The Modular µSiM Instrumented: Integration of Impedance Sensing for Real-Time Monitoring of Barrier Function

Introduction: : Tissue barriers, such as the vascular endothelium, play a crucial role in maintaining physiological homeostasis by regulating the selective exchange of substances between the bloodstream and tissue compartments. Disrupted barrier integrity can contribute to the development of various pathological conditions, including neuroinflammation, acute lung injury, and others. Consequently, physiologically relevant in vitro barrier models are vital for systematically studying these barriers in both healthy and diseased contexts. Microfluidic systems are becoming increasingly popular because they offer excellent control over the microenvironment cues needed to maintain barrier tissues. We have previously introduced the modular µSiM platform, that features 200 nm thick permeable silicon culture membranes and enables real-time imaging of tissue barriers. The modular design of the system allows the simple introduction of physiological flows to condition the barrier and introduce secondary cell populations through stainless steel syringe needles. Barrier integrity is assessed using various techniques, including permeability assays, imaging, and electrical measurements. TEER is a conventional single-frequency measure that provides global insight into the tightness of the barrier. However, impedance spectroscopy (IS) offers advantages over traditional TEER by providing insights into the paracellular and transcellular pathways and capacitance of the barriers, which is crucial because distinguishing between these specific pathways (transport between cells vs. through cells) and understanding membrane characteristics (via capacitance) provides a much deeper, mechanistic understanding of barrier function and changes compared to the single, global tightness value offered by TEER.

Materials and

Methods: : To enable real-time electrical measurements of barrier tissues, we designed and fabricated an impedance measurement module that magnetically integrates with the m-µSiM platform. This modular approach allows for the addition and removal of impedance measurement capabilities as needed. The module utilizes the same stainless steel needles used in flow as electrodes for real-time electrical measurements in both static and dynamic culture environments. The performance of the module was characterized using human umbilical vein endothelial cells (HUVECs) and human-induced pluripotent stem cell-derived brain microvascular endothelial cells (hiPSC-BMECs). To develop a more comprehensive understanding of the electrical behavior of the barrier, we constructed an equivalent resistor-capacitor circuit model that captures the key electrical characteristics of the cellular layer. This model was used to thoroughly analyze the electrical impedance spectroscopy data acquired over the frequency range of 2.5 Hz to 10 kHz. Immunocytochemistry for junctional proteins was performed, and sampling-based and in situ small molecule permeability assays were conducted using published techniques. Barrier disruption was assessed by exposing cells to lipopolysaccharide (LPS) and monitoring impedance changes over 24 hours.

Results, Conclusions, and Discussions:: We first focused on the design and electrical modeling of the barrier impedance measurement module. The magnetically sealed module uses stainless steel needles as electrodes to provide real-time electrical measurements of endothelial barrier integrity. Finite element modeling and experimental validation confirmed the importance of precise electrode alignment for accurate impedance readings, with less than 5% variation across devices. Real-time monitoring capabilities were demonstrated by observing lipopolysaccharide (LPS)-induced barrier disruption. Impedance spectroscopy showed a decline in paracellular resistance (RPar), indicating compromised barrier function, which correlated with increased molecular permeability and disrupted VE-Cadherin junctions observed through immunostaining. The platform also allowed assessment of shear flow effects on iPSC-BMEC monolayers, showing cell alignment and significantly increased impedance, suggesting enhanced barrier function under flow conditions. Our system also successfully supported the establishment of functional barriers within biomimetic 3D hyaluronic acid hydrogel environments in the bottom channel. These 3D cultures showed increased net impedance and maintained selective permeability comparable to planar cultures. In conclusion, this work presents a versatile tool for dynamic barrier studies. The µSiM platform, and impedance sensing module, offers real-time, label-free monitoring and is compatible with various experimental setups, including flow and 3D matrices. Its compatibility with standard biological assays like permeability tests and immunostaining enables multi-pronged characterization and validation studies. This platform advances in vitro barrier modeling, particularly for studying barrier function in complex microenvironments and under dynamic conditions like inflammation or shear stress. Specifically, the impedance spectroscopy measurements provided insights beyond the single global resistance value offered by traditional TEER. For instance, by resolving the impedance into components like paracellular resistance (RPar), the study directly demonstrated that LPS-induced disruption primarily affected the integrity of the junctions between the cells. This level of detail, pinpointing the specific pathway affected, is crucial for mechanistic understanding and is not available from TEER alone. Thus, IS allows for a more nuanced interpretation of barrier dynamics, distinguishing between effects on intercellular junctions versus the cells themselves under different conditions. The modular nature and use of readily available components also contribute to its potential for broader adoption and impact in the research community.

Acknowledgements and/or References (Optional)::

References:
1. McCloskey, M. C., Kasap, P., Ahmad, S. D., Su, S., Chen, K., Mansouri, M., Ramesh, N., Nishihara, H., Belyaev, Y., Abhyankar, V. V., Begolo, S., Singer, B. H., Webb, K. F., Kurabayashi, K., Flax, J., Waugh, R. E., Engelhardt, B., & McGrath, J. L. (2022). The Modular µSiM: A Mass Produced, Rapidly Assembled, and Reconfigurable Platform for the Study of Barrier Tissue Models In Vitro. In Advanced Healthcare Materials (Vol. 11, Issue 18). https://doi.org/10.1002/adhm.202200804
2. Mansouri, M., Ahmed, A., Ahmad, S. D., McCloskey, M. C., Joshi, I. M., Gaborski, T. R., Waugh, R. E., McGrath, J. L., Day, S. W., & Abhyankar, V. V. (2022). The Modular µSiM Reconfigured: Integration of Microfluidic Capabilities to Study In Vitro Barrier Tissue Models under Flow. In Advanced Healthcare Materials (Vol. 11, Issue 21). Wiley. https://doi.org/10.1002/adhm.202200802