The human gut microbiome is crucial to hosting physiology and health. Therefore, stable in vitro coculture of primary human intestinal cells with a microbiome community is essential for understanding intestinal disease progression and revealing novel therapeutic targets. Here, a three‐dimensional scaffold system is presented to regenerate an in vitro human intestinal epithelium that recapitulates many functional characteristics of the native small intestines. The epithelium, derived from human intestinal enteroids, contains mature intestinal epithelial cells and possesses selectively permeable barrier functions. Importantly, by properly positioning the scaffolds cultured under normal atmospheric conditions, two physiologically relevant oxygen gradients, a proximal‐to‐distal oxygen gradient along the gastrointestinal (GI) tract, and a radial oxygen gradient across the epithelium, are distinguished in the tissues when the lumens are faced up and down in cultures, respectively. Furthermore, the presence of the low oxygen gradients supported the coculture of intestinal epithelium along with a complex living commensal gut microbiome (including obligate anaerobes) to simulate temporal microbiome dynamics in the native human gut. This unique silk scaffold platform may enable the exploration of microbiota‐related mechanisms of disease pathogenesis and host‐pathogen dynamics in infectious diseases including the potential to explore the human microbiome‐gut‐brain axis and potential novel microbiome‐based therapeutics.

Crypt-villus architecture in the small intestine is crucial for the structural integrity of the intestinal epithelium and maintenance of gut homeostasis. We utilized three-dimensional (3D) printing and inverse molding techniques to form threedimensional (3D) spongy scaffold systems that resemble the intestinal crypt-villus microarchitecture. The scaffolds consist of silk fibroin protein with curved lumens with rows of protruding villi with invaginating crypts to generate the architecture. Intestinal cell (Caco-2, HT29-MTX) attachment and growth, as well as long-term culture support were demonstrated with cell polarization and tissue barrier properties compared to two-dimensional (2D) Transwell culture controls. Further, physiologically relevant oxygen gradients were generated in the 3D system. The various advantages of this system may be ascribed to the more physiologically relevant 3D environment, offering a system for the exploration of disease pathogenesis, host−microbiome interactions, and therapeutic discovery.

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