Name: an in-house-built and light-emitting-diode-based photodynamic therapy device for enhancing verteporfin cytotoxicity in a 2d cell culture model
Uploaded: 2023-01-13T12:40:00
Description: Watch this Scientific Journal Video about An In-House-Built and Light-Emitting-Diode-Based Photodynamic Therapy Device for Enhancing Verteporfin Cytotoxicity in a 2D Cell Culture Model at JoVE.com

We thank Arthur Henrique Gomes de Oliveira and Lucas Julian Cruz Gomes for helping with the filming process. This project was supported by the Brazilian Research Council (CNPq, grant numbers 400953/2016-1-404286/2021-6) and Funda&#231;&#227;o Arauc&#225;ria-PPSUS 2020/2021 (SUS2020131000003). This study was also financed in part by Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior-Brasil (CAPES)-Finance Code 001.

NOTE: See the Table of Materials for details related to all materials, reagents, and software used in this protocol.
1. Device construction
<ol>
	<li>Saw 3 mm thickn medium-density fiberboards (MDF) to obtain pieces with the dimensions shown in Figure 1A. 
	NOTE: Use the vector file (Supplemental File 1) for computer numerical control (CNC) cutting.</li>
	<li>Build two boxes with the following dimensions (l...

<ol>
	<li>Ferlay, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+agency+for+research+on+cancer.">International agency for research on cancer.</a> Global Cancer Observatory: Cancer Today. 23 (7), 323-326 (2018).</li><li>Gottesman, M. M., Fojo, T., Bates, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidrug+resistance+in+cancer:+role+of+Atp-dependent+transporters.">Multidrug resistance in cancer: role of Atp-dependent transporters.</a> Nature Reviews Cancer. 2 (1), 48-58 (2002).</li><li>Szakacs, G., Paterson, J. K., Ludwig, J. A., Boothe-Genthe, C., Gottesman, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+multidrug+resistance+in+cancer.">Targeting multidrug resistance in cancer.</a> Nature Reviews Drug Discovery. 5 (3), 219-234 (2006).</li><li>Ackroyd, R., Kelty, C., Brown, N., Reed, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+history+of+photodetection+and+photodynamic+therapy.">The history of photodetection and photodynamic therapy.</a> Photochemistry and Photobiology. 74 (5), 656-669 (2001).</li><li>Hamblin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+for+cancer:+what's+past+is+prologue.">Photodynamic therapy for cancer: what's past is prologue.</a> Photochemistry and Photobiology. 96 (3), 506-516 (2020).</li><li>Barr, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contrasting+mechanisms+of+colonic+collagen+damage+between+photodynamic+therapy+and+thermal+injury.">The contrasting mechanisms of colonic collagen damage between photodynamic therapy and thermal injury.</a> Photochem Photobiol. 46 (5), 795-800 (1987).</li><li>Algorri, J. F., Ochoa, M., Rold&#225;n-Varona, P., Rodr&#237;guez-Cobo, L., L&#243;pez-Higuera, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy:+A+compendium+of+latest+reviews.">Photodynamic therapy: A compendium of latest reviews.</a> Cancers. 13 (17), 4447 (2021).</li><li>Aniogo, E. C., Plackal, B., George, B. P. A., Abrahamse, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+photodynamic+therapy+on+multidrug+resistant+breast+cancer.">The role of photodynamic therapy on multidrug resistant breast cancer.</a> Cancer Cell International. 19, 91 (2019).</li><li>Spring, B. Q., Rizvi, I., Xu, N., Hasan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+photodynamic+therapy+in+overcoming+cancer+drug+resistance.">The role of photodynamic therapy in overcoming cancer drug resistance.</a> Photochemical &amp; Photobiological Sciences. 14 (8), 1476-1491 (2015).</li><li>Dougherty, T. J., Grindey, G. B., Fiel, R., Weishaupt, K. R., Boyle, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoradiation+therapy.+II.+Cure+of+animal+tumors+with+hematoporphyrin+and+light.">Photoradiation therapy. II. Cure of animal tumors with hematoporphyrin and light.</a> Journal of the National Cancer Institute. 55 (1), 115-121 (1975).</li><li>Etcheverry, M. E., Pasquale, M. A., Garavaglia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+of+HeLa+cell+cultures+by+using+LED+or+laser+sources.">Photodynamic therapy of HeLa cell cultures by using LED or laser sources.</a> Journal of Photochemistry and Photobiology B: Biology. 160, 271-277 (2016).</li><li>Guo, Q., Dong, B., Nan, F., Guan, D., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Aminolevulinic+acid+photodynamic+therapy+in+human+cervical+cancer+via+the+activation+of+microRNA-143+and+suppression+of+the+Bcl-2/Bax+signaling+pathway.">5-Aminolevulinic acid photodynamic therapy in human cervical cancer via the activation of microRNA-143 and suppression of the Bcl-2/Bax signaling pathway.</a> Molecular Medicine Reports. 14 (1), 544-550 (2016).</li><li>Mroz, P., Yaroslavsky, A., Kharkwal, G. B., Hamblin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+pathways+in+photodynamic+therapy+of+cancer.">Cell death pathways in photodynamic therapy of cancer.</a> Cancers. 3 (2), 2516-2539 (2011).</li><li>Mahalingam, S. M., Ordaz, J. D., Low, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+a+photosensitizer+to+the+mitochondrion+enhances+the+potency+of+photodynamic+therapy.">Targeting of a photosensitizer to the mitochondrion enhances the potency of photodynamic therapy.</a> ACS Omega. 3 (6), 6066-6074 (2018).</li><li>Granville, D. J., Levy, J. G., Hunt, D. W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+treatment+with+benzoporphyrin+derivative+monoacid+ring+A+produces+protein+tyrosine+phosphorylation+events+and+DNA+fragmentation+in+murine+P815+cells.">Photodynamic treatment with benzoporphyrin derivative monoacid ring A produces protein tyrosine phosphorylation events and DNA fragmentation in murine P815 cells.</a> Photochemistry and Photobiology. 67 (3), 358-362 (1998).</li><li>Castano, A. P., Demidova, T. N., Hamblin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+in+photodynamic+therapy:+part+two+-+cellular+signaling,+cell+metabolism+and+modes+of+cell+death.">Mechanisms in photodynamic therapy: part two - cellular signaling, cell metabolism and modes of cell death.</a> Photodiagnosis Photodynamic Therapy. 2 (1), 1-23 (2014).</li><li>Detty, M. R., Gibson, S. L., Wagner, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+clinical+and+preclinical+photosensitizers+for+use+in+photodynamic+therapy.">Current clinical and preclinical photosensitizers for use in photodynamic therapy.</a> Journal of Medicinal Chemistry. 47 (16), 3897-3915 (2004).</li><li>Allison, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy:+oncologic+horizons.">Photodynamic therapy: oncologic horizons.</a> Future Oncology. 10 (1), 123-142 (2014).</li><li>Chepurna, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+with+laser+scanning+mode+of+tumor+irradiation.">Photodynamic therapy with laser scanning mode of tumor irradiation.</a> Optical Fibers and Their Applications 2015. 9816, 323-326 (2015).</li><li>Huang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+progress+in+clinical+photodynamic+therapy.">A review of progress in clinical photodynamic therapy.</a> Technology in Cancer Research and Treatment. 4 (3), 283-293 (2005).</li><li>Chepurna, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LED-based+portable+light+source+for+photodynamic+therapy.">LED-based portable light source for photodynamic therapy.</a> Optics in Health Care and Biomedical Optics. 11190, 109-115 (2019).</li><li>Hasson, O., Wishkerman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CultureLED:+A+3D+printer-based+LED+illumination+cultivation+system+for+multi-well+culture+plates.">CultureLED: A 3D printer-based LED illumination cultivation system for multi-well culture plates.</a> HardwareX. 12, 00323 (2022).</li><li>Wu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localised+light+delivery+on+melanoma+cells+using+optical+microneedles.">Localised light delivery on melanoma cells using optical microneedles.</a> Biomedical Optics Express. 13 (2), 1045-1060 (2022).</li><li>Erkiert-Polguj, A., Halbina, A., Polak-Pacholczyk, I., Rotsztejn, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-emitting+diodes+in+photodynamic+therapy+in+non-melanoma+skin+cancers-own+observations+and+literature+review.">Light-emitting diodes in photodynamic therapy in non-melanoma skin cancers-own observations and literature review.</a> Journal of Cosmetic and Laser Therapy. 18 (2), 105-110 (2016).</li><li>Neupane, J., Ghimire, S., Shakya, S., Chaudhary, L., Shrivastava, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+light+emitting+diodes+in+the+photodynamic+therapy+of+rheumatoid+arthritis.">Effect of light emitting diodes in the photodynamic therapy of rheumatoid arthritis.</a> Photodiagnosis and Photodynamic Therapy. 7 (1), 44-49 (2010).</li><li>Lins, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+785-nm+laser+diode-based+system+for+standardization+of+cell+culture+irradiation.">A novel 785-nm laser diode-based system for standardization of cell culture irradiation.</a> Photomedicine and Laser Surgery. 31 (10), 466-473 (2013).</li><li>Hopkins, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+vitro+cell+irradiation+protocol+for+testing+photopharmaceuticals+and+the+effect+of+blue,+green,+and+red+light+on+human+cancer+cell+lines.">An In vitro cell irradiation protocol for testing photopharmaceuticals and the effect of blue, green, and red light on human cancer cell lines.</a> Photochemical and Photobiological Sciences. 15 (5), 644-653 (2016).</li><li>Zhang, K., Waguespack, M., Kercher, E. M., Spring, B. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+and+stable+LED+array+illumination+system+for+multiwell+plate+cell+culture+photodynamic+therapy+experiments.">An automated and stable LED array illumination system for multiwell plate cell culture photodynamic therapy experiments.</a> Research Square. , 1-18 (2022).</li><li>G&#225;lvez, E. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+and+evaluation+of+the+operational+characteristics+of+a+new+photodynamic+therapy+device.">Analysis and evaluation of the operational characteristics of a new photodynamic therapy device.</a> Photodiagnosis and Photodynamic Therapy. 37, 102719 (2022).</li><li>Bretin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+activity+of+new+human+colorectal+cancer.">Photodynamic therapy activity of new human colorectal cancer.</a> Cancers. 11 (10), 1474 (2019).</li><li>. T. SketchUp Available from: <a target="_blank" href="https://www.sketchup.com/">https://www.sketchup.com/</a> (2022)</li><li>LCDR PhotoDynamic Therapy (PDT) Equipment Repository. GitHub, Inc Available from: <a target="_blank" href="https://github.com/PhotoDynamicTherapy">https://github.com/PhotoDynamicTherapy</a> (2022)</li><li>W3C CSS Color Module Level 3. W3C, Inc Available from: <a target="_blank" href="https://www.w3.org/TR/css-color-3/#SRGB">https://www.w3.org/TR/css-color-3/#SRGB</a> (2022)</li></ol>

The final PhotoACT device was convenient to construct with commercially available, low-cost components at a total cost of less than $50. Additional advantages include low maintenance demands, the capacity to irradiate multiple types of culture plates, the simultaneous use of up to four units per assay, low weight (2 kg)/size (44 cm3) that allows portability, accurate and reproducible irradiation (data not shown), and a user-friendly and simple setup interface that does not require connection to computers or ot...

Among the most lethal noncommunicable diseases, cancer represents a global leading cause of premature death. It accounted for nearly 10 million deaths in 2020, representing about one in six deaths worldwide1. Additionally, the multidrug resistance (MDR) phenomenon represents a tremendous public health threat, as approved chemotherapeutic protocols fail to reach remission stages for this clinical condition2. Cancer cells can develop resistance to chemotherapy through several mechanisms; however, the overexpression of some ATP-binding cassette (ABC) transporters - ATP-dependent efflux pumps - is considered the main cause of MDR development within a tumor microenvironment3. In addition to MDR, other cancer complications, such as recurrence and metastasis, reinforce the urgent demand to develop and improve therapeutic approaches to overcome this oncological challenge.
The curative utilization of light has been practiced for centuries4, and photodynamic therapy (PDT) represents a clinically approved therapeutic approach for solid tumors. PDT combines the administration of a photosensitizer (PS) followed by light irradiation to generate reactive oxygen species (ROS) to exert selective cytotoxicity in tumor cells. This therapeutic approach is superior to conventional methods, including surgery, radiation, and chemotherapy5; it is a minimally invasive technique showing lower cytotoxicity in connective tissues6. The light application and PS accumulation directly in the tumor or its microenvironment ensure precise targeting and, consequently, minor, undesirable systemic side effects7 and the possibility of repeated treatment at the same site. Moreover, the cost is lower than that of other approaches. Owing to its promising features, PDT can be considered an appropriate option for both single, especially in the case of inoperable tumors, or adjuvant cancer treatment7, and represents an alternative for MDR related to chemotherapy8,9.
The first report showing a high objective response rate using PDT was described in 1975 in a mouse and rat model10. Since then, studies have been conducted using PDT with positive outcomes7 both in vivo and in vitro with human tumor cell lines in 2D cell culture11,12. Considering the broad applicability of clinically approved PS, regardless of their specific accumulation pathways and wavelength ranges of absorption peaks, the general process is as follows: (i) PS uptake, (ii) peaking of PS concentration at the tumor or its microenvironment, (iii) light application, (iv) PS-light interaction, (v) transfer of PS excited-state energy to either tissue substrate or surrounding oxygen molecules, (vi) ROS production involving singlet oxygen or superoxide anion, (vii) tumor cell death via, essentially, necrosis or apoptosis (direct death), autophagy (cytoprotective mechanism), tissue ischemia (vascular damage), immune modulation, or an overlap of these mechanisms7. In this final stage, the activation of a specific cell death pathway depends on many factors, such as cell characteristics, experimental design and, most importantly, PS intracellular localization and PDT-related targeted damage13.
Verteporfin is a second-generation PS, approved by regulatory agencies for clinical use in Norway and China to treat age-related macular degeneration7. After dose delivery, this prodrug was reported to partially accumulate in mitochondria14 and induce cellular protein tyrosine phosphorylation and DNA fragmentation, leading to tumor cell apoptosis15,16. After 24 h incubation for verteporfin internalization, a PDT protocol using a 690 nm wavelength setup is recommended to achieve effective levels of electromagnetic radiation transfer to adjacent molecules7,17.
Regarding the light source for PDT, the classical diode laser systems are usually expensive, technically complicated, oversized, and thus unportable18,19. As a consequence of its single-wavelength profile, which can also be observed in LED-based PDT equipment, the demand for independent units for each photosensitizer application makes the utilization of diode laser systems even more complex and economically unfeasible20,21. Therefore, the utilization of LED machinery is considered the most promising alternative to solve not only costs22 and maintenance issues, but also to provide high power output and less harmful23 and wider illuminating capability24,25,26,27.
Despite the potential contribution that LED-based equipment can offer to PDT experiments28, most commercial options still possess drawbacks such as a lack of portability, high cost, and complex construction projects and operation29. The main objective of this work was to offer a simple and reliable tool for in vitro PDT assays. This paper describes PhotoACT, an in-house-built LED-based PDT device, which is inexpensive, user-friendly, and portable. As a proof of concept, this device is shown to enhance the cytotoxicity of verteporfin in a 2D cell culture model and, therefore, can be used as a research tool in PDT experiments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5% Trypsin-EDTA (10x)</td>
			<td>Gibco</td>
			<td>15400054</td>
			<td>Mammalian cell culture dissociation reagent</td>
		</tr>
		<tr>
			<td>3D printer</td>
			<td>Flashforge</td>
			<td></td>
			<td>Finder model</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td></td>
			<td></td>
			<td>Non-sterile, polystyrene, and high-binding surface plates with flat bottom wells used for 2D cell culture</td>
		</tr>
		<tr>
			<td>Arduino</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brightness sensor</td>
			<td></td>
			<td></td>
			<td>TSL2561 model with 0.1-40.000+ lux detection levels and I2C interface</td>
		</tr>
		<tr>
			<td>Buttons</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buzzer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture Flasks</td>
			<td></td>
			<td></td>
			<td>Sterile, polystyrene, rectangular bottom flask with Tissue Culture (TC)-treated surface, canted neck and vent cap (sizes)</td>
		</tr>
		<tr>
			<td>Centrifuge Tubes</td>
			<td></td>
			<td></td>
			<td>Sterile, polypropylene tubes with 15/50 mL capacity used for cell culture dilution at seeding step of the assay</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Controller board</td>
			<td></td>
			<td></td>
			<td>ESP32</td>
		</tr>
		<tr>
			<td>Design Software</td>
			<td>Trimble</td>
			<td></td>
			<td>SketchUp</td>
		</tr>
		<tr>
			<td>DMEM High Glucose</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td>DMEM (Dulbecco&#39;s Modified Eagle Medium) is a widely used basal medium for supporting the growth of many different mammalian cells.</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D4540-500ML</td>
			<td>Dimethyl sulfoxide, &#8805;99.5% (GC), suitable for plant cell culture</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>Gibco</td>
			<td>12657029</td>
			<td>FBS provides the best value by delivering consistency of cell growth over time and passages.</td>
		</tr>
		<tr>
			<td>Gentamicin (50 mg/mL)</td>
			<td>Gibco</td>
			<td>15750060</td>
			<td>Water-soluble antibiotic drug originally purified from the fungus&#160;Micromonospora purpurea. Gentamicin acts by preventing cell culture contamination</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td></td>
			<td></td>
			<td>Neubauer patterned chamber used for cell counting at seeding step of the assay</td>
		</tr>
		<tr>
			<td>Inverted Laboratory Microscope</td>
			<td>Leica</td>
			<td></td>
			<td>DM IL LED</td>
		</tr>
		<tr>
			<td>Laminar Flow Hood</td>
			<td></td>
			<td></td>
			<td>Cabin designed to protect the working environment from contaminants by maintaining a constant, unidirectional flow of HEPA-filtered air over the work area. Used at several steps of cell cultivation and treatment procedures</td>
		</tr>
		<tr>
			<td>LCD display</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LED RGB WS2812</td>
			<td></td>
			<td></td>
			<td>5050 RGB SMD model with a built-in processor. Tape with 30 LEDs, 1 meter length and 9 watts</td>
		</tr>
		<tr>
			<td>MDF fiberboards</td>
			<td></td>
			<td></td>
			<td>3mm thickness medium-density fiberboards</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes</td>
			<td></td>
			<td></td>
			<td>Sterile, polypropylene tubes with safety lid and 1.5/2.0 mL capacity. Convenient tools for manipulating small volumes at treatment step of the assay</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>ThermoFischer</td>
			<td></td>
			<td>Multiskan FC Microplate Photometer designed to detect a broad wavelength range of absorbance (340-850 nm). The equipment was used to evaluate cell viability after MTT incubation.</td>
		</tr>
		<tr>
			<td>MTT Reagent</td>
			<td>Invitrogen</td>
			<td>M6494</td>
			<td>3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Used for cell viability assays</td>
		</tr>
		<tr>
			<td>Operational System</td>
			<td>Real Time Engineers ltd.</td>
			<td></td>
			<td>FreeRTOS</td>
		</tr>
		<tr>
			<td>P10 micripipette</td>
			<td></td>
			<td></td>
			<td>Non-electronic, single-channel, 1-10 &#956;L capacity</td>
		</tr>
		<tr>
			<td>P1000 micropipette</td>
			<td></td>
			<td></td>
			<td>Non-electronic, single-channel, 10-1000 &#956;L capacity</td>
		</tr>
		<tr>
			<td>P200 micropipette</td>
			<td></td>
			<td></td>
			<td>Non-electronic, single-channel, 20-200 &#956;L capacity</td>
		</tr>
		<tr>
			<td>PDT Equipment</td>
			<td>LumaCare</td>
			<td></td>
			<td>Model LC-122</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline pH 7.4</td>
			<td>Gibco</td>
			<td>10010031</td>
			<td>Balanced salt formulation used for washing cells during cultivation and assay procedures</td>
		</tr>
		<tr>
			<td>Potentiometers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tips</td>
			<td></td>
			<td></td>
			<td>Non-sterile, universal fit, 10/200/1000 &#956;L maximum volumes</td>
		</tr>
		<tr>
			<td>Verteporfin</td>
			<td>Sigma-Aldrich</td>
			<td>SML0534-5MG</td>
			<td>Verteporfin, &#8805;94% (HPLC)</td>
		</tr>
	</tbody>
</table>

an in-house-built and light-emitting-diode-based photodynamic therapy device for enhancing verteporfin cytotoxicity in a 2d cell culture model

This paper describes a novel, simple, and low-cost device to perform in vitro photodynamic therapy (PDT) assays, named the PhotoACT. The device was built using a set of conventional programmable light-emitting diodes (LEDs), a liquid crystal display (LCD) module, and a light sensor connected to a commercial microcontroller board. The box-based structure of the prototype was made with medium-density fiberboards (MDFs). The internal compartment can simultaneously allocate four cell culture multiwell microplates.
As a proof of concept, we studied the cytotoxic effect of the photosensitizer (PS) verteporfin against the HeLa cell line in two-dimensional (2D) culture. HeLa cells were treated with increasing concentrations of verteporfin for 24 h. The drug-containing supernatant medium was discarded, the adherent cells were washed with phosphate-buffered saline (PBS), and drug-free medium was added. In this study, the effect of verteporfin on cells was examined either without light exposure or after exposure for 1 h to light using red-green-blue (RGB) values of 255, 255, and 255 (average fluence of 49.1 &#177; 0.6 J/cm2). After 24 h, the cell viability was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Experimental results showed that exposure of cells treated with verteporfin to the light from the device enhances the drug&#39;s cytotoxic effect via a mechanism mediated by reactive oxygen species (ROS). In addition, the use of the prototype described in this work was validated by comparing the results with a commercial PDT device. Thus, this LED-based photodynamic therapy prototype represents a good alternative for in vitro studies of PDT.

The final PDT device, named the PhotoACT, included a dark chamber to allocate up to four multiwell microplates, with its upper interior surface equipped with a set of 30 scattered LEDs programmed to emit distinct spectrums of visible light (Figure 3 and Supplemental File 6). The device was built using two associated boxes: an internal box designed as a dark chamber for the PDT assays, and an external box to cover the internal chamber and hold the control unit (<strong class=...

Watch this Scientific Journal Video about An In-House-Built and Light-Emitting-Diode-Based Photodynamic Therapy Device for Enhancing Verteporfin Cytotoxicity in a 2D Cell Culture Model at JoVE.com

An In-House-Built and Light-Emitting-Diode-Based Photodynamic Therapy Device for Enhancing Verteporfin Cytotoxicity in a 2D Cell Culture Model

Here, we describe a novel, simple, and low-cost device to successfully perform in vitro photodynamic therapy (PDT) assays using two-dimensional HeLa cell culture and verteporfin as a photosensitizer.

This paper describes a novel, simple, and low-cost device to perform in vitro photodynamic therapy (PDT) assays, named the PhotoACT. The device was built ...

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