Platform for Drug Testing and Studying Rapid-Onset Signaling and Induction of Cellular Phenotypes Ex Vivo

The development of drugs and the assessment of their efficacy require reliable platforms that can mimic in vivo conditions. Our research has demonstrated that ex vivo skin exhibits numerous core phenotypes following injury that serve as subjects for research and targets for drug development, including cellular senescence, apoptosis, inflammation, mTOR and Erk signaling pathways, among others. In this context, we present a protocol for establishing an ex vivo skin platform, including skin preparation, biopsy collection, targeted drug application, tissue incubation, fixation, and the evaluation of drug effects on signaling pathways and cellular phenotypes. Although fundamentally simple in nature, our protocol is designed to be accessible to any laboratory interested in studying fundamental tissue damage responses and the mobilization of cells for repair. We believe this protocol will serve as a cost-effective, reliable, and reproducible tool consistent with the 3Rs methodology for research, clinical applications, and industry use.