Fig. 1. Engineering microstructured scaffold. A. Scaffold microfabrication: PDMS (mold 2) replica of the micromilled brass mold is microfabricated. This PDMS replica is then non-covalently sealed with a second PDMS mold (mold 1) that consists in an open chamber (previously treated with APTES and glutaraldehyde). Collagen I solution is then injected within this closed device using a syringe. After collagen polymerization at 37 °C, the top part of the device is removed and organoids are seeded on top of the collagen 3D scaffold. B. Schematic organization of the small intestine. Simple columnar epithelium (blue) covers finger-like projections – villi and invaginations – crypts. Epithelial cells are attached to the basement membrane. Stroma, mostly made of collagen type I (gray), contains fibroblasts (red and green). C. Schematics of the brass mold used to prepare the PDMS intermediate replica. Villus height 350 μm, crypt depth 150 μm. Inset, arrays of villi and crypts. D. PDMS mold coated with laminin showing one unit consisting of one villus surrounded by six crypts. Scale bar, 150 μm. E. A scaffold made of TAMRA-labelled collagen type I (red) coated with Cy3-labeled laminin (green). Side view. Cross-sections at the top (1) and middle (2) of the villi and plateau/opening of the crypt (3). Scale bar, 50 μm. F. Collagen cross-linking preserves the scaffold structure. Quantification of the height of the scaffold (villus plus crypt) after 14 days. The initial height of the scaffold made of polymerized 10 mg ml⁻¹ collagen I. Scaffold was either treated with threose or left untreated, and then populated with epithelial cells (organoids) or organoids and primary intestinal fibroblasts. n = 20 units, N = 3. Mean ± SEM, t-test **, p < 0.001; *, p < 0.05.