Assessment of epithelial barrier integrity in cellular monolayer models using low cost, clinically approved fluorescent contrast agents

Epithelial barriers serve as the interface between body tissue and the external environment, maintaining tissue homeostasis by regulating the transport of various cells, molecules and microbes between these compartments. In particular, the epithelial barrier of the gut is a key component in numerous physiological processes that collectively define human health, such as nutrient absorption, microbiota regulation and neuroimmune communication. As such, gut epithelial barrier dysfunction, also known as ‘leaky gut’ syndrome has been associated with a variety of different conditions such as inflammatory bowel disease, type II diabetes, HIV and undernutrition. A diverse range of in vitro models have therefore been developed to better understand epithelial barrier function in different disease contexts. To study gut permeability in vitro , typical measurements include transepithelial electrical resistance (TEER) and the use of fluorescent contrast agents such as FITC-dextran to assess barrier integrity and permeability, with microfluidic ‘gut-on-a-chip’ models exclusively using FITC-dextran. However, utilising FITC-dextran in this context has multiple drawbacks for translational gastroenterology research. Namely, FITC-dextran is expensive, not clinically approved, and there is no agreed molecular weight ‘cut-off’ to determine barrier damage. The molecular weight of FITC-dextran also does not mimic substances used for barrier assessment in clinical settings (e.g., lactulose) nor the molecules thought to drive enteropathy in vivo (e.g., LPS and other pathogen associated molecular patterns (PAMPs)). Therefore, the clinically approved fluorescent contrast agents fluorescein and methylene blue (which have molecular weights comparable to substances used in the clinic) were explored in cellular monolayer models of the gut epithelium as low-cost alternatives for fluorescent assessment of epithelial barrier integrity. Both fluorescein and methylene blue exhibited analogous behavior to FITC-dextran, indicating suitability for use in fluorescent epithelial barrier assays. Hence, wider adoption of fluorescein and methylene may provide cost savings for in vitro epithelial barrier assessment assays as well as opportunities for more advanced, coherent and translational assessment of epithelial barrier function in health and disease.