INTRODUCTION:

The intestinal epithelium forms a selective barrier that protects the host from pathogens while permitting nutrient uptake. Giardia intestinalis is an extracellular protozoan that adheres to and proliferates in the intestinal lumen. Its extracellular vesicles (EVs) modulate immune responses and parasite–host communication. Conventional two-dimensional (2D) cultures fail to reproduce the structural and functional complexity of the gut, whereas three-dimensional (3D) cocultures provide a more physiological environment. This study aims to develop a 3D Caco- 2/HT-29 epithelial model, compare it to the 2D system during G. intestinalis infection, assess epithelial barrier alterations, and characterize the impact of extracellular vesicles on epithelial integrity and host cell responses.

MATERIALS AND METHODS:

Caco-2 and HT-29 cells (ratio 9:1) were seeded on gelatin-coated Transwell® inserts and cultured for 10 days. Barrier formation was monitored by TEER and FITC-dextran flux. EVs from parasite-free and infected cultures were isolated by differential centrifugation and analysed by nanoparticle tracking analysis and protein quantification. 2D monolayers and 3D epithelium were infected with G. intestinalis WB trophozoites; adhesion, tight-junction protein levels (ZO-1, claudin-1, occludin), reactive-oxygen species and apoptosis were quantified.

RESULTS AND DISCUSSION:

The 3D model formed multilayered epithelium with mucus and functional tight junctions (high TEER; low FITC-dextran flux). Compared with 2D cultures, 3D cells secreted more EVs enriched in adhesion-and redox-related proteins. G. intestinalis adhered rapidly in 3D but poorly in 2D. After 48 hours, infection provoked a TEER drop (≈40 % after 6 h), a four-fold rise in FITC-dextran passage, down-regulation of ZO-1/occludin, and increased ROS and apoptosis of host cells. EVs collected during infection accelerated these effects, highlighting a vesicle-mediated mechanism for epithelial disruption.

CONCLUSIONS:

The Caco-2/HT-29 3D model faithfully reproduces key features of the intestinal barrier and reveals EV-dependent modulation of epithelial integrity during G. intestinalis infection. By coupling electrical, molecular and redox readouts, it provides a powerful platform to dissect host-parasite communication and to screen therapeutic interventions aimed at preserving epithelial homeostasis.