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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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 ± 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'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.

Introduction

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 o....

Protocol

NOTE: See the Table of Materials for details related to all materials, reagents, and software used in this protocol.

1. Device construction

  1. 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.
  2. Build two boxes with the following dimensions (l.......

Representative Results

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 (

Discussion

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.......

Acknowledgements

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 Fundação Araucária-PPSUS 2020/2021 (SUS2020131000003). This study was also financed in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES)-Finance Code 001.

....

Materials

NameCompanyCatalog NumberComments
0.5% Trypsin-EDTA (10x)Gibco15400054Mammalian cell culture dissociation reagent
3D printerFlashforgeFinder model
96-well platesNon-sterile, polystyrene, and high-binding surface plates with flat bottom wells used for 2D cell culture
Arduino
Brightness sensorTSL2561 model with 0.1-40.000+ lux detection levels and I2C interface
Buttons
Buzzer
Cell culture FlasksSterile, polystyrene, rectangular bottom flask with Tissue Culture (TC)-treated surface, canted neck and vent cap (sizes)
Centrifuge TubesSterile, polypropylene tubes with 15/50 mL capacity used for cell culture dilution at seeding step of the assay
CO2 Incubator
Controller boardESP32
Design SoftwareTrimbleSketchUp
DMEM High GlucoseGibco11965092DMEM (Dulbecco's Modified Eagle Medium) is a widely used basal medium for supporting the growth of many different mammalian cells.
DMSOSigma-AldrichD4540-500MLDimethyl sulfoxide, ≥99.5% (GC), suitable for plant cell culture
Fetal Bovine Serum Gibco12657029FBS provides the best value by delivering consistency of cell growth over time and passages.
Gentamicin (50 mg/mL)Gibco15750060Water-soluble antibiotic drug originally purified from the fungus Micromonospora purpurea. Gentamicin acts by preventing cell culture contamination
HemocytometerNeubauer patterned chamber used for cell counting at seeding step of the assay
Inverted Laboratory MicroscopeLeicaDM IL LED
Laminar Flow HoodCabin 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
LCD display
LED RGB WS28125050 RGB SMD model with a built-in processor. Tape with 30 LEDs, 1 meter length and 9 watts
MDF fiberboards3mm thickness medium-density fiberboards
Microcentrifuge TubesSterile, polypropylene tubes with safety lid and 1.5/2.0 mL capacity. Convenient tools for manipulating small volumes at treatment step of the assay
Microplate readerThermoFischerMultiskan 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.
MTT ReagentInvitrogenM64943-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Used for cell viability assays
Operational SystemReal Time Engineers ltd.FreeRTOS
P10 micripipetteNon-electronic, single-channel, 1-10 μL capacity
P1000 micropipetteNon-electronic, single-channel, 10-1000 μL capacity
P200 micropipetteNon-electronic, single-channel, 20-200 μL capacity
PDT EquipmentLumaCareModel LC-122
Phosphate-Buffered Saline pH 7.4Gibco10010031Balanced salt formulation used for washing cells during cultivation and assay procedures
Potentiometers
TipsNon-sterile, universal fit, 10/200/1000 μL maximum volumes
VerteporfinSigma-AldrichSML0534-5MGVerteporfin, ≥94% (HPLC)

References

  1. Ferlay, J., et al. International agency for research on cancer. Global Cancer Observatory: Cancer Today. 23 (7), 323-326 (2018).
  2. Gottesman, M. M., Fojo, T., Bates, S. E. Multidrug resistance in cancer: role of Atp-dependent transporters. ....

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Photodynamic TherapyPDTLight emitting DiodeLEDVerteporfinCytotoxicity2D Cell CultureIn house built DevicePhotoActLow costHeLa CellsDulbecco s Modified Eagle MediumCell Culture

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