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

This article describes a new photodynamic therapy (PDT) device for evaluating the efficacy of Rose Bengal (RB)-mediated PDT in vitro. The device, called CELL-LED-550/3, consists of two 3D-printed parts, one generating a 550 nm green light from a single LED (the light source part) and the other distributing this light to a 96-well cell culture plate (the light distributor part). The light source part is controlled by a driver with three different modes, enabling the light distributor part to deliver three different irradiance levels to the bottom exterior surface of the wells: 0.02 mW/cm², 0.23 mW/cm², or 0.62 mW/cm². The light distributor part was designed to illuminate the wells individually and simultaneously. To demonstrate the relevance of the CELL-LED-550/3 device, its ability to induce cell death by RB-mediated PDT on HepG2 hepatocellular carcinoma cells was evaluated. First, HepG2 cells were treated for 2 h with increasing concentrations of Rose Bengal, a photosensitizer with an absorption peak (the highest one) at around 550 nm. The treated cells were then illuminated using the CELL-LED-550/3 device set to its maximum irradiance level at light doses of 0.3 J/cm², 0.6 J/cm², and 1.2 J/cm² (respectively, with illumination times of 8 min and 4 s, 16 min and 8 s, and 32 min and 16 s). Finally, viability was measured using Cell-Titer Glo at 24 h post-PDT. The experimental results show that RB-mediated PDT using the CELL-LED-550/3 device is capable of inducing a decrease in the viability of HepG2 cells, in a manner dependent both on the concentration of Rose Bengal and on the administered dose of light. Based on the proof of concept presented in this article, the CELL-LED-550/3 device adds a further tool to the study of RB-mediated PDT.