The gastrointestinal (GI) mucus layer plays a pivotal role in tissue homoeostasis and functionality of the gut. However, due to the shortage of affordable, realistic in vitro mucus models, studies with deeper insights into its structure and characteristics are rare. To obtain an improved mucus model, we developed a reusable culture chamber facilitating the application of physiologically relevant GI shear stresses (0.002-0.08 dyn/cm2) to cells in a bioreactor system. Differentiation of a confluent monolayer of human mucus-producing epithelial HT29-MTX cells was monitored under dynamic and static culture conditions. Cells under flow remained highly proliferative and analysis via confocal microscopy revealed superior reorganization into 3-dimensional villi-like structures compared to static culture (up to 120 vs. 80 µm in height). Additionally, the median mucus thickness was significantly increased under dynamic conditions compared to static culture (41±14 vs. 29±14 µm) with a simultaneous drastic reduction of culture time from three to two weeks for sufficient maturation into goblet-like cells. We demonstrated the impact of culture conditions on the differentiation of HT29-MTX cells, revealing outstanding in vivo like reorganization of cells and the production of thick adherent mucus networks when cultured under physiological shear stress using our newly designed culture chamber.