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Reconstruction of Ultra-thin Alveolar-capillary Basement Membrane Mimics
Alveolar-capillary basement membrane (BM) is ultra-thin (<2 µm) extracellular matrix that maintains integral epithelial-endothelial cell layers. In vitro reconstructions of alveolar-capillary barrier supported on synthetic scaffolds closely resembling the fibrous and ultra-thin natural BM are essential in mimicking the lung pathophysiology. Although BM topology and dimensions are well known to significantly influence cellular behavior, conventionally used BM mimics fail to recreate this natural niche. To overcome this, electrospun ultra-thin 2 µm poly(caprolactone) (PCL) nanofibrous mesh is used to establish an alveolar-capillary barrier model of lung endothelial/epithelial cells. Transepithelial electrical resistance (TEER) and permeability studies reveal integral tight junctions and improved mass transport through the highly porous PCL meshes compared to conventional dense membranes with etched pores. The chemotaxis of neutrophils is shown across the barrier in presence of inflammatory response that is naturally impeded in confined regions. Conventional requirement of 3 µm or larger pore size can lead to barrier disruption due to epithelial/endothelial cell invasion. Despite high porosity, the interconnected BM mimic prevents barrier disruption and allows neutrophil transmigration, thereby demonstrating the physiological relevance of the thin nanofibrous meshes. It is envisioned that these bipolar cultured barriers would contribute to an organ-level in vitro model for pathological disease, environmental pollutants, and nanotoxicology. © 2021 The Authors. Advanced Biology published by Wiley-VCH GmbH
3D‐Printed Bioreactor with Integrated Impedance Spectroscopy for Cell Barrier Monitoring
Cell culture experiments often suffer from limited commercial availability of laboratory-scale bioreactors, which allow experiments to be conducted under flow conditions and additional online monitoring techniques. A novel 3D-printed bioreactor with a homogeneously distributed flow field enabling epithelial cell culture experiments and online barrier monitoring by integrated electrodes through electrical impedance spectroscopy (EIS) is presented. Transparent and conductive indium tin oxide glass as current-injecting electrodes allows direct visualization of the cells, while measuring EIS simultaneously. The bioreactor's design considers the importance of a homogeneous electric field by placing the voltage pick-up electrodes in the electrical field. The device's functionality is demonstrated by the cultivation of the epithelial cell line Caco-2 under continuous flow and monitoring of the cell layer by online EIS. The collected EIS data were fitted by an equivalent electric circuit, resulting in the cell layer's resistance and capacitance. This data is used to monitor the cell layer's reaction to ethylene glycol-bis-(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid and forskolin. These two model substances show the power of impedance spectroscopy as a non-invasive way to characterize cell barriers. In addition, the bioreactor design is available as a print-ready file in the Appendix, enabling its use for other scientific institutions.