Development of a gut-on-a-chip microfluidic device with three-dimensionally printed human intestinal tissue for studying human-microbe interactions

The human small intestinal epithelium features villi protruding into the gut lumen, and crypts invaginating toward the gut exterior, forming a microarchitecture critical for intestinal homeostasis and renewal. Reproducing this complex geometry at physiological dimensions and pliability, while achieving cell compatibility, remains challenging. Here, we developed three-dimensionally (3D) printed gelatin methacryloyl (GelMA) crypt-villus scaffolds and a gut-on-a-chip microfluidic device integrating dynamic fluid flow control, oxygen/pH regulation, and continuously sampled gut effluent collection. Using our custom biological projection micro-stereolithography (BioPμSL) system, we fabricated physiologically relevant crypt-villus scaffolds with physiological dimensions and softness within 30 minutes. We demonstrated that microbial transglutaminase (TG) enzyme could stably link proteins to GelMA, significantly improving Caco-2 cell adhesion to the GelMA surface. Moreover, we show stable protein density gradients could be created by allowing the mixture of TG and proteins to diffuse into hydrogels. Human intestinal cells seeded on 3D printed intestinal tissues exhibited robust adhesion, proliferation, and maturation into an apico-basal polarized monolayer. By integrating the crypt-villus scaffolds into the microfluidic platform, we demonstrated its potential for co-culturing epithelial cells with gut-relevant microbes, enabling monitoring of oxygen and pH levels and analysis of microbial growth during co-culture and assessment of cell viability afterward. This innovative platform holds promise for investigating human-microbiome interactions, advancing disease diagnosis/prevention, and facilitating drug screening applications.