Silk Scaffold Crypt‐Villus Geometry Combined With L‐ <scp>WRN</scp> Feeder Cells Sustains Spatially Organized Epithelial Proliferation and Multilineage Differentiation

The crypt-villus axis of the small intestine provides essential structural and biochemical cues that govern epithelial renewal, barrier function, and lineage specification. However, most existing in vitro models rely on flat monolayers or villus-only hydrogels, limiting their ability to capture stem cell niches and spatial organization. Here, we report silk fibroin scaffolds engineered with physiologically scaled crypt-villus topography through a combination of 3D-printed molds, vacuum-assisted silk infiltration, and water annealing. This approach reproducibly generated scaffolds with well-defined crypt and villus features and an open design that permitted visual inspection prior to seeding. When seeded with human intestinal organoid-derived monolayers, the scaffolds supported polarized epithelia with apical ZO-1, organized F-actin, and E-cadherin localization along villus axes. Differentiation protocols yielded all major intestinal lineages, including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells, confirmed by immunostaining and qRT-PCR. Ultrastructural analysis further revealed dense brush-border microvilli on villi, indicative of absorptive maturation. Incorporation of L-WRN feeder cells within the scaffold bulk sustained Ki-67 positive proliferative zones and increased LGR5 transcript expression, consistent with prolonged proliferative activity. Collectively, these results establish silk-based crypt-villus scaffolds as a reproducible and versatile platform for generating spatially organized human intestinal epithelium, with potential applications in epithelial biology, host-microbe studies, and intestinal disease modeling.