High-fidelity digital light processed villi-crypt scaffold-on-chip for functional intestinal epithelium modelling

Introduction

The small intestine is essential for nutrient absorption and barrier integrity, characterized by its villi-crypt epithelial architecture. Conventional in vitro models often fail to replicate these complex physiological features, and they may rely on complex synthesis and processing procedures involving toxic solvents. Gelatin is a biobased polymer derived from the partial hydrolysis of collagen, a structural protein abundantly found in animal connective tissues such as skin, bones, and cartilage. Its natural origin and wide availability make it a sustainable material widely used in biomedical, pharmaceutical, and food applications. Digital light processing (DLP) is an additive manufacturing technique that uses projected light to selectively polymerize photosensitive precursors layer by layer. It enables high-resolution fabrication without the use of toxic solvents, making it a cleaner and more precise method for producing complex microstructures in biomedical and engineering applications.

In this study, we introduce a novel DLP 3D-printed microfluidic device that incorporates a biomimetic villi-crypt scaffold, based on a blend of gelatin methacryloyl (GelMA) and poly(ethylene glycol) diacrylate (PEGDA).

Experimental

A blend of home-synthetized GelMA and commercial PEGDA, dissolved in Dulbecco's Modified Eagle Medium (DMEM), was employed. The production parameters such as GelMA methacrylation degree, pre-hydrogel solution composition (polymers, photoinitiatior and photoabsorber concentrations), layer thickness and exposure time, and power intensity were optimized. Different 3D-printed GelMA:PEGDA formulations were analyzed for mechanical properties using a rheometer, to find the optimal balance between stiffness mimicry and printing fidelity. Porosities of the printed hydrogels were observed on lyophilized samples by scanning electron microscopy. Analysis of the fluid shear stress exerted during perfusion was performed by computational fluid dynamics (CFD) using COMSOL Multiphysics.

Cytocompatibility of the printed materials was evaluated using the Caco-2 cell line. The resazurin reduction assay was employed to assess cellular metabolic activity, and the structure of the formed epithelial layer was observed by confocal fluorescence microscopy of the nuclei (DAPI) and F-actin (phalloidin). Gene expression analysis was performed by real-time polymerase chain reaction (rt-PCR), and protein quantification by nano-flow Liquid Chromatography coupled with Tandem Mass Spectrometry.

Results and Discussion

By optimizing the GelMA–PEGDA formulation, we achieved well-defined intestinal microarchitectures with minimal swelling or deformation. The resulting structures included parabolic villi (433 ± 15 µm height; 320 ± 20 µm base) and cylindrical crypts (141 ± 6 µm height; 134 ± 10 µm diameter). Among the tested compositions, the Villi-(R)-Crypt scaffold (9 wt% GelMA: 4.5 wt% PEGDA) exhibited a shear elastic modulus of 21.9 ± 1.3 kPa at 1 Hz, while the Villi-(F)-Crypt scaffold (4.5 wt% GelMA: 4.5 wt% PEGDA) displayed 7.0 ± 0.4 kPa. The former demonstrated superior structural integrity and lower porosity, whereas the latter offered greater flexibility while still supporting cell growth. The closed devices were finally produced with compliant squared channels of 500 µm side, and their perfusability was assessed over a 0 – 100 µL/min range of flow rates. Computational fluid dynamics (CFD) analysis confirmed that a perfusion flow rate of 20 µL/min generates physiological shear stress levels (0.001–0.02 dyne/cm²) within the microfluidic platform.

Cytocompatibility assessments showed that both scaffold types supported epithelial cell attachment, proliferation, and differentiation. Metabolic activity assays and Phalloidin/DAPI fluorescence microscopy revealed a slightly enhanced cell adhesion and growth on the Villi-(R)-Crypt scaffold. Finally, gene expression and protein quantification analyses indicated successful epithelial differentiation on both scaffold compositions, with some differences in expression timing and protein levels.

Conclusions

Using GelMA with a high degree of substitution (DoS) and PEGDA, we successfully optimized DLP-3D printing to fabricated high-fidelity structures able to sustain the attachment, growth and differentiation of the Caco-2 cells.