OBJECTIVES/GOALS: Our goal is to explore the extent to which organoids can serve as models for the protective mechanisms of the stomach–the mucus barrier and the pH gradient across it. We aim to first optimize and validate an organoid-based model of the gastric mucus layer, and then define the cellular mechanisms by which the gastric pH gradient is maintained across it. METHODS/STUDY POPULATION: We have developed a method for the in vitro engineering of gastric mucus by growing epithelial cells at the air-liquid interface (ALI). We use microrheology with fluorescent microspheres to define and compare the biophysical and viscoelastic properties of our lab-grown mucus to those of native mucus. We will perform CryoFE-SEM to compare the internal heterogeneity of our lab-grown mucus to fresh mucus obtained from patient tissue. For our mechanistic studies, we will use a pH-sensitive dye (methyl red) to assess the ability of our lab-grown mucus to maintain an artificial pH gradient in a microfluidic device. Next, we will use a pH microelectrode to measure proton flux through our mucus in vitro, investigating the potential for a physiological gradient in both 2D and 3D organoid models. RESULTS/ANTICIPATED RESULTS: Here we show that gastric organoids and their corresponding epithelial monolayers produce a mucus gel that does indeed mimic in vivo functions. Immunohistochemical staining, electron microscopy, microrheology, and particle tracking analyses revealed that our gastric organoid mucus is viscoelastic and structurally heterogeneous–both properties that are crucial to the stomach’s mucosal first line of defense. Mechanically similar mucus was also engineered using two-dimensional air-liquid interface cultures of the same epithelia. Lastly, live confocal imaging revealed that H. pylori motility–an important virulence factor–was drastically hindered by our lab- grown mucus. DISCUSSION/SIGNIFICANCE: We describe a novel method for the in vitro engineering of gastric mucus and highlight biophysical properties that contribute to our stomach’s defense against pathogens. This work will lead to an improved understanding of gastric physiology and may contribute to the development of novel drug delivery systems to tackle diseases of the gastric mucosa.