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

Authors

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

Abstract

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.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 12 Jun 2026.

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