Development of an Innovative LED-based Illumination Device for In Vitro Application of Photodynamic Therapy with Rose Bengal

The scope of our research is to develop fundamental and translational projects aiming to characterize the effect of photodynamic therapy in the treatment of cancer with no effective therapeutic options. The current experimental challenges are to develop the technologies necessary for the implementation of photodynamic therapy from in vitro to clinic application, including new photosensitizers and new illumination devices. Compared to other techniques, our protocol involves a low-cost and homemade device, enabling a more generous illumination of a 96-well plate and the physiological condition inside a cell culture incubator.

To begin, remove the cultured HepG2 cells from the incubator and place the flask under the pre-cleaned microbiological safety station. Using a pipette, remove the culture medium from the culture flask. To eliminate residual medium, rinse with phosphate-buffered solution while gently mixing, then carefully remove the buffer.

Then, apply three milliliters of trypsin solution containing 0.25%trypsin and 0.53 millimolar EDTA to the cells and place the culture back in the incubator at 37 degrees Celsius for five minutes to detach the cells. After five minutes, add seven milliliters of culture medium to the flask to neutralize the trypsin and suspend the cells. Now, transfer the trypsin, medium, and detached cells into a 15-milliliter centrifuge tube.

To count the cells, pipette 20 microliters of the cell solution into a clean tube, and add 20 microliters of trypan blue solution. After mixing, load the stained mixture onto the counting slide and insert it into the cell counter. Next, add culture medium to a tube, then introduce cells to reach a final concentration of 150, 000 cells per milliliter.

Seed 1.5 times 10 to the power of four cells per well into a white 96-well plate with a clear, flat bottom. Prepare two plates for each viability reading time. Incubate the plates for 24 hours before treatment to let the cells attach.

To prepare a stock solution of rose bengal, dissolve it in 10%saline solution. Dilute the rose bengal treatment solutions at concentrations ranging from zero to 100 micromolar in cell culture medium. Now, remove the culture medium from the cell plates and add 100 microliters of the prepared rose bengal treatment solutions to each well.

Incubate the cells with rose bengal for two hours to allow internalization. After two hours, remove the rose bengal solution from each well, wash the cells two times with PBS, aspirate the PBS and add 100 microliters per well of rose bengal free culture medium. Cover the microplates with aluminum foil to protect them from light, and set aside half of the plates for illumination during the photodynamic therapy assay.

Connect the male connector on the light distributor to the female connector of the light source. Remove the photodynamic therapy and dark condition microplates from the incubator. Unwrap the photodynamic therapy plate.

Then, set the dimmer on the LED driver to the maximum level to deliver an average irradiance of 0.62 milliwatt per square centimeter across the 96 wells. After that, place the plate on the light distributor. Illuminate the microplate until the desired light dose on the cells is achieved.

Once the desired light dose is reached, turn off the device. Rewrap the photodynamic therapy microplate in aluminum foil and return it along with the control plate to the incubator until the viability test is performed. After completing the photodynamic therapy assay, place the illuminated plate in the incubator for 24 hours to allow for post-treatment cellular response.

After the incubation period, retrieve one illuminated and one non-illuminated plate. Add 100 microliters of reagent from the cell viability assay kit into each well to measure mitochondrial metabolism and ATP production. Incubate the plates in the dark for 10 minutes before proceeding with luminescence measurement.

After 10 minutes, read the luminescence in each well using a multimodal reader. Consider the luminescence from the untreated control wells as representing 100%viability. Normalize the luminescence values from the treated wells to this control to calculate the percentage of viability for each treatment condition.

Repeat the same protocol for cell viability measurement at additional post-treatment time points, such as 24 hours, 48 hours, and 72 hours. Illumination alone without rose bengal did not alter HepG2 cell viability at any of the tested light doses, and rose bengal alone without light also caused no significant change across all concentrations. Rose bengal-mediated photodynamic therapy using CELL-LED-550/3 significantly reduced HepG2 cell viability at all light doses, showing a strong cytotoxic effect.

The overlap between the LED emission profile and the absorption spectrum of rose bengal confirmed the spectral compatibility of the CELL-LED-550/3 device with the photosensitizer. In the future, one of our main objectives is to develop new PDT packages each comprising both photosensitive compound enabling specific cancer cell targeting and an associated illumination device.