Developing an advanced gut on chip model enabling the study of epithelial cell/fibroblast interactions

Abstract

Organoids are widely used as a model system to study gut pathophysiology; however, they fail to fully reproduce the complex, multi-component structure of the intestinal wall. We present here a new gut on chip model that allows the co-culture of primary epithelial and stromal cells. The device has the topography and dimensions of the mouse gut and is based on a 3D collagen I scaffold. The scaffold is coated with a thin layer of laminin to mimic the basement membrane. To maintain the scaffold structure while preserving its cytocompatibility, the collagen scaffold was rigidified by threose-based post-polymerization treatment. This treatment being cytocompatible enabled the incorporation of primary intestinal fibroblasts inside the scaffold, reproducing the gut stromal compartment. We observed that mouse organoids, when deposited into crypts, opened up and epithelialized the scaffold, generating a polarized epithelial monolayer. Proper segregation of dividing and differentiated cells along the crypt-villus axis was achieved under these conditions. Finally, we show that the application of fluid shear stress allows the long-term culture of this intestinal epithelium. Our device represents a new biomimetic tool that captures key features of the gut complexity and could be used to study gut pathophysiology.

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.

Fig. 2. Epithelialization of the scaffolds. A. The unit consists of one villus and six crypts. Three hours after seeding the organoids on the collagen scaffold in the presence of mouse intestinal fibroblasts. Collagen type I (TAMRA-labeled, red); F-actin (phalloidin, green), DNA (DAPI, blue). Top view, maximal projection of 343 μm. Scale bar, 150 μm. Insets, higher magnification of one crypt containing an organoid. Single plane (i), maximal projection of 152 μm (ii). Scale bar, 20 μm. B. Organoids expressing LifeAct-GFP (F-actin label) spreading over collagen scaffolds. Time in hours. Top view. The dashed line outlines the base of the villus. Scale bar, 200 μm.

Fig. 3. Cell morphology on the microdevice resembles the in vivo epithelium. A. A continuous monolayer of epithelial cells over the collagen scaffold four days after seeding the organoids, in the presence of mouse intestinal fibroblasts. x/y, top view, maximal projection of 433 μm. B. Orthogonal section through one villus and two crypts. Scale bar, 150 μm. C. Cross-section through the villus and crypt and top view of the region between villi and crypts (plateau) on collagen scaffolds. Collagen type I (TAMRA-labeled, pink); F-actin (phalloidin, green), DNA (DAPI, red). Scale bar, 20 μm. Insets, higher magnification of boxed regions. D. A monolayer of epithelial cells (F-actin labeled, red) four days after seeding the organoids over the collagen scaffold containing fibroblasts (expressing αSMA, green). A cross-section through the crypt region, 348 μm from the top of the scaffold showing fibroblasts in contact with crypts. Scale bar, 150 μm. E. Side view of the crypt. Epithelial cells and mouse intestinal fibroblasts (F-actin, green; DNA, DAPI, red), collagen (TAMRA-labelled, pink). Scale bar, 50 μm. F. Comparison of the epithelial cell height on the chip and in vivo in villi and crypts, n = 10–60 cells, from N = chips or mice.

Fig. 4. Shear stress improves epithelial integrity. A. Epithelial cells (F-actin, phalloidin) 14 days after seeding the organoids over the collagen scaffold containing mouse intestinal fibroblasts with fluid shear stress (right panel, dynamic conditions) and without shear stress (right panel, static conditions). Top view, maximal projection. Scale bar, 150 μm. B. Percentage of units covered with epithelial cells after 14 days after seeding the organoids on collagen scaffolds containing mouse intestinal fibroblasts under static conditions (stat) and with fluid shear stress (dynamic conditions, Dyn). n = 128 units, N = 3 independent experiments. Mean ± SEM, t-test ***, p < 0.0001.

Fig. 5. Segregation of proliferative and differentiated cells. A. Percentage of the chip covered with epithelial cells 14 days after seeding the organoids on collagen scaffolds, in the absence or presence of fibroblasts. n = 128 units, N = 3 independent experiments. Mean ± SEM, t-test **, p < 0.001; *, p < 0.05. B. Left: Dividing (EdU positive cells) in the monolayer of epithelial cells stained 14 days after seeding the organoids over the collagen scaffold containing fibroblasts with fluid shear stress. Top view, maximal projection of 493 μm. Scale bar, 100 μm. Right: Percentage of the surface area (2D maximal projection) occupied with dividing, EdU positive cells, in crypts and villi, in the absence or presence of fibroblasts. n = 10 units (unit: 1 villi surrounded by 6 crypts), N = 3. Mean ± SEM, t-test **, p < 0.001; *, p < 0.05. C. Left: Fully differentiated enterocytes labeled with anti-L-FABP antibodies (red) in the epithelial monolayer 14 days after seeding the organoids over the scaffold containing primary mouse intestinal fibroblasts with continuous fluid shear stress. F-Actin (green), DNA (DAPI, blue). Left panel, maximal projection of 95 μm from the top of the villus. Scale bar 20 μm. Right panel, a cross-section through the villus. Right: The number of enterocytes in crypts and villi, in the absence or presence of fibroblasts. n = 12 units, N = 2. Mean ± SEM, t-test **, p < 0.001; *, p < 0.05. D. Left: Fully differentiated goblet cells labeled with anti-mucin2 antibodies (red) in the epithelial monolayer 14 days after seeding the organoids over the scaffold containing primary mouse intestinal fibroblasts with continuous fluid shear stress. F-Actin (green), DNA (DAPI, blue). Left panel, maximal projection of 95 μm from the top of the villus. Scale bar 20 μm. Right panel, a cross-section through the villus. Right: The number of goblet cells in crypts and villi, in the absence or presence of fibroblasts. n = 15 units, N = 2. Mean ± SEM, t-test **, p < 0.001; *, p < 0.05. E. Left: Fully differentiated Paneth cells labeled with anti-lysozyme antibodies (red) in the epithelial monolayer 14 days after seeding the organoids over the scaffold containing primary mouse intestinal fibroblasts with continuous fluid shear stress. F-Actin (green), DNA (DAPI, blue). Left panel, maximal projection of 55 μm from the bottom of the crypt. Scale bar 20 μm. Right panel, a cross-section through the crypt. Right: The number of Paneth cells in crypts and villi, in the absence or presence of fibroblasts. n = 10 units, N = 2. Mean ± SEM, t-test **, p < 0.001; *, p < 0.05.