Exploring Barrier Function with TEER Assays in Organ-on-Chip Models

Understanding barrier function and monitoring barrier integrity is fundamental for unraveling the complexities of physiological processes and disease mechanisms in the human body. Barriers play a particularly crucial role in drug discovery as they are often responsible for drug transport into the cell or are part of the drug target mechanism. However, traditional models, such as 2D cell cultures or animal models, often have fallen short in relevance, reproducibility, and translatability. This has spiked the emergence of microphysiological systems, also called organ-on-chip models, replicating human organs in vitro. Those platforms provide a much more physiologically relevant environment for studying barrier functions.

In this blog we will delve into the significance of organ models in studying barrier function, focusing on transendothelial/transepithelial electrical resistance (TEER) assay, exploring its essential features, and showcasing examples of groundbreaking research in various organ systems.

Why Organ Models Trump Traditional 2D Models for Studying Barrier Function

Mimicking the in vivo microenvironment

Traditional 2D models, such as Transwell systems, often fail to replicate the complex microenvironment of human organs accurately. In contrast, organ-on-chip models closely mimic the three-dimensional architecture, cell-cell interactions, the immune or microbial component and mechanical forces such as tissue-specific perfusion present in vivo. On a technical level, biochips for cultivation of organ models are typically multilayered microdevices that can include porous membranes, which are particularly suitable for building epithelial/endothelial interfaces (Ballerini 2022 et al.). The physiological conditions combined with the mechanical structure provided by the organ-on-chip systems is vital for studying barrier function, able to mirror human organ function.

Cellular complexity

Barrier function is often a result of intricate interactions among multiple cell types within an organ. Organ-on-chip models, by incorporating various cell types relevant to the target organ, or the integration of a more realistic extracellular matrix, including proteins like collagen IV, laminin, and fibronectin better replicate the multicellular complexity compared to the simplicity of monolayer cultures. Among the proteins that can be better replicated in organ models are specific membrane transporters, including P-glycoprotein (P-gp) that regulate efflux of potentially harmful agents, including lipophilic agents (van der Helm 2016 et al.). P-gp efflux transporters were found to be upregulated in a microfluidics-based microvasculature model compared to transwells (Prabhakarpandian 2013 et al.). In blood-brain barrier (BBB) models, the expression of specific markers such as adherens and tight junction proteins can be tested (van der Helm 2016 et al.).

The mechanism of recruitment and the extravasation of leukocytes during bacterial infection can be studied in BBB and lung-on-a-chip models, respectively. Which is not possible in this way in Transwell cultures (Huh 2010 et al.). Equally, BBBs-on-chips have shown an increase in barrier tightness when astrocytes and pericytes are included (van der Helm 2016 et al.). Those are just some examples of how the increased complexity in organ models leads to a more comprehensive understanding of barrier function and regulation.

Perfusion in organ models

A hallmark of physiological organ models is the integration of perfusion, reproducing flow conditions specific to the organ environment studied, a feature most 2D cultures lack. The integration of perfusion into a model system mimics the blood flow in vivo, which enables physiological assembly and formation of cellular tissue. This is particularly important for proper barrier function dynamics.  In vascularized organs, the impact of shear stress on barrier function cannot be overlooked (Maurer 2019 et al.). Flow furthermore enables physiological interaction of immune cells with the barrier. In contrast to static conditions, where immune cells tend to interact with the barrier also without inflammatory triggers, just due to gravitational forces settling the cells (Raasch 2015 et al.).

Furthermore, Rinkenauer 2015 et al. compared static and dynamic in vitro cell culture and was able to show that shear stress caused by perfusion significantly altered the efficiency of the uptake of different nanoparticles by the barrier. Including perfusion adds an additional layer of complexity to the assessment of barrier function, leading to more comprehensive models resembling the situation in vivo.

Measurement in real-time and for long timeframes

The ability to monitor barrier function in real-time is a distinctive advantage of organ-on-chip models over traditional 2D systems. The chopstick assay traditional employed to measure TEER in Transwells requires manual insertion of electrodes which must be removed afterwards. Assays can therefore not be conducted in incubators or in real-time over longer timeframes. In contrast, TEER assays in organ models are often performed through electrodes that are fixed within the biochip used to culture the organ model. TEER assays in organ models are therefore non-invasive and real-time.

Handling and Reproducibility

Fixed electrodes offer the added advantage of having almost no detectable measurement bias caused by electrode movement. Due to the manual handling of electrodes in traditional TEER assays for 2D cell culture electrodes can be misplaced and have freedom to move making measurement less comparable. In contrast multiple models under different conditions could be analyzed using TEER assays withing organ-on-chip models making results more reproducible. Additionally, as less manual handling is involved in the TEER assay it reduces the chance for handling mistakes potentially damaging the tissue culture.

Technical features to consider in TEER assays

Electrodes in organ-on-chip TEER assays are most commonly made of gold or platinum. One of such model systems is our Dynamic TEER chip which includes gold-plated electrodes in the inside of the biochip top and bottom layer.

The materials are favorable as they are inert and biocompatible. However, they can suffer from a high electrode-electrolyte interface impedance due to their polarizability, which can cover up small changes in the TEER (Holzreuter 2024 et al.). That said, they are still favorable compared to their alternatives, such as Ag/AgCl. Those compounds have shown cytotoxic effects, making them unsuitable especially for TEER assays over longer timeframes (Holzreuter 2024 et al.).

Apart from material considerations there are also measurement specificities that need to be taken into account when using TEER assays in organ-on-chip models. When calculating TEER for a specific tissue, the blank resistance of the biochip with culture medium is subtracted from the total resistance with the tissue culture measured. Due to their long channels combined with small cross-sectional areas, biochips have an inherently high blank resistance compared to the values measured with tissue. This can be illustrated by the formular used to calculate the resistance of the blank biochip:

R_{medium =} ρ * ( l_ch / A_ch )

R_medium is the medium resistance, ρ is the specific resistance of the medium, l_ch is the length of the channel, and A_ch is the cross-sectional area of the channel (Holzreuter 2024 et al.).

Examples of Research Studying Barrier Function Using Organ Models and TEER Assays

Lung-on-Chip Models

A study by Jabbar 2022 et al. looked at pulmonary disease condition induced by particulate matter (PM10) using on-chip healthy human lung distal airway model including a TEER sensor.

A study by Gabela-Zuniga 2024 et al. developed a humanized in vitro ventilator-on-a-chip (VOC) model of the lung microenvironment simulating the different injurious forces generated in the lung during mechanical ventilation. They furthermore used TEER measurements to investigate how application of different mechanical forces alters real-time changes in barrier integrity during and after injury.

Intestinal-on-Chip Models

Gijzen 2020 et al. developed an immunocompetent 3D perfused intestine-on-a-chip model to study intestinal inflammation. The inflammation process was simulated through exposure with tumor necrosis factor-α and interleukin-1β and quantified via measurement of TEER and proinflammatory cytokine secretion.

Vera 2024 et al. developed a 3D bioprinted hydrogel gut-on-chip with integrated electrodes for TEER measurements.

Morelli 2024 et al. developed an intestine-on-chip platform to study the effects of enterotoxins on the gut. Particularly, they measured the dose-dependent reduction in barrier permeability using TEER.

Blood-Brain Barrier (BBB) Models

Florez 2023 et al. developed a BBB-on-chip model with integrated TEER electrodes to assess the barrier permeability of gold nanorods against Alzheimer’s disease.

Nair 2023 et al. demonstrated barrier disruption, endothelial inflammation, and T cell migration under neuroinflammatory conditions in BBB-on-a-chip model comprising human brain microvascular endothelial cells and using TEER measurements.

Ohbuchi 2024 et al. developed a human BBB-on-a-chip to model barrier dysfunction and immune cell migration. TEER measurements showed that barrier integrity increased under co-culture with pericytes and a decreased caused by EDTA and anti-Claudin-5 neutralizing antibody.

Conclusion

In conclusion, the shift from traditional 2D models, such as Transwell cultures, to organ-on-chip models for studying barrier function represents a paradigmatic advancement in biomedical research. The physiological relevance, multicellular complexity, dynamic microfluidic environment, real-time monitoring capabilities, and improved reproducibility make organ-on-chip models with TEER assays indispensable tools. The examples discussed underscore the superior performance of organ-on-chip systems in providing more accurate insights into barrier function across various organ systems. As the field continues to evolve, organ-on-chip models are poised to lead the way in unraveling the intricacies of barrier function and advancing our understanding of physiological and pathological processes.

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