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In vitro  models of the small intestine are crucial tools for the prediction of drug absorption. The Caco-2 monolayer transwell model has been widely employed to assess drug absorption across the intestine. However, it is now well-established that 3D  in vitro  models capture tissue-specific architecture and interactions with the extracellular matrix and therefore better recapitulate the complex  in vivo  environment. However, these models need to be characterized for barrier properties and changes in gene expression and transporter function. Here, we report that geometrically controlled self-assembling multicellular intestinal Caco-2 spheroids cultured using Sacrificial Micromolding display reproducible intestinal features and functions that are more representative of the  in vivo  small intestine than the widely used 2D transwell model. We show that Caco-2 cell maturation and differentiation into the intestinal epithelial phenotype occur faster in spheroids and that they are viable for a longer period of time. Finally, we were able to invert the polarity of the spheroids by culturing them around Matrigel beads allowing superficial access to the apical membrane and making the model more physiological. This robust and reproducible  in vitro  intestinal model could serve as a valuable system to expedite drug screening as well as to study intestinal transporter function.

Human intestinal spheroids cultured using Sacrificial Micromolding as a model system for studying drug transport