EnView: a tunable microphysiological platform with human crypt-scale topography and independent apical-basal flow for...

Microphysiological systems (MPS) can improve intestinal epithelial modelling by combining 3D topographical cues, extracellular matrix mechanics and controlled apical-basal access. We present the EnView system, an imaging-compatible microphysiological platform configured to integrate human colon-inspired crypt-scale topography, tunable matrix stiffness and independent perfusion of apical/luminal and basal/stromal compartments. Crypt-like structures were molded in an interpenetrating polyacrylamide/collagen type I hydrogel, generating soft and stiff matrices with bulk stiffness values of 3.8 ± 1.8 kPa and 26.2 ± 8.4 kPa. Molecular transport through the hydrogel was characterized using FITC-dextrans of increasing molecular weight, and the experimentally derived diffusion coefficients were implemented in a numerical diffusion model to estimate the time required for soluble mediators to reach defined regions of the epithelial interface. Human colonic epithelial Caco-2 cells were cultured under continuous microfluidic perfusion for up to 21 days. Cells colonized the patterned surface, formed polarized epithelial monolayers and displayed stiffness-dependent morphology, with a more columnar organization on softer matrices highlighting the importance of matrix stiffness on cell morphology. Relative to standard 2D Transwell® conditions, these cultures exhibited increased gene expression of enterocyte markers such as FABP1 (fatty acid binding protein-1), ALPI (intestinal alkaline phosphatase), KRT20 (cytokeratin-20) and VIL1 (villin-1), indicating that the EnView environment supports better epithelial polarization and maturation-associated features compared with conventional Transwell® culture. Basal/stromal TNF-α stimulation induced an apical/luminal measurable epithelial IL8 secretion, demonstrating the capacity of the system for basal stimulation and apical sampling. Our results show the relevance of considering matrix stiffness when modelling the human colon epithelium. This innovative MPS, recapitulating 3D topography, tunable matrix stiffness and continuous microfluidic perfusion, represents a powerful platform for long term culture and in situ imaging of epithelial constructs enabled by active microfluidic control of the apical/luminal and basal/stromal compartments.