Designing a Suitable Model for Evaluating the Penetration Depth of Cold Atmospheric Plasma by Implanting B16F10 Melanoma Cells on Polycaprolactone Nanofibrous Scaffolds

ABSTRACT Cold atmospheric plasma (CAP) is increasingly recognized for its potential in melanoma cancer treatment due to its ability to induce cell death selectively in cancer cells while sparing normal L929 fibroblast cells. In this study, culturing B16‐F10 melanoma and L929 fibroblast cell lines on polycaprolactone (PCL) nanofiber scaffolds, the penetration depth of CAP effects, especially cytotoxicity and selectivity, in a three‐dimensional (3D) scaffold has been examined. Results from MTT assays, ROS measurements, and flow cytometry confirmed that CAP effectively reduces the viability of melanoma cells across scaffold layers, with a higher impact on surface layers and a gradual decline in deeper layers. ROS production followed a similar trend, demonstrating CAP penetration through multiple layers while retaining its selective cytotoxic effects. Flow cytometry analysis further supported CAP‐induced apoptosis in melanoma cells, with reduced effectiveness in deeper layers. Finally, a strong linear regression model ( R 2 > 0.97) enabled extrapolation of cell viability across scaffold layers, yielding an estimated effective plasma penetration depth of 116–166 µm based on the 50% viability threshold (IC 50 ). These findings highlight the suitability of PCL‐based 3D scaffolds as in vitro models for studying CAP penetration and reinforce CAP's potential as a targeted therapy for melanoma.