Engineering a Biomimetic Intestinal Scaffold with Tunable Villi Architecture via DLP 3D Printing

The human gut is a highly complex and dynamic organ, central to nutrient absorption, immune modulation, and drug metabolism [1,2]. Conventional in vitro models often fail to fully replicate the gut’s architectural, mechanical, and biochemical microenvironment features, thus limiting their utility in translational research [3]. In this work, we present a proof-of-concept biomimetic scaffold designed to emulate villi-crypt structures of the human small intestine. The system was fabricated using digital light processing (DLP) 3D printing and PEGDA-based photopolymer resin. The 3D-printed scaffold is designed to comprise four zones, three of which contain villi of varying sizes, and a fourth that blends all three, enabling spatially resolved cellular interactions. Printing was conducted using optimized DLP parameters (50 µm layer height, 20 mW/cm² intensity, 3 s exposure), and ensuring high resolution and reproducibility [4]. The villi-crypt scaffold architecture was tailored to support compatibility with standard cell culture protocols. Scanning electron microscopy (SEM) confirmed the fidelity of the villi-crypt structures and the dimensional variation across zones (493 µm, 628 µm, and 778 µm). Additionally, surface modification with poly-D-lysine via electrostatic adsorption introduced a positively charged interface to promote cell adhesion.

The in vitro models were seeded with Caco-2 cells to evaluate cell adhesion and cytocompatibility. Confocal microscopy confirmed epithelial monolayer formation and successful cellular attachment to the scaffold. After 21 days of culture, cells exhibited polarization. Live/dead assays and TEER measurements demonstrated the formation of a viable, functionally active epithelial layer, highlighting the system’s potential for long-term intestinal studies.

Compared to conventional Transwell™ cultures, the scaffold offers superior imaging accessibility, scalability, and microenvironmental control. The use of commercial PEGDA resins enables rapid prototyping and future customization with functional biomaterials or patient-derived samples. This system also lays the foundation for co-culture integration, colorectal cancer (CRC) modeling, and personalized medicine approaches.

Acknowledgements:

This research acknowledges the funding provided by the UID/50026:3B’s-Biomaterials, Biodegradables and Biomimetics Research Group, University of Minho (3B's Res. Group/UMinho), CAN-TARGET (ref.NORTE2030-FEDER-02705300, CCDR-N), and the EU-EC through the ONCOSCREEN (ID: 101097036) and EngVIPO (ID: 101183041) projects.

References:

[1] Yilmaz, B. et al.,  Nature Reviews Gastroenterology & Hepatology, 2025. doi:10.1038/s41575-024-01000-4

[2] Doenyas, C. et al., Scientific Reports, 2025. doi:10.1038/s41598-025-86858-3

[3] Ozkan, A. et al., Nature Reviews Gastroenterology & Hepatology, 2024. doi: 10.1038/s41575-024-00968-3

[4] Chang, S.-Y., Materials Today: Proceedings, 2022. doi:10.1016/j.matpr.2022.09.017