Summary

Organoids and microphysiological systems, such as organs-on-a-chip, have emerged as powerful tools for modeling human gut physiology and disease in vitro . However, although physiologically relevant, these systems often lack the environmental milieu, spatial organization, cell-type diversity, and maturity necessary for mimicking adult human intestinal mucosa. To instead generate models closely resembling the in vivo cell-type composition and spatial compartmentalization, we herein integrated organoid and organ-on-a-chip technology to develop a primary human stem–cell-derived organoid model, called ‘mini-colons’. The luminal access and flow in human mini-colons removes shed cells to greatly enhance tissue longevity and differentiation over physically inaccessible human intestinal organoids that accumulate trapped cellular debris and waste. By establishing a gradient of growth factors, we replicated and sustained in vivo -like cell fate patterning and concurrent differentiation to secretory cell types and colonocytes. These long-lived human mini-colons contain abundant mucus-producing Goblet cells that lubricate the colonic epithelial lining. The stem and proliferative progenitor cells are also realistically confined to the crypts, facilitating stable homeostatic tissue turnover and preserving tissue integrity for several weeks. Also signifying mini-colon in vivo -like maturation, single-cell RNA sequencing showed emerging mature colonocytes and absorptive BEST4 + colonocytes. This methodology could be expanded to generate microtissues derived from the small intestine and incorporate additional microenvironmental components, thus emulating the intricate complexity of the native gut in an in vitro setting. Our bioengineered human organoids provide a highly accurate, long-lived, functional platform to systematically study human gut physiology and pathology, and for the development of novel therapeutic strategies.