Photopatterned Membranes and Chemical Gradients Enable Scalable Phenotypic Organization of Primary Human Colon Epithelial Models

Biochemical gradients across the intestinal epithelium play a major role in governing intestinal stem cell compartmentalization, differentiation dynamics, and organ-level self-renewal. Advances in primary cell-derived in vitro models, in which a full suite of stem and differentiated cell types are present, have vastly accelerated our understanding of intestinal homeostasis and disease. However, scalable platforms that recapitulate the architecture and gradients present in vivo are absent. We present a platform in which individually addressable arrays of chemical gradients along the crypt long axis can be generated, enabling scalable culture of in vitro colonic epithelial replicas. The platform utilizes standardized well plate spacing, maintains access to basal and luminal compartments, and relies on a photopatterned porous membrane to act as diffusion windows while supporting the in vitro crypts. Simultaneous fabrication of 3,875 crypts over a single membrane was developed. Growth factor gradients were modelled and then experimentally optimized to promote long-term health and self-renewal of the crypts which were assayed in situ by confocal fluorescence microscopy. The cultured in vitro crypt arrays successfully recapitulated the architecture, stem/proliferative and differentiated cell compartmentalization, and luminal-to-basal polarity observed in vivo. Furthermore, known signaling regulators produced measurable and predictable effects on the proliferative and differentiated cell compartments. This platform is readily adaptable to the screening of tissue from individual patients to assay the impact of food and bacterial metabolites and/or drugs on colonic crypt dynamics. Importantly, the cassette is compatible with a wide range of sensing/detection modalities, and the developed fabrication methods should find applications for other cell and tissue types.