Protocol for developing a mucus-producing gut-on-a-chip model from Caco-2 and HT29-MTX-E12 cells

Organ-on-chip (OoC) technology offers a more relevant gut model in comparison with static in vitro gut models from stable cell lines by replicating the 3D structure, mucus production, fluid dynamics, and mechanical forces, closely simulating in vivo conditions. Here, we present a protocol for developing a mucus-producing gut-on-a-chip model from Caco-2 and HT29-MTX-E12 cells. We describe steps for assessing model viability, evaluating barrier integrity, and employing different microscopy techniques, including confocal microscopy.