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An In-House-Built and Light-Emitting-Diode-Based Photodynamic Therapy Device for Enhancing Verteporfin Cytotoxicity in a 2D Cell Culture Model
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Summary January 13th, 2023
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.
Transcript
Photodynamic therapy, PDT, offers several advantage to cancer treatment, and its efficacy depends on a light source to activate a photosensitizer. Although recent advances in the field, there is a lack of access to an expensive and reproducible device for PTD for in vitro models. In order to achieve this demand, this work describes a novel, simple, and low-cost device to perform PDT assays on cell cultures, named PhotoAct.
To start the construction, saw three millimeters thickness medium density fiber boards, MDF, to obtain pieces with the following dimensions. Build two boxes with the following dimensions. Drill the back of the bigger box to install a barrel jack connector.
Also drill the top of the bigger box, the top and the bottom of the smaller box to provide a passage for electric cables. Paint all the internal surfaces with black ink to promote homogeneous light incidence. Attach in parallel three LED tapes with 10 LEDs each at the upper interior surface of the smaller box.
Additionally, install a brightness sensor at the center of the bottom interior surface of the smaller box. Print the structure of the control unit using the supplemental 3D printing file. Install all components, power button, potentiometers, time start touchpad, LEDs, brightness sensor, LCD, buzzer, and power supply, and the parts of an ESP-32 controller board mounted at the control unit interior.
Upload the programming code available at supplemental file, and run a test to check that all connections are working. Assemble the boxes and fix them together to avoid gaps and consequently external lighting interference and immediate light loss. Attach the mounted control unit to the drilled area at the top of the prototype.
Build a front door of the same material in the following dimensions, and fix it on the outer box with hinges and Velcro tapes to ensure chamber closure and uninterrupted assays. Also install a handle to manipulate the front door with ease and precision. Attach four rubber foot pads at the bottom of the prototype to ensure more stability during the operations.
Cultivate HeLa cell line in Dulbecco's Modified Eagle Medium low glucose with 10%of fetal bovine serum and 1%of gentamycin. Keep the culture flasks at 5%of carbon dioxide and 37 Celsius degrees. Manage and inspect the cell culture until reaching 80 to 90%of confluence.
Start the cell viability protocol with the seeding process. Remove the medium from the flask with confluent HeLa cell culture. Wash the flask with phosphate buffer saline, PBS, and detach the culture with trypsin following highlighted details.
Count the resuspend cells with a hemocytometer and seed them into a multi-well microplate at concentration of 20, 000 cells per well. Prepare two plates for dark and light conditions of treatment and incubate them for 24 hours to cell attachment. To proceed with treatment with a photosensitizer, remove the medium from both plates and treat the cells with 100 microliters of increasing concentrations of Verteporfin.
Keep the cells in treatment for 24 hours to allow Verteporfin internalization. After incubation, remove the treatment, wash the cells with PBS and add drug-free medium. Cover one microplate with aluminum foil to protect it from light exposure and incubate it for 24 hours.
This microplate will offer control data for further analysis of PDT results. The other microplate will be utilized on light exposure condition at the PhotoAct. To operate the equipment, plug it into the outlet and turn it on, pressing the power button.
Place the multi-well microplate at the PDT chamber and close the equipment by fastening the frontal door with the side Velcro tapes. To set up the equipment, use the potentiometers to adjust RGB configuration of light emission. Press plus/minus touchpad to adjust time configuration and set the assay duration.
Check if the correct information about the assay is shown on the display and make final adjustments if necessary. Press the start touchpad to initiate the assay. A one-beep buzzer must be heard at the beginning of the experiment.
During the experiment, progress information can be observed at the display, such as irradiance and time left. Do not open the front door or change any configuration during the PDT assay. At the end of the assay, a four-beep buzzer must be heard and the electronic system will turn all the LEDs off.
A finished message and the final amount of energy expanded during the experiment can be observed at the display. The fluence final value is calculated according to the highlighted equation. Cover the microplate that suffered light exposure and proceed with the 24-hour incubation.
After incubation period, remove medium from both plates, wash the monolayer of cells with PBS, and add MTT solution. Incubate both plates dark and light conditions for four hours to allow formazan crystals formation. Remove the MTT solution carefully and dissolve the purple crystals with a DMSO and ethanol solution.
After complete crystals dissolution, carry out the absorbance measurement using a microplate reader at 595 nanometers. The final product consists of a dark chamber with its upper interior surface equipped with a set of 30 scattered light emitting diodes, LEDs, programmed to emit distinct spectrums of visible light. A homogeneous light incidence is established due to the low reflectivity of interior surfaces and uniform distribution of LEDs configuration.
The setup interface is user-friendly and the settled experimental condition was reproducible. As a proof of concept, the device was used to enhance the cytotoxic effect of Verteporfin in 2D HeLa cell culture after light exposure. As shown in the figure, the GI50 value was 3.1 micromolar for the light condition.
and 13.8 micromolar for the dark condition Therefore, the increase of more than fourfold in efficiency comparing the conditions confirms the use of Verteporfin as a photosensitizer and the applicability of PhotoAct on PDT assays. To validate the use of the prototype described in this work, a commercial PDT device was used under the same experimental conditions, including photosensitizer, cells, and fluence, and the results were compared. As shown in the figure, both devices photoactivated Verteporfin equally, enhancing the cytotoxic effect.
Finally, the ROS-mediated cell death triggered by Verteporfin after light exposure was confirmed by flow cytometry using DCFDA assay. In summary, the device was easily built with commercially available low-cost components with a total cost of less than 50. The main other advantage of the device include portability, low maintenance demand, capacity to irradiate multiple types of culture plates, the simultaneous use of up to four units per assay, accurate and reproducible irradiation, user-friendly and simple setup interface that does not require connection to computers or other machines.
In addition, a decision flow chart is presented to provide a systematic problem-solving approach to find and correct problems or errors during the operation. These findings allow to extend the benefits of PhotoAct to facilitate PDT to scientific research, exploring the mechanism of action of photosensitizers and their clinical applications.
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