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 um, 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.
Ota, S., SHUKLA, P., Otaki, N., Taylor, E. J., Hattori, K.
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