Microfluidic Core–Shell Encapsulation Enables Scalable Generation of Apical-Out Intestinal Spheroids

Apical-out intestinal spheroids provide direct access to the lumen-facing epithelial surface, making them attractive three-dimensional models for studying epithelial barrier function, nutrient uptake, and luminal exposure. However, existing polarity-reversal methods typically require releasing spheroids from surrounding ECM gels and culturing them in suspension, which can compromise matrix-derived cues, promote fusion, increase size heterogeneity, and limit scalability. Here, we develop a microfluidic core-shell encapsulation strategy to scalably produce apical-out intestinal spheroids within uniform hydrogel microcapsules. These microcapsules consist of a Matrigel core surrounded by an agarose shell. Flow-focusing microfluidics first confines Caco-2 cells in Matrigel cores that provide instructive extracellular matrix cues, and particle-templated emulsification subsequently encloses each core within an inert agarose shell that prevents spheroid fusion and preserves batch uniformity. The method generated >100,000 microcapsules per experiment, with a mean shell diameter of 117 µm, a coefficient of variation below 9%, and >90% single-spheroid formation efficiency. The resulting spheroids established apical–basolateral polarity, organised tight junctions, formed a dextran-excluding epithelial barrier, and exhibited fatty-acid uptake. This core–shell strategy provides an experimentally tractable platform for scalable intestinal epithelial modelling and may be extensible to other epithelial microtissue systems.