Exploring digital light processing 3D printing to produce villi-crypt scaffold-on-chip mimicking the intestinal epithelium

In vitro models are crucial for studying the intricate interplay between the intestinal epithelium and microbiota in a controlled environment. This work focuses on developing a 3D in vitro model using DLP-3D printing to mimic the complex intestinal crypt-villi system within a perfusion microfluidic device. A blend of home-produced gelatin methacrylate (GelMA) and low molecular weight polyethylene glycol diacrylate (PEGDA), dissolved in DMEM, was employed. The production parameters such as GelMA methacrylation, pre-hydrogel solution composition (polymers, photoinitiatior and photoabsorber concentrations), layer thickness and exposure time, and power intensity were optimized. Different formulations were analyzed for mechanical properties using a rheometer, to find the optimal balance between stiffness mimicry and printing fidelity, and were evaluated for cytocompatibility using Caco-2 cells. The desired architectures were achieved, having parabolic villi (433 ± 15 µm height; 320 ± 20 µm base), and cylindrical crypts (141 ± 6 µm height; 134 ± 10 µm diameter). GelMA with high degree of substitution was successfully produced, achieving high printing fidelity, long-term stability and cytocompatibility. The chosen GelMA/PEGDA compositions 9.0/4.5 (w%/w%) and 4.5/4.5 (w%/w%) displayed a shear elastic modulus of 21.9 ± 1.3 kPa and 7.0 ± 0.4 kPa, respectively. 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. Both the compositions revealed good cell attachment and metabolic activity of Caco2 cells. At 24 hours, the Resazurin reduction assay revealed consistent metabolic activity across the two compositions, whether in their original form or when coated with gelatin. Overall, we successfully developed a closed microfluidic device, comprising the desired internal villi-crypt like morphology, with tunable mechanical properties and able to sustain the attachment and growth of the Caco-2 cell line.