Objectives/Goals: This collaboration explores magnetic-responsive scaffolds toward modeling Salmonella infection via the small extracellular vesicle (sEV) route. We aim to elucidate the mechanism by which sEVs reflect host–pathogen interactions to inform new Salmonella therapeutics. Methods/Study Population: Human intestinal epithelial cells (INT-407) are being cultured on magnetic-responsive scaffolds under controlled mechanical stimulation and infected with Salmonella Typhimurium 12023 WT. Kinetic assays are assessed via confocal microscopy, flow cytometry, and qPCR. sEV isolation is being performed by NanoPoms immunomagnetic capture and characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blotting for canonical markers (CD9, CD63, and CD81). Additionally, sEVs derived from human THP-1 cells are being used to treat naïve INT-407, in which cytokine responses and proteomic shifts are analyzed. Results/Anticipated Results: We anticipate that INT-407 in 3D magnetic scaffolds will shift the Salmonella infection kinetics compared to the unstimulated controls and traditional 2D culture. We expect the sEVs from infected cells to be enriched in antigenic proteins and canonical sEV markers. Furthermore, sEVs from infected THP-1 cells are anticipated to induce pro-inflammatory cytokine release and proteomic shifts toward intracellular signaling and inflammatory processing in naïve INT-407 following treatment. Taken together, these findings will elucidate the mechanism by which magnetic-responsive scaffolds better recapitulate the gut microenvironment for modeling host–pathogen interactions. Discussion/Significance of Impact: This project’s findings will indicate if 3D magentic scaffolds provide a sound model for Salmonella infection. Analysis of sEVs derived from INT-407 and THP-1 will provide insight into the functional characteristics sEVs have in the gastrointestinal environment upon pathogen insult.