Culture-specific transcriptional drifts limit the fidelity of organoid infection models.

Intestinal organoids are powerful tools for modeling host-pathogen interactions, yet culture-induced artifacts remain poorly defined. Here, we performed time-course transcriptomic profiling of patient-derived ileal organoids microinjected with Mycobacterium avium subsp. paratuberculosis (MAP) or treated with the swelling agent forskolin (FSK) and compared expression changes to untreated controls. Our analysis revealed that culture-associated transcriptional drift was the primary driver of gene expression changes over the 48-hour time course, likely obscuring pathogen-specific responses. This drift involved progressive downregulation of metabolic genes accompanied by the upregulation of genes with high GC content. Although FSK treatment improved. microinjection efficiency, it also introduced additional confounding effects, including transient activation of apoptotic pathways and upregulation of inflammatory genes that resolved within two days. While MAP infection did not produce significant single-gene responses, gene set analysis revealed significant infection-associated effects. Spatial transcriptomics further demonstrated that multiple enterocyte gene sets became downregulated during culture, independent of their normal crypt-villus expression pattern. Gene sets typically expressed at the crypt-villus junction showed the greatest downregulation. Notably, MAP infection selectively counteracted these culture-induced changes, preserving gene expression patterns characteristic of the upper villus epithelium. These findings indicate that both metabolic stress and pharmacological interventions can substantially confound organoid-based infection models. More broadly, they underscore the importance of spatially resolved transcriptomics and appropriate temporal controls to distinguish genuine pathogen responses from culture-related artifacts. To our knowledge, no previous studies have used human intestinal organoids to model MAP infection or characterized the epithelial transcriptomic response. This work establishes critical frameworks for investigating MAP's role in Crohn's disease pathogenesis.