Assessment of epithelial barrier integrity in cellular monolayer models using low cost, clinically approved fluorescent contrast agents
6 affiliations
- 1Helen Hamlyn Centre, Institute of Global Health Innovation, Department of Surgery & Cancer, Bessemer Building, Imperial College London, London, United Kingdom
- 2Blizard Institute, Queen Mary University of London, London, United Kingdom
- 3Tropical Gastroenterology and Nutrition Group, University of Zambia School of Medicine, Lusaka, Zambia
- 4Division of Digestive Diseases, Department of Metabolism, Digestion and Reproduction, St Mary’s Hospital, Imperial College London, London, United Kingdom
- 5School of Electronic and Electrical Engineering, University of Leeds, Woodhouse, Leeds, United Kingdom
- 6School of Infection & Immunity, College of Veterinary and Life Sciences, University of Glasgow, Sir Graeme Davies Building, 120 University Place, Glasgow, United Kingdom
Abstract
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.
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BibTeX
@article{sanchez2026assessment,
title = {{Assessment of epithelial barrier integrity in cellular monolayer models using low cost, clinically approved fluorescent contrast agents}},
author = {Elena Monfort Sanchez and Aaron F. Watson and Tafhima Haider and Despoina Chrysostomou and Maelle Storder and Nilanjan Mandal and Paul Kelly and Julian R. Marchesi and James Avery and Claire D. Bourke and Alex J. Thompson},
journal = {PLOS One},
year = {2026},
month = oct,
doi = {10.1371/journal.pone.0358746},
url = {https://doi.org/10.1371/journal.pone.0358746},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Assessment of epithelial barrier integrity in cellular monolayer models using low cost, clinically approved fluorescent contrast agents
AU - Elena Monfort Sanchez
AU - Aaron F. Watson
AU - Tafhima Haider
AU - Despoina Chrysostomou
AU - Maelle Storder
AU - Nilanjan Mandal
AU - Paul Kelly
AU - Julian R. Marchesi
AU - James Avery
AU - Claire D. Bourke
AU - Alex J. Thompson
PY - 2026
DA - 2026/10/01
JO - PLOS One
DO - 10.1371/journal.pone.0358746
UR - https://doi.org/10.1371/journal.pone.0358746
AB - 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.
SN - 1932-6203
ER -