PURPOSE: The effects of conventional (CONV) and FLASH proton therapy on primary bronchial epithelial cell (PBEC) organoids from individuals with chronic obstructive pulmonary disease (COPD) were investigated. The primary objective was to compare the effect of FLASH and CONV on COPD PBEC organoids with a focus on DNA damage, organoid formation, and gene expression. METHODS: PBECs were obtained from six COPD donors, cultured as three-dimensional (3D) organoids and exposed to 2 and 8 Gy CONV and FLASH proton radiation at the Holland Proton Therapy Center. DNA damage was assessed by γH2AX staining. Organoid formation capacity was assessed by counting the organoids formed after reseeding irradiated cells at 24 h and 7 days. Bulk RNA sequencing (RNAseq) and qPCR analyses were performed to identify pathways and differences in the radiation response. RESULTS: γH2AX foci analysis showed a significant dose-dependent increase in DNA damage at 1 h for both CONV and FLASH treatments, without differences between the two modalities. Organoid formation assays revealed a dose-dependent decrease in organoid formation capacity at 24 h for both treatments. At 7 days, 2 Gy FLASH-treated samples showed significantly reduced organoid formation compared to 2 Gy CONV (p = 0.008). RNAseq identified CONV and FLASH-induced changes in expression of DNA-damage response and apoptosis pathway genes. A dose-dependent upregulation of MDM2, GDF15, DDB2, BAX, P21, AEN and a decrease in MKi67 expression was confirmed by qPCR analysis. CONCLUSION: No significant differences were found in DNA damage or gene expression profiles between CONV and FLASH. The organoid formation assay showed a prolonged detrimental effect in the FLASH-treated organoids, suggesting a more complex interaction of FLASH with lung epithelial cells. The results of this study contribute to the advancement of robust