Konstanze Brandauer, Alexandra Lorenz, Silvia Schobesberger, Patrick Schuller, Martin Frauenlob, Sarah Spitz, and Peter Ertl

2025, Lab on a Chip

Background and Objectives:

It is widely recognized that various gastrointestinal disorders (e.g., inflammatory bowel disease) and non-gastrointestinal conditions (e.g., Parkinson’s disease, rheumatoid arthritis) may originate in the gastrointestinal tract and are linked to intestinal barrier dysfunction. Additionally, the prevalence of these diseases increases with age, suggesting that aging influences gut barrier function. However, the effect of aging on the gastrointestinal barrier still remains unclear.

While animal studies suggest that aging increases intestinal permeability, human studies remain inconclusive, with factors like microbiome diversity and immune dysfunction complicating the identification of “healthy” aging-related changes. To better study age-related gut barrier dysfunction, researchers are turning to organ-on-a-chip models, which emulate the human gastrointestinal microenvironment more accurately than traditional 2D cultures. These systems integrate shear stress, nutrient gradients, and advanced sensing strategies, allowing real-time, non-invasive, and in situ monitoring of cellular functions. This way, an impaired gut barrier can be monitored by transepithelial electrical resistance (TEER). However, common TEER methods, such as chopstick electrodes, have limitations, e.g., measurement inaccuracies and cell damage during the insertion process.

In this study, a membrane-based impedance sensor tailored for a gut-on-a-chip platform was developed to dynamically monitor the barrier integrity of a senescent gut model. The system was optimized to streamline the fabrication, improve handling, and align the apical cultivation areas with the dimension of a 24-well Transwell (TW) setup.

 

Results

Initial characterization of the integrated porous membrane-based impedance sensor

The microfluidic gut-on-a-chip system integrates interdigitated gold electrodes on a porous PET membrane to enable continuous, in situ monitoring of cell attachment, proliferation, differentiation, and gut barrier integrity. The electrodes, covering ~57% of the apical cell culture area, ensure broad detection capabilities while maintaining direct contact with epithelial cells for reliable impedance measurements. The PDMS-based microfluidic platform replicates the in vivo gut microenvironment, aligning with TW systems for direct comparability.

To validate the functionality and stability of the system, impedance spectroscopy was conducted:

• Collagen coating improved cell adherence and increased impedance values, confirming its impact on surface modification.
• Electrode stability tests exhibited a low relative standard deviation (RSD) demonstrating high reproducibility.
• Continuous impedance monitoring for 60 hours confirmed robustness in a humidified incubator environment.
• Batch-to-batch variation testing showed a low RSD, indicating a consistent fabrication process.
• Salt concentration experiments confirmed that barrier integrity measurements remain unaffected at 20 kHz, ensuring reliable impedance readings.
• Fluorescence diffusion studies verified uninterrupted nutrient diffusion across the membrane, confirming pore integrity and suitability for epithelial cell polarization.

Sensor validation in a gut model resembling key gastrointestinal features

Next, the system was evaluated for biocompatibility, electrode performance, barrier formation, barrier functionality, and application versatility using Caco-2 intestinal epithelial cells:

• Biocompatibility tests were set out to investigate potentially harmful effects mediated by incomplete removal of cytotoxic resist components during the manufacturing process. Cytotoxicity assessments confirmed high cell viability and metabolic activity across the entire membrane area.
• Impedance measurements were carried out to assess whether epithelial cellular barriers affect electrode performance and maintain stable impedance values throughout extended cultivation periods. The system showed stable impedance after 14 days of cell culturing, with cell detachment restoring baseline impedance values, proving that cell adherence does not affect electrode performance.
• Impedance-time trace measurements and frequency analysis confirmed successful barrier formation in the system.
• Barrier functionality was validated by a FITC-dextran assay, demonstrating the sensor’s functionality in monitoring the barrier formation and establishing a tight barrier model.
• To show the application versatility for other intestinal in vitro models, impedance measurements of a direct epithelial co-culture (HT29-MTX with Caco-2) and an indirect endo- and epithelial co-culture (HUVECs with Caco-2) were successfully conducted

In order to prove the physiological relevance of the simplified gut model cultured in the sensor-integrated platform, the epithelial functionality, mucus secretion, ZO1 production, and microvilli formation were investigated.

• Intestinal functionality of epithelial Caco-2 cells was assessed by measuring aminopeptidase activity, a key enzyme of the apical brush border. After 7 days of cultivation, Caco-2 cells exhibited high enzymatic activity, which is 3.5x higher compared to the traditional methods.
• Alcian blue staining revealed a pronounced mucus layer in the on-chip model, indicating high secretion of mucus in vitro.
• Cellular expression of ZO1 reveals the presence of tight junctions in the cell barrier.
• The microvilli structure in the intestine is crucial for nutrient absorption, secretion, and mechanotransduction and, therefore, has to be considered in an intestinal in vitro Scanning electron microscopy (SEM) showed densely packed microvilli across the apical surface, confirming structural resemblance to the human intestine.

Sensor application for monitoring barrier integrity in a senescent gut model

To assess the capability of the gut-on-a-chip platform in monitoring senescence-mediated changes in human intestinal epithelial cells, cells were exposed to doxorubicin (DXR), a well-known inducer of cellular senescence that causes DNA damage and oxidative stress. As a result of senescence, significant molecular and morphological alterations were observed in DXR-treated cells. The analysis revealed an increase in senescence-associated β-galactosidase activity, confirming the presence of aged cells. Microscopic evaluation further showed enlarged cell sizes, a common characteristic of senescent cells. Impedance measurements uncovered enhanced barrier integrity in the senescent gut in vitro model. At the gene expression level, changes associated with aging were evident. While no significant increase in expression of the gene encoding for ZO1 was detected, indicating that the tight junction structure remained unaltered, the gene encoding for claudin-2, a pore-forming protein linked to reduced barrier integrity, was slightly overexpressed. Moreover, the expression of the gene encoding for p21, a key marker of cell cycle arrest, was significantly elevated, further supporting the establishment of a senescent state. Similarly, expression of the gene encoding for CCL2 was markedly increased, reflecting the activation of inflammatory pathways commonly associated with aging cells.

These findings confirm that the sensor-integrated gut-on-a-chip platform successfully established a senescent intestinal in vitro model.

 

Conclusion:

The current study demonstrates the applicability of a gut-on-chip system for real-time, in situ, and label-free monitoring of cellular barrier function and morphological changes with high stability, reproducibility, and robustness. Unlike traditional chopstick electrodes, the fixed impedance sensor eliminates positioning errors and offers higher sensitivity. In addition, it was shown that the system can mimic key human gastrointestinal features, as well as its capability to monitor senescence-mediated changes.

Read the full publication: Sensor-integrated gut-on-a-chip for monitoring senescence-mediated changes in the intestinal barrier

 

Glossary:

Alcian blue staining: a histological technique used to detect mucus and connective tissues by binding to their key components and producing a blue stain.

Aminopeptidase: an enzyme located in the apical brush border of intestinal epithelial cells, playing a crucial role in protein digestion, aiding in nutrient absorption and gut functionality.

Apical cultivation: refers to the growth of cells on the upper surface of a membrane or culture system, mimicking the natural orientation of epithelial tissues, where the apical side faces the lumen (e.g., the intestinal or airway surface) for barrier function and nutrient absorption studies.

Batch-to-batch variation testing: a quality control process that evaluates the consistency and reproducibility of a product, experiment, or fabrication process by comparing multiple production batches to ensure minimal variability and reliable performance across different sets.

Biocompatibility:  refers to the ability of a material or device to interact with biological systems without causing toxic, harmful, or adverse immune responses, ensuring its safety and functionality for medical or biological applications.

Brush border: the dense layer of microvilli on the apical surface of epithelial cells in organs like the intestine and kidney, increasing surface area for nutrient absorption, enzymatic activity, and transport functions.

Caco-2: a human epithelial cell line derived from colorectal adenocarcinoma (colorectal cancer cells), widely used as an in vitro model of the intestinal barrier due to its ability to differentiate into enterocyte-like cells, forming tight junctions, microvilli, and expressing brush border enzymes.

C-C Motif Chemokine Ligand 2 (CCL2): a pro-inflammatory cytokine that is part of the senescence-associated secretory phenotype and therefore serves as a marker for senescence.

Cell polarisation: the asymmetric organisation of cellular components, including the cytoskeleton, organelles, and membrane proteins, enabling specialised functions such as directed transport, signal transduction, and barrier formation, essential in epithelial cells, neurons, and immune cells.

Claudin-2:  a pore-forming tight junction protein that regulates paracellular permeability, allowing the passage of small ions and water between epithelial cells, and is often upregulated in inflammatory conditions, contributing to increased intestinal permeability.

Direct epithelia co-culture: refers to a cell culture technique where two or more epithelial cell types are grown in direct contact within the same environment, allowing cell-cell interactions, signaling, and functional integration, often used to mimic physiological tissue conditions in vitro.

Doxorubicin (DXR): a chemotherapeutic drug that leading to DNA damage, oxidative stress, and apoptosis, and is also used to induce cellular senescence in research models.

FITC-dextran: a fluorescently labeled polysaccharide used as a tracer molecule to assess permeability and barrier integrity in biological systems, commonly applied in intestinal and blood-brain barrier studies to measure paracellular transport and leakage.

Impedance spectroscopy: a non-invasive analytical technique that measures the electrical impedance of biological or material systems across different frequencies, providing insights into cellular properties, barrier integrity, and electrochemical interactions in applications such as biosensing, tissue engineering, and gut-on-a-chip models.

In situ: refers to the observation, measurement, or experimentation of a process or phenomenon directly in its original location or natural environment, without removing or altering the subject, commonly used in biological, medical, and engineering research.

In vitro: refers to experiments or procedures performed outside a living organism, typically in a controlled laboratory environment, such as in cell cultures, test tubes, or microfluidic systems, to study biological processes under reproducible conditions.

In vivo: refers to experiments or studies conducted within a living organism, such as humans, animals, or plants, to observe biological processes, disease progression, or drug effects in their natural physiological environment

Indirect epithelia co-culture: a cell culture technique where different cell types are grown in the same system but physically separated, typically using porous membranes or Transwell inserts, allowing paracrine signaling and molecular interactions without direct cell-to-cell contact.

Microvilli: tiny, finger-like projections on the apical surface of epithelial cells, primarily in the intestine and kidney, that increase surface area for enhanced absorption, secretion, and cellular interactions.

Organ-on-a-chip: a microfluidic cell culture platform that mimics the structure and function of human organs, integrating living cells, nutrient gradients, and mechanical cues to model physiological processes, disease conditions, and drug responses in a controlled in vitro environment.

p21: a cyclin-dependent kinase inhibitor that regulates the cell cycle, playing a key role in cellular senescence, DNA damage response, and tumor suppression.

Polyethylene Terephthalate (PET): a durable, lightweight, and biocompatible polymer commonly used in medical devices, biosensors, and membrane-based cell culture systems due to its chemical stability, transparency, and mechanical strength.

Polydimethylsiloxane (PDMS): a biocompatible, flexible, and transparent silicone-based polymer widely used in microfluidics and organ-on-a-chip devices due to its gas permeability, ease of molding, and optical clarity.

Senescence: a state of permanent cell cycle arrest that occurs in response to cellular stress, DNA damage, or aging, where cells remain metabolically active but no longer divide, often contributing to tissue dysfunction, inflammation, and age-related diseases.

Senescence-associated β-galactosidase (SA-β-gal): an enzyme marker commonly used to identify senescent cells, as its activity increases in lysosomes during cellular aging, serving as a reliable indicator of the senescence phenotype in vitro and in vivo.

Scanning electron microscopy (SEM): an imaging technique that uses a focused beam of electrons to scan a specimen’s surface, producing high-resolution, three-dimensional images that reveal fine structural details at the micro- to nanoscale.

Tight junctions: specialised connections between adjacent epithelial or endothelial cells that form a selective barrier, regulating the paracellular transport of ions and molecules and maintaining cell polarity and tissue integrity.

Transepithelial electrical resistance (TEER): a quantitative, non-invasive method used to measure the integrity and permeability of cell layers, especially epithelial or endothelial barriers, by assessing the electrical resistance across the cell monolayer.

Transwell setup: an in vitro cell culture system consisting of a permeable membrane insert within a multiwell plate, allowing the study of barrier function, cell migration, and transport between apical and basolateral compartments.

Zonula Occludens-1 (ZO1): a protein found at tight junctions in epithelial and endothelial cells, where it connects transmembrane proteins like claudins and occludin to the actin cytoskeleton, playing a key role in maintaining cell polarity, barrier integrity, and paracellular transport regulation.