Experimental context determines repeated UVB responses and photoageing-associated phenotypes in human dermal fibroblasts

Abstract Repeated ultraviolet B (UVB) exposure of dermal fibroblasts is widely used to investigate photoaging-associated cellular stress responses; however, substantial variation in experimental design limits reproducibility and complicates comparison across studies. Here, We systematically evaluated how experimental context—including seeding density, cumulative UVB dose, irradiation schedule and experimental time—collectively shapes repeated UVB responses in human dermal fibroblasts. Full-factorial analysis demonstrated that cumulative UVB dose and experimental time accounted for the largest proportion of biological variation, while Dose × Day, Dose × Schedule, and Dose × Density interactions were also substantial. Experimental observations confirmed that identical cumulative UVB doses produced distinct proliferative outcomes depending on irradiation schedule, culture density and recovery time. Experimental validation showed that initial cell density reshaped the apparent dose-response relationship, with low-density cultures exhibiting greater UVB sensitivity but reduced experimental stability, whereas intermediate-density cultures revealed distinct low- and high-response states. Furthermore, equivalent cumulative UVB doses delivered under different irradiation schedules produced distinct proliferative responses, demonstrating that recovery interval modifies cumulative UVB responses independently of total dose. A representative repeated-UVB condition reproduced canonical photoaging-associated phenotypes, including recurrent oxidative stress, impaired migration, extracellular matrix remodeling, and activation of stress- and senescence-associated transcriptional programs. Collectively, these findings demonstrate that repeated UVB responses are determined not only by cumulative UVB dose but also by experimental context. Considering seeding density, experimental time, and recovery dynamics alongside UVB dose provides a practical framework for improving the reproducibility, interpretation, and comparison of fibroblast-based photoaging models.