Hypothesis: The development of advanced oral delivery systems for bioactive compounds requires the fundamental understanding of the digestion process within the gastrointestinal tract. Towards this goal, dynamic in vitro digestion models, capable of characterising the molecular as well as colloidal aspects of food, together with their biological interactions with relevant in vitro cell culture models, are essential. Experiments: In this study, we demonstrate a novel digestion model that combines flow-through time resolved small angle X-ray scattering (SAXS) with an in vitro Caco-2/HT-29 cell co-culture model that also contained a mucus layer. This set-up allows the dynamic in situ characterisation of colloidal structures and their transport across a viable intestinal cell layer during simulated digestion. Findings: An integrated online SAXS - in vitro cell co-culture model was developed and applied to study the digestion of nature's own emulsion, milk. The impact of the in vitro cell culture on the digestion- triggered formation and evolution of highly ordered nanostructures in milk is demonstrated. Reported is also the crucial role of the mucus layer on top of the cell layer, protecting the cells from degradation by digestive juice components such as lipase. The novel model can open unique possibilities for the dynamic investigation of colloidal structure formation during lipid digestion and their effect on the uptake of bioactive molecules by the cells. (C) 2020 The Authors. Published by Elsevier Inc.