D)">A combination of high dose rate (10X FFF/2400 MU/min/10 MV X-rays) and total low dose (0.5 Gy) induces a higher rate of apoptosis in melanoma cells in vitro and superior preservation of normal melanocytes.</a> Melanoma Research. 25 (5), 376-389 (2015).</li>
<li>Hao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effects+of+extra+high+on+glioma+stem-like+cells%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">The effects of extra high on glioma stem-like cells.</a> PLoS One. 13 (8), 0202533(2018).</li>
<li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Radiation-induced+G2%2FM+arrest+rarely+occurred+in+glioblastoma+stem-like+cells%5BTitle%5D)+AND+%22International+Journal+of+Radiation+Biology%22%5BJournal%5D)">Radiation-induced G2/M arrest rarely occurred in glioblastoma stem-like cells.</a> International Journal of Radiation Biology. 94 (4), 394-402 (2018).</li>
<li>Mcdermott, P., et al. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMcdermott%2C+P+%22The+Physics+and+Technology+of+Radiation+Therapy%22">The Physics and Technology of Radiation Therapy</a>. , Medical Physics Pub Corp. Madison WI. (2010).</li>
<li>Lohse, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+high+dose+per+pulse+flattening+filter-free+beams+on+cancer+cell+survival%5BTitle%5D)+AND+%22Radiotherapy+%26+Oncology%22%5BJournal%5D)">Effect of high dose per pulse flattening filter-free beams on cancer cell survival.</a> Radiotherapy & Oncology. 101 (1), 226-232 (2011).</li>
</ol>

The authors have nothing to disclose.

Radiotherapy is an integral part of cancer management. Ongoing efforts seek to improve the efficacy and efficiency of radiation treatment. Advancements in linear accelerator technology have provided the opportunity to treat patients with unprecedented accuracy and safety. Because most patients are treated with high energy X-rays from linear accelerators, studies examining the biologic effects of a large range of dose rates performed on linear accelerators may be readily applied to patients. There have been several reports applying linear accelerators to radiation biology research, but results are mixed and additional studies are needed13,14,15,16,19.
Linear accelerators are relatively safe in the sense that after the prescribed dose is delivered there will be no X-rays produced. This is in contrast to radioisotopes where radiation is constantly emitted. Moreover, use of linear accelerators to deliver radiation permits the use of sophisticated beam arrangements to control the shape of the target volume with a large range of doses and dose rates. The disadvantage of this method is that the availability of the linear accelerator may be limited due to patient use and therefore requires advanced planning with clinicians and medical physicists.
In our protocol we describe the basic procedure for irradiation of cells. This method can be tailored to meet other research needs. For example, it can be combined with drug treatment to investigate the effect of combined chemotherapy and radiotherapy. When applying this method to a specific cell line or assay following the radiation, some modifications should be expected. For example, in order to investigate early changes in biomarkers after irradiation, cells may be collected soon after the irradiation.
Because the dose delivered is dependent on beam energy and the deposited dose varies as a function of depth of penetration, the amount of media and water-equivalent bolus needed to achieve the desired dose is critical. In the method described above, we use 6 MV photons in a single anterior-posterior beam and ensure that the cell media is at 1 cm in height in the culture dish. We use an additional 1 cm of water-equivalent material on top of the cell culture dish to build up dose to the cells. To achieve very high dose rates, we raise the couch on which the cells are placed. Because the field size gets smaller as the couch is raised, we use a 35 mm culture dish and ensure that the entire dish is within the irradiation field, as demarcated by the light field. To achieve low dose rates, we lower the treatment couch or place cell culture dish on the floor of the room to increase the source to surface distance. Design of treatment fields for animals, which is more complex, is beyond the scope of this article. The techniques described here will aid researchers in understanding how to use a linear accelerator for biologic assays with cell lines grown in suspension or attached to dishes. Close collaboration between biologists, clinicians and medical physicists will ensure successful execution of the radiation studies.

Radiotherapy is an effective treatment for many types of cancer1,2,3,4. Extra high dose rate irradiation is relatively new in radiation therapy and is made possible by recent technological advances in linear accelerators5. Clinical advantages of extra high dose rate over standard dose rate irradiation include shortened treatment time and improved patient experience. Linear accelerators also provide a clinical setting for cell culture based radiation biology studies. The biological and therapeutic implications of radiation dose and dose rates have been a focus of interest of radiation oncologists and biologists for decades6,7,8. But, the radiobiology of extra high dose rate irradiation and flash irradiation - an extremely high dose rate of radiation - has yet to be thoroughly investigated.
Gamma ray irradiation is widely used in cell culture based radiation biology9,10,11. Radiation is achieved by gamma-rays emitted from decaying radioactive isotope sources, typically Cesium-137. Use of radioactive sources is highly regulated and often restricted. With source-based irradiation, it is challenging to test a wide range of dose rates, limiting its utility in the analysis of the biologic effects of clinical achievable dose rates12.
There have been several studies that illustrate both dose and dose rate effects12,13,14,15,16,17. In these studies, both gamma-irradiation generated from radioactive isotopes or X-rays generated from linear accelerators were used. A variety of cell lines representing lung cancer, cervical cancer, glioblastoma, and melanoma were used. Radiation effects on cell survival, cell cycle arrest, apoptosis and DNA damage were evaluated as readouts12,13,14,15,16,17. Here, we describe a method to define the biological effects of clinically relevant radiation dose and dose rates by delivering X-ray based radiation using a linear accelerator. These studies should be performed with close collaboration between the biologist, radiation oncologist and medical physicist.

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>glioma stem-like cell 387</td><td></td><td></td><td>gift from Dr. Jeremy Rich</td></tr><tr><td>293 cells</td><td>ATCC</td><td>CRL-1573</td><td></td></tr><tr><td>neuron stem cell culture media</td><td>Thermo Fisher Scientific</td><td>21103049</td><td>Neurobasal&lt;sup&gt;TM&lt;/sup&gt; media</td></tr><tr><td>DMEM</td><td>Thermo Fisher Scientific</td><td>10569044</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher Scientific</td><td>16000044</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>Recombinant Human EGF Protein</td><td>R&amp;amp;D Systems</td><td>236-EG-01M</td><td></td></tr><tr><td>Recombinant Human FGF basic</td><td>R&amp;amp;D Systems</td><td>4114-TC-01M</td><td></td></tr><tr><td>B-27&amp;trade; Supplement</td><td>Thermo Fisher Scientific</td><td>17504044</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Thermo Fisher Scientific</td><td>11360070</td><td></td></tr><tr><td>L-Glutamine</td><td>Thermo Fisher Scientific</td><td>25030164</td><td></td></tr><tr><td>Tripsin-EDTA</td><td>Thermo Fisher</td><td>25200056</td><td></td></tr><tr><td>extracellular proten matrix</td><td>Corning</td><td>354277</td><td>Matrigel&lt;sup&gt;TM&lt;/sup&gt;</td></tr><tr><td>Ethanol</td><td>Fisher chemical</td><td>A4094</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10 cm cell culture dish</td><td>Denville</td><td>T1110</td><td></td></tr><tr><td>3.5 cm cell culture dish</td><td>USA Scientific Inc.</td><td>CC7682-3340</td><td></td></tr><tr><td>22x22mm glass cover slip</td><td>electron microscopy sciences</td><td>72210-10</td><td></td></tr><tr><td>15 ml centrifuge tube</td><td>Thomas Scientific</td><td>1159M36</td><td></td></tr><tr><td>50 ml centrifuge tube</td><td>Thomas Scientific</td><td>1158R10</td><td></td></tr><tr><td>5 ml Pipette</td><td>Fisher Scientific</td><td>14-955-233</td><td></td></tr><tr><td>pipet aid</td><td>Fisher Scientific</td><td>13-681-06</td><td></td></tr><tr><td>Vortex mixer</td><td>Fisher Scientific</td><td>02-215-414</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Linear Accelerator</td><td>Varian</td><td>n/a</td><td></td></tr><tr><td>water equivalent material</td><td>Sun Nuclear corporation</td><td>557</td><td>Solid water&lt;sup&gt;TM&lt;/sup&gt;</td></tr><tr><td>&lt;strong&gt;Reagent preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM media</td><td>10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/mL penicillin G, 100 &amp;micro;g/mL streptomycin in 500 ml DMEM media</td><td></td><td></td></tr><tr><td>stem cell culture media</td><td>10 ml B27 supplement, 20 &amp;micro;g hFGF, 20 &amp;micro;g hEGF, 2 mM L-glutamine, 100 units/mL penicillin G, 100 &amp;micro;g/mL streptomycin in 500 ml Neurobasal media</td><td></td><td></td></tr></tbody></table>

use of a linear accelerator for conducting in vitro radiobiology experiments

Radiation therapy remains one of the cornerstones of cancer management. For most cancers, it is the most effective, nonsurgical therapy to debulk tumors. Here, we describe a method to irradiate cancer cells with a linear accelerator. The advancement of linear accelerator technology has improved the precision and efficiency of radiation therapy. The biological effects of a wide range of radiation doses and dose rates continue to be an intense area of investigation. Use of linear accelerators can facilitate these studies using clinically relevant doses and dose rates.

To investigate the cell cycle effect of standard dose rate and extra high dose rate irradiation by a linear accelerator, three samples of glioma stem-like cells were prepared using this protocol and collected 24 h after irradiation17: one control sample that was not irradiated (Figure 2A), one sample irradiated with 400 MU/min (monitor unit, 4.2 Gy/min standard dose rate, Figure 2B) to 4 Gy, and another sample irradiated with 2100 MU/min (21.2 Gy/min extra high dose rate, Figure 2C) to 4 Gy. Cell cycle profiles are shown with the percentages of cells in different phases of the cell cycle.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59514/59514fig2.jpg" /> 
Figure 2: Example of cell cycle analysis after 4 Gy irradiation of linear accelerator. G2 cell cycle arrest was observed after irradiation of glioma stem cells with either a standard dose rate (400 MU/min) (B) or an extra high dose rate (2100 MU/min) (C) compared with non-irradiated control cells (A). <a href="https://www.jove.com/files/ftp_upload/59514/59514fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>DoseRate (MU/MIN)</td>
			<td>SSD (CM)</td>
			<td>Energy</td>
			<td>MU</td>
		</tr>
		<tr>
			<td>20</td>
			<td>250</td>
			<td>6X</td>
			<td>2380*</td>
		</tr>
		<tr>
			<td>400</td>
			<td>100</td>
			<td>6X</td>
			<td>390*</td>
		</tr>
		<tr>
			<td>2100</td>
			<td>80</td>
			<td>6FFF</td>
			<td>260*</td>
		</tr>
	</tbody>
</table>
Table 1: Setup for dose rates used in experiments, assuming 4 Gy dose. MU can be scaled linearly for other required doses. *These are example MU for the LINAC we used. The MU should be calculated for the user&#8217;s specific LINAC using the equation above.

Watch this Scientific Journal Video about Use of a Linear Accelerator for Conducting In Vitro Radiobiology Experiments at JoVE.com

Use of a Linear Accelerator for Conducting In Vitro Radiobiology Experiments

Clinical linear accelerators can be used to determine biologic effects of a wide range of dose rates on cancer cells. We discuss how to set up a linear accelerator for cell-based assays and assays for cancer stem-like cells grown as tumorspheres in suspension and cell lines grown as adherent cultures.

Radiation therapy remains one of the cornerstones of cancer management. For most cancers, it is the most effective, nonsurgical therapy to debulk tumors. ...

use-linear-accelerator-for-conducting-vitro-radiobiology

Universidad Científica del Sur

Research

JoVE Journal

Medicine

7.5K Views.  Cleveland Clinic. Clinical linear accelerators can be used to determine biologic effects of a wide range of dose rates on cancer cells. We discuss how to set up a linear accelerator for cell-based assays and assays for cancer stem-like cells grown as tumorspheres in suspension and cell lines grown as adherent cultures.

Video: Use of a Linear Accelerator for Conducting In Vitro Radiobiology Experiments

Characterization of Recombination Effects in a Liquid Ionization Chamber Used for the Dosimetry of a Radiosurgical Accelerator

Most modern radiation therapy devices allow the use of very small fields, either through beamlets in Intensity-Modulated Radiation Therapy (IMRT) or via stereotactic radiotherapy where positioning accuracy allows delivering very high doses per fraction in a small volume of the patient. Dosimetric measurements on medical accelerators are conventionally realized using air-filled ionization chambers. However, in small beams these are subject to nonnegligible perturbation effects. This study focuses on liquid ionization chambers, which offer advantages in terms of spatial resolution and low fluence perturbation. Ion recombination effects are investigated for the microLion detector (PTW) used with the Cyberknife system (Accuray). The method consists of performing a series of water tank measurements at different source-surface distances, and applying corrections to the liquid detector readings based on simultaneous gaseous detector measurements. This approach facilitates isolating the recombination effects arising from the high density of the liquid sensitive medium and obtaining correction factors to apply to the detector readings. The main difficulty resides in achieving a sufficient level of accuracy in the setup to be able to detect small changes in the chamber response.

A growing number of radiation therapy devices offer the advantage of delivering the dose through very small beams to the tumor, allowing for increased conformity and higher doses per fraction. Many different detectors can be used for the dosimetry of these small fields. In the present study, the effect of ion recombination is investigated for a liquid ionization chamber using a stereotactic radiation therapy system.

Most modern radiation therapy devices allow the use of very small fields, either through beamlets in Intensity-Modulated Radiation Therapy (IMRT) or via ...

Engineering

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without the ability to target a specific or well-delineated tumor volume. The delivery of radiation was achieved using fixed radiation sources or linear accelerators producing megavoltage (MV) X-rays. These devices are unable to achieve sub-millimeter precision required for small animals. Furthermore, the high doses delivered to healthy surrounding tissue hamper response assessment. To increase the translation between small animal studies and humans, our goal was to mimic the treatment of human glioblastoma in a rat model. To enable a more accurate irradiation in a preclinical setting, recently, precision image-guided small animal radiation research platforms were developed. Similar to human planning systems, treatment planning on these micro-irradiators is based on computed tomography (CT). However, low soft-tissue contrast on CT makes it very challenging to localize targets in certain tissues, such as the brain. Therefore, incorporating magnetic resonance imaging (MRI), which has excellent soft-tissue contrast compared to CT, would enable a more precise delineation of the target for irradiation. In the last decade also biological imaging techniques, such as positron emission tomography (PET) gained interest for radiation therapy treatment guidance. PET enables the visualization of e.g., glucose consumption, amino-acid transport, or hypoxia, present in the tumor. Targeting those highly proliferative or radio-resistant parts of the tumor with a higher dose could give a survival benefit. This hypothesis led to the introduction of the biological tumor volume (BTV), besides the conventional gross target volume (GTV), clinical target volume (CTV), and planned target volume (PTV).
At the preclinical imaging lab of Ghent University, a micro-irradiator, a small animal PET, and a 7 T small animal MRI are available. The goal was to incorporate MRI-guided irradiation and PET-guided sub-volume boosting in a glioblastoma rat model.

In the past, small animal irradiation was usually performed without the ability to target a well-delineated tumor volume. The goal was to mimic the treatment of human glioblastoma in rats. Using a small animal irradiation platform, we performed MRI-guided 3D conformal irradiation with PET-based sub-volume boosting in a preclinical setting.

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

Cancer Research

Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation can induce a progressive and debilitating decline in cognition that may, in part, be caused by the depletion of neural stem cells. Given the increased survival of patients diagnosed with brain cancer, quality of life in terms of cognitive health has become an increasing concern, especially in the absence of any satisfactory long-term treatments.
To address this serious health concern we have used stem cell replacement as a strategy to combat radiation-induced cognitive decline. Our model utilizes athymic nude rats subjected to cranial irradiation. The ionizing radiation is delivered as either whole brain or as a highly focused beam to the hippocampus via linear accelerator (LINAC) based stereotaxic radiosurgery. Two days following irradiation, human neural stem cells (hNSCs) were stereotaxically transplanted into the hippocampus. Rats were then assessed for changes in cognition, grafted cell survival and for the expression of differentiation-specific markers 1 and 4-months after irradiation. Our cognitive testing paradigms have demonstrated that animals engrafted with hNSCs exhibit significant improvements in cognitive function. Unbiased stereology reveals significant survival (10-40%) of the engrafted cells at 1 and 4-months after transplantation, dependent on the amount and type of cells grafted. Engrafted cells migrate extensively, differentiate along glial and neuronal lineages, and express a range of immature and mature phenotypic markers.
Our data demonstrate direct cognitive benefits derived from engrafted human stem cells, suggesting that this procedure may one day afford a promising strategy for the long-term functional restoration of cognition in individuals subjected to cranial radiotherapy. To promote the dissemination of the critical procedures necessary to replicate and extend our studies, we have provided written and visual documentation of several key steps in our experimental plan, with an emphasis on stereotaxic radiosurgey and transplantation.

Brain tumor patients routinely undergo cranial radiotherapy, and while beneficial, this treatment often results in debilitating cognitive dysfunction. This serious unresolved problem has at present, no clinical recourse, and has driven our efforts to devise stem cell based therapies for the recovery of radiation-induced cognitive decrements.

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation ...

Positron Emission Tomography-based Dose Painting Radiation Therapy in a Glioblastoma Rat Model using the Small Animal Radiation Research Platform

A rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was previously established. Similar to the clinical treatment, computed tomography (CT) and magnetic resonance imaging (MRI) were combined during the treatment-planning process. Positron emission tomography (PET) imaging was subsequently added to implement sub-volume boosting using a micro-irradiation system. However, combining three imaging modalities (CT, MRI, and PET) using a micro-irradiation system proved to be labor-intensive because multimodal imaging, treatment planning, and dose delivery have to be completed sequentially in the preclinical setting. This also results in a workflow that is more prone to human error. Therefore, a user-friendly algorithm to further optimize preclinical multimodal imaging-based radiation treatment planning was implemented. This software tool was used to evaluate the accuracy and efficiency of dose painting radiation therapy with micro-irradiation by using an in silico study design. The new methodology for dose painting radiation therapy is superior to the previously described method in terms of accuracy, time efficiency, and intra- and inter-user variability. It is also an important step towards the implementation of inverse treatment planning on micro-irradiators, where forward planning is still commonly used, in contrast to clinical systems.

Here we present a protocol to perform preclinical positron emission tomography-based radiotherapy in a rat glioblastoma model using algorithms developed in-house to optimize the accuracy and efficiency.

A rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was previously established. Similar to the clinical ...

Radiation Response in Patient-Derived Tumor Organoids Using a Clonogenic Assay

Live Imaging to Quantify Cellular Radiosensitivity in Patient-Derived Tumor Organoids

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, often (but not always) because of differences in the ability of malignant cells to repair oxidative DNA damage as elicited by ionizing rays. Clonogenic assays have been employed for decades to assess the sensitivity of cultured cancer cells to ionizing irradiation, largely because irradiated cancer cells often die in a delayed manner that is difficult to quantify with short-term flow cytometry- or microscopy-assisted techniques. Unfortunately, clonogenic assays cannot be employed as such for more complex tumor models, such as patient-derived tumor organoids (PDTOs). Indeed, irradiating established PDTOs may not necessarily abrogate their growth as multicellular units, unless their stem-like compartment is completely eradicated. Moreover, irradiating PDTO-derived single-cell suspensions may not properly recapitulate the sensitivity of malignant cells to RT in the context of established PDTOs. Here, we detail an adaptation of conventional clonogenic assays that involves exposure of established PDTOs to ionizing radiation, followed by single-cell dissociation, replating in suitable culture conditions and live imaging. Non-irradiated (control) PDTO-derived stem-like cells reform growing PDTOs with a PDTO-specific efficiency, which is negatively influenced by irradiation in a dose-dependent manner. In these conditions, PDTO-forming efficiency and growth rate can be quantified as a measure of radiosensitivity on time-lapse images collected until control PDTOs achieve a predefined space occupancy.

Author Spotlight: Radiotherapy and Clonogenic Assays for Advancing Cancer Research and Personalized Medicine

Here, we detail a straightforward live imaging approach for quantifying the sensitivity of patient-derived tumor organoids to ionizing radiation.

Radiation therapy (RT) is one of the mainstays of modern clinical cancer management. However, not all cancer types are equally sensitive to irradiation, ...

Dynamic Lung Tumor Tracking for Stereotactic Ablative Body Radiation Therapy

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable risks for normal tissue injury and the hazards of physical tumor miss. A first-of-its-kind SBRT platform achieves high-precision ablative radiation treatment through a combination of versatile real-time imaging solutions and sophisticated tumor tracking capabilities. It uses dual-diagnostic kV x-ray units for stereoscopic open-loop feedback of cancer target intrafraction movement occurring as a consequence of respiratory motions and heartbeat. Image-guided feedback drives a gimbaled radiation accelerator (maximum 15 x 15 cm field size) capable of real-time &#177;4 cm pan-and-tilt action. Robot-driven &#177;60&#176; pivots of an integrated &#177;185&#176; rotational gantry allow for coplanar and non-coplanar accelerator beam set-up angles, ultimately permitting unique treatment degrees of freedom. State-of-the-art software aids real-time six dimensional positioning, ensuring irradiation of cancer targets with sub-millimeter accuracy (0.4 mm at isocenter). Use of these features enables treating physicians to steer radiation dose to cancer tumor targets while simultaneously reducing radiation dose to normal tissues. By adding respiration correlated computed tomography (CT) and 2-[18F] fluoro-2-deoxy-&#7429;-glucose (18F-FDG) positron emission tomography (PET) images into the planning system for enhanced tumor target contouring, the likelihood of physical tumor miss becomes substantially less1. In this article, we describe new radiation plans for the treatment of moving lung tumors.

Stereotactic ablative body radiation therapy (SBRT) involves the precise delivery of high-dose radiation to cancer tumor targets. A novel SBRT platform offers a first-of-its-kind gimbaled radiation accelerator mounted within a pivoting O-ring gantry capable of dynamic image-guided tumor tracking. Here, we describe dynamic tumor tracking for lung targets.

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable ...

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the safety of stem cell transplantation. Although the transplantation of differentiated rather than undifferentiated cells is common practice, teratomas can still arise from the presence of residual undifferentiated stem cells at the time of transplant or from spontaneous mutations in differentiated cells. Because stem cell therapies are often delivered into anatomically sensitive sites, even small tumors can be clinically devastating, resulting in blindness, paralysis, cognitive abnormalities, and cardiovascular dysfunction. Surgical access to these sites may also be limited, leaving patients with few therapeutic options. Controlling stem cell misbehavior is, therefore, critical for the clinical translation of stem cell therapy.
External beam radiation offers an effective means of delivering targeted therapy to decrease the teratoma burden while minimizing injury to surrounding organs. Additionally, this method avoids genetic manipulation or viral transduction of stem cells-which are associated with additional clinical safety and efficacy concerns. Here, we describe a protocol to create pluripotent stem cell-derived teratomas in mice and to apply external beam radiation therapy to selectively ablate these tumors in vivo.

Research on treatment strategies for pluripotent stem cell-derived teratomas is important for the clinical translation of stem cell therapy. Here, we describe a protocol to, first, generate stem cell-derived teratomas in mice and, then, to selectively target and treat these tumors in vivo using a small-animal irradiator.

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the ...

Proton Therapy Delivery and Its Clinical Application in Select Solid Tumor Malignancies

Radiation therapy is a frequently used modality for the treatment of solid cancers. Although the mechanisms of cell kill are similar for all forms of radiation, the in vivo properties of photon and proton beams differ greatly and maybe exploited to optimize clinical outcomes. In particular, proton particles lose energy in a predictable manner as they pass through the body. This property is used clinically to control the depth at which the proton beam is terminated, and to limit radiation dose beyond the target region. This strategy can allow for substantial reductions in radiation dose to normal tissues located just beyond a tumor target. However, the degradation of proton energy in the body remains highly sensitive to tissue density. As a consequence, any changes in tissue density during the course of treatment may significantly alter proton dosimetry. Such changes may occur through alterations in body weight, respiration, or bowel filling/gas, and may result in unfavorable dose deposition. In this manuscript, we provide a detailed method for the delivery of proton therapy using both passive scatter and pencil beam scanning techniques for prostate cancer. Although the described procedure directly pertains to prostate cancer patients, the method may be adapted and applied for the treatment of virtually all solid tumors. Our aim is to equip readers with a better understanding of proton therapy delivery and outcomes in order to facilitate the appropriate integration of this modality during cancer therapy.

The fundamentals of radiation planning and delivery for proton therapy using prostate cancer as a model are presented. The application of these principles to other selected disease sites highlights how proton radiotherapy may enhance clinical outcomes for cancer patients.

Radiation therapy is a frequently used modality for the treatment of solid cancers. Although the mechanisms of cell kill are similar for all forms of ...

Dosimetry for Cell Irradiation using Orthovoltage (40-300 kV) X-Ray Facilities

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose determination using low energy X-ray facilities, but depending on the irradiation configurations, samples, materials or beam quality, it is sometimes difficult to know which protocol is the most appropriate to employ. We, therefore, propose a dosimetry protocol for cell irradiations using low energy X-ray facility. The aim of this method is to perform the dose estimation at the level of the cell monolayer to make it as close as possible to real cell irradiation conditions. The different steps of the protocol are as follows: determination of the irradiation parameters (high voltage, intensity, cell container etc.), determination of the beam quality index (high voltage-half value layer couple), dose rate measurement with ionization chamber calibrated in air kerma conditions, quantification of the attenuation and scattering of the cell culture medium with EBT3 radiochromic films, and determination of the dose rate at the cellular level. This methodology must be performed for each new cell irradiation configuration as the modification of only one parameter can strongly impact the real dose deposition at the level of the cell monolayer, particularly involving low energy X-rays.

Dosimetry for Cell Irradiation using Orthovoltage 40-300 kV X-Ray Facilities

This document describes a new dosimetry protocol for cell irradiations using low energy X-ray equipment. Measurements are performed in conditions simulating real cell irradiation conditions as much as possible.