A dynamic 5D microfluidic model of the microbiota-gut-brain axis to investigate biological barrier dysfunction in Autism Spectrum Disorder

Autism Spectrum Disorder (ASD) is a multifactorial neurodevelopmental condition characterized by heterogeneous features that encompasses not only core symptoms of impaired social communication and restricted, repetitive behaviors, but also a high prevalence of gastrointestinal manifestations, including abdominal pain, altered intestinal motility and increased intestinal permeability, commonly referred to as "leaky gut". This co-occurrence of neurological and gastrointestinal symptoms suggests the involvement of shared pathophysiological mechanisms, among which the microbiota-gut-brain axis (MGBA) has emerged as a critical bidirectional communication network linking the intestinal environment to the central nervous system. In this framework, the compromise of the intestinal epithelial barrier (IEB) and the blood-brain barrier (BBB) may allow the translocation of pro-inflammatory mediators and neurotoxic molecules into the systemic circulation, promoting neuroinflammation and potentially exacerbating both the neurological and gastrointestinal features of ASD. Despite growing interest, these mechanistic links remain difficult to study in conventional static models, which fail to recapitulate the dynamic, multicellular and mechanobiological complexity of the in vivo environment. To overcome these limitations, a 5D dynamic in vitro model of the MGBA is currently under development on IVTech LiveBox2 microfluidic bioreactors, enabling the simultaneous culture of gut and brain cellular compartments under physiologically relevant perfusion flow. The IEB compartment was successfully established through a co-culture of NCM460 and HT29-MTX human intestinal cell lines on collagen-coated PET membranes, providing a physiologically relevant mucus layer that more faithfully reproduces the in vivo intestinal environment. Barrier integrity was validated by transepithelial electrical resistance (TEER) measurements, which showed a progressive increase over time, and by Lucifer Yellow paracellular permeability assays, yielding a passage rate consistently below 5%, collectively confirming the establishment of a structurally and functionally competent barrier model. The modulatory effects of food-derived Lactiplantibacillus plantarum strains on IEB permeability, tight junction protein expression and inflammatory cytokine profile are currently under investigation within this system, to evaluate their potential as microbiota-targeted interventions along the MGBA. The BBB compartment will be developed through a tri-culture of hCMEC/D3 human cerebral endothelial cells, astrocytes and pericytes representing the neurovascular unit, complemented by THP-1 monocytes to simulate circulating immune cell trafficking. Once both compartments are established, they will be connected under continuous perfusion flow to model the bidirectional gut-brain communication and to study the downstream impact of intestinal hyperpermeability and microbial interventions on barriers integrity. Downstream molecular analyses will employ nCounter® gene expression profiling and multiplex ELISA to simultaneously quantify tight junction transcripts, mucin components and inflammatory mediators across both compartments. This platform represents a reproducible, translational and ethically advantageous tool for the identification of novel biomarkers and therapeutic targets at the gut-brain interface in ASD.