Epithelial barriers are crucial for protecting the body from external environments. Disruptions in these barriers, like "leaky gut," are implicated in various chronic inflammatory diseases. Current methods for studying barrier integrity, such as Transwell assays and Ussing chambers, have limitations. Transwells offer simplicity and high throughput but lack physiological realism. Ussing chambers, while using *ex vivo* tissue, suffer from short viability times and static media. Organ-on-a-chip (OoC) devices improve upon this, incorporating microfluidics and engineered tissues, but still fall short of representing the in vivo complexity of barrier tissues. This study aims to overcome these limitations by creating a microphysiological system that maintains the *in vivo* cellular complexity of the intestinal barrier while enabling real-time TEER measurements over extended periods.

Traditional methods for assessing epithelial barrier integrity include fluorescently labeled probes to visualize paracellular flux. However, real-time monitoring is increasingly desired, leading to the adoption of TEER measurements. While Transwells are simple and cost-effective, they lack physiological relevance due to the absence of media flow. OoC devices improve this by integrating microfluidics and engineered tissues, but they do not fully replicate the complexity of *in vivo* barrier tissue. *Ex vivo* tissue explant models, such as the Ussing chamber, offer greater physiological accuracy but are often limited by short tissue viability (<3h), static media, and high costs. Existing *ex vivo* devices capable of longer-term viability (>48h) usually lack real-time barrier integrity measurements, requiring tissue removal at multiple time points. This necessitates the use of more animals and increases experimental time and labor.

The researchers designed a microfluidic chamber using CAD and fabricated it using a 3D printer. The chamber consists of two halves, each containing a PCB, a gold electrode chip, and a PDMS layer. The tissue explant is secured between the two halves. Media is pumped through the chamber via Luer lock connectors using syringe pumps, allowing for independent control of luminal and serosal media. 3D fluid simulations ensured uniform media flow over the tissue. The integrated TEER electrodes connect to a custom-built electronic system housed in a metal enclosure. This system features a Howland current source for constant-current TEER measurement, signal conditioning circuitry, an ADC, and a microcontroller interfacing with a custom GUI via USB. The system can accommodate up to three chambers simultaneously. Electrodes were fabricated using photolithography and gold deposition on a glass substrate. The system was sterilized using a multi-step protocol. Experiments used male C57BL/6 mice. Colon tissue was dissected, and the muscle layer was either left intact or removed. Custom-made Adult Neurobasal media with supplements, glucose, HEPES buffer, and inulin (for microbiome maintenance) was used. Luminal media optionally included collagenase or HCl for barrier disruption experiments. TEER measurements were performed every 2 hours using both sinusoidal (5kHz) and square wave stimuli. Post-experiment, tissue was processed for histochemical analysis. TEER data processing involved noise reduction, curve fitting, drift correction, and baseline subtraction to isolate the epithelial barrier resistance. Statistical analyses were used to assess differences in TEER values.

The microphysiological system successfully maintained mouse colon tissue viability for up to 72 hours. Histological analysis showed preserved mucosal architecture, goblet cells, and claudin-1 expression, indicating maintained barrier integrity. TEER measurements correlated with observed physiological changes in barrier function. Treatment with collagenase induced a reduction in TEER, accompanied by changes in goblet cell morphology and claudin-1 expression, consistent with barrier impairment. Acidic media (pH 2) treatment caused significant TEER reduction, along with substantial damage to the epithelial layer, including goblet cell loss and dramatic reduction in claudin-1. The system accurately distinguished differences in TEER based on the presence or absence of the muscle layer, showing a 39% decrease in TEER with muscle removal. It also distinguished between proximal and distal colon regions, with the distal colon showing approximately 19% lower TEER. The system demonstrated high electrical performance with a bandwidth of 47.5 kHz and a signal-to-noise ratio (SNR) of 28.14 dB. A comparison of the microphysiological system to existing systems highlighted its advantages in terms of tissue viability, real-time TEER measurement, throughput, and integrated design.

This study successfully addressed the limitations of existing systems for studying epithelial barrier function. The ability to maintain *ex vivo* tissue viability for 72 hours while performing real-time TEER measurements is a significant advancement. The correlation between TEER measurements and histological findings confirms the system's accuracy in assessing barrier integrity. The ability to distinguish between different tissue types and treatments showcases the system's sensitivity and applicability to various research questions. This microphysiological system offers a more physiologically relevant model compared to in vitro systems, enabling more accurate investigations into the mechanisms of barrier dysfunction and the effects of various stimuli or treatments.

This research presents a highly integrated microphysiological system for studying live tissue barrier permeability. Its key features include extended tissue viability (up to 72 hours), real-time TEER measurement, high throughput potential, and a compact, user-friendly design. The system offers a significant improvement over existing methods, opening new possibilities for investigating barrier health and developing therapies for related diseases. Future research could explore the system's application to different tissue types and the incorporation of additional sensors to provide a more comprehensive assessment of barrier function.

The current study focuses on mouse colon tissue. Further validation is needed to confirm the system's applicability to other tissues and species. While the system is designed to be scalable, the current iteration accommodates only three chambers simultaneously. The use of a custom-built system may present a barrier to widespread adoption, although the modular design could facilitate adaptation and replication.