Online monitoring of epithelial barrier kinetics and cell detachment during cisplatin-induced toxicity of renal proximal tubule cells.

Real-time and non-invasive assessment of tissue health is crucial for maximizing the potential of microphysiological systems (MPS) for drug-induced nephrotoxicity screening. Although impedance has been widely considered as a measure of the barrier function, it has not been incorporated to detect cell detachment in MPS with top and bottom microfluidic channels separated by a porous membrane. During cell delamination from the porous membrane, the resistance between both channels decreases, while capacitance increases, allowing the detection of such detachment. Previously reported concepts have solely attributed the decrease in the resistance to the distortion of the barrier function, ignoring the resistance and capacitance changes due to cell detachment. Here, we report a two-channel MPS with integrated indium tin oxide (ITO) electrodes capable of measuring impedance in real time. The trans-epithelial electrical resistance (TEER) and tissue reactance (capacitance) were extracted from the impedance profiles. We attributed the anomalous initial increase observed in TEER, upon cisplatin administration, to the distortion of tight junctions. Cell detachment was captured by sudden jumps in capacitance. TEER profiles illuminated the effects of cisplatin and cimetidine treatments in a dose-dependent and polarity-dependent manner. The correspondence between TEER and barrier function was validated for a continuous tissue using the capacitance profiles. These results demonstrate that capacitance can be used as a real-time and non-invasive indicator of confluence and will support the accuracy of the drug-induced cytotoxicity assessed by TEER profiles in the two-channel MPS for the barrier function of a cell monolayer.

Cells sorted off hiPSC-derived kidney organoids coupled with immortalized cells reliably model the proximal tubule.

Of late, numerous microphysiological systems have been employed to model the renal proximal tubule. Yet there is lack of research on refining the functions of the proximal tubule epithelial layer-selective filtration and reabsorption. In this report, pseudo proximal tubule cells extracted from human-induced pluripotent stem cell-derived kidney organoids are combined and cultured with immortalized proximal tubule cells. It is shown that the cocultured tissue is an impervious epithelium that offers improved levels of certain transporters, extracellular matrix proteins collagen and laminin, and superior glucose transport and P-glycoprotein activity. mRNA expression levels higher than those obtained from each cell type were detected, suggesting an anomalous synergistic crosstalk between the two. Alongside, the improvements in morphological characteristics and performance of the immortalized proximal tubule tissue layer exposed, upon maturation, to human umbilical vein endothelial cells are thoroughly quantified and compared. Glucose and albumin reabsorption, as well as xenobiotic efflux rates through P-glycoprotein were all improved. The data presented abreast highlight the advantages of the cocultured epithelial layer and the non-iPSC-based bilayer. The in vitro models presented herein can be helpful in personalized nephrotoxicity studies.

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

Electrical stimulation of microengineered skeletal muscle tissue: Effect of stimulus parameters on myotube contractility and maturation.

Skeletal muscle tissues engineered in vitro are aneural, are short in the number of fibres required to function properly and degenerate rapidly. Electrical stimulation has been widely used to compensate for such a lack of neural activity, yet the relationship between the stimulation parameters and the tissue response is subject to debate. Here we studied the effect of overnight electrical stimulation (training) on the contractility and maturity of aligned C2C12 myotubes developed on micropatterned gelatin methacryloyl (GelMA) substrates. Bipolar rectangular pulse (BRP) trains with frequency, half-duration and applied pulse train amplitudes of f = 1 Hz, ton  = 0.5 ms and Vapp  = {3 V, 4 V, 4.5 V}, respectively, were applied for 12 h to the myotubes formed on the microgrooved substrates. Aligned myotubes were contracting throughout the training period for Vapp  ≥ 4 V. Immediately after training, the samples were subjected to series of BRPs with 2 ≤ Vapp  ≤ 5 V and 0.2 ≤ ton  ≤ 0.9 ms, during which myotube contraction dynamics were recorded. Analysis of post-training contraction revealed that only the myotubes trained at Vapp  = 4 V displayed consistent and repeatable contraction profiles, showing the dynamics of myotube contractility as a function of triggering pulse voltage and current amplitudes, duration and imposed electrical energy. In addition, myotubes trained at Vapp  = 4 V displayed amplified expression levels of genes pertinent to sarcomere development correlated with myotube maturation. Our findings are imperative for a better understanding of the influence of electrical pulses on the maturation of microengineered myotubes.

Carbon nanotubes embedded in embryoid bodies direct cardiac differentiation.

We embedded carbon nanotubes (CNTs) in mouse embryoid bodies (EBs) for modulating mechanical and electrical cues of the stem cell niche. The CNTs increased the mechanical integrity and electrical conductivity of the EBs. Measured currents for the unmodified EBs (hereafter, EBs) and the EBs-0.25 mg/mL CNTs were 0.79 and 26.3 mA, respectively, at voltage of 5 V. The EBs had a Young's modulus of 20.9 ± 6.5 kPa, whereas the Young's modulus of the EB-0.1 mg/mL CNTs was 35.2 ± 5.6 kPa. The EB-CNTs also showed lower proliferation and greater differentiation rates compared with the EBs as determined by the expression of pluripotency genes and the analysis of EB sizes. Interestingly, the cardiac differentiation of the EB-CNTs was significantly greater than that of the EBs, as confirmed by high-throughput gene analysis at day 5 of culture. Applying electrical stimulation to the EB-CNTs specifically enhanced the cardiac differentiation and beating activity of the EBs.

Macroporous mesh of nanoporous gold in electrochemical monitoring of superoxide release from skeletal muscle cells.

Real-time monitoring of metabolically relevant biochemicals released in minuscule amounts is of utmost diagnostic importance. Superoxide anion as a primary member of reactive oxygen species, has physiological and pathological effects that depend on its concentration and release rate. Here we present fabrication and successfully testing of a highly sensitive electrochemical biosensor featuring a three-dimensional macroporous mesh of nanoporous gold tailored to measure the dynamics of extracellular superoxide concentration. Wide and accessible surface of the mesh combined with high porosity of the thin nanoporous gold coating enables capturing the analyte in pico- to nano-molar ranges. The mesh is functionalized with cytochrome-c (cyt-c) and incorporated as a working electrode to measure the release rate of drug-induced superoxides from C2C12 cells through a porous membrane. The device displays a considerably improved superoxide sensitivity of 7.29nAnM-1cm-2 and a low level of detection of 70pM. Such sensitivity is orders of magnitude higher than any similar enzyme-based electrochemical superoxide sensor and is attributed to the facile diffusion of the analyte through the well-spread nanofeatured gold skin. Superoxide generation rates captured from monolayer myoblast cultures containing about 4×104 cells, varied from 1.0 to 9.0nMmin-1 in a quasi-linear fashion as a function of drug concentration. This work provides a platform for the development of highly sensitive molecular electrochemical biosensors.

A microfluidic optical platform for real-time monitoring of pH and oxygen in microfluidic bioreactors and organ-on-chip devices.

There is a growing interest to develop microfluidic bioreactors and organ-on-chip platforms with integrated sensors to monitor their physicochemical properties and to maintain a well-controlled microenvironment for cultured organoids. Conventional sensing devices cannot be easily integrated with microfluidic organ-on-chip systems with low-volume bioreactors for continual monitoring. This paper reports on the development of a multi-analyte optical sensing module for dynamic measurements of pH and dissolved oxygen levels in the culture medium. The sensing system was constructed using low-cost electro-optics including light-emitting diodes and silicon photodiodes. The sensing module includes an optically transparent window for measuring light intensity, and the module could be connected directly to a perfusion bioreactor without any specific modifications to the microfluidic device design. A compact, user-friendly, and low-cost electronic interface was developed to control the optical transducer and signal acquisition from photodiodes. The platform enabled convenient integration of the optical sensing module with a microfluidic bioreactor. Human dermal fibroblasts were cultivated in the bioreactor, and the values of pH and dissolved oxygen levels in the flowing culture medium were measured continuously for up to 3 days. Our integrated microfluidic system provides a new analytical platform with ease of fabrication and operation, which can be adapted for applications in various microfluidic cell culture and organ-on-chip devices.

Hydrogels containing metallic glass sub-micron wires for regulating skeletal muscle cell behaviour.

Hydrogels with tunable electrical and mechanical properties have a wide range of biological applications in tissue engineering, biosensing, and biorobotics. In this work, palladium-based metallic glass sub-micron wires (PdMGSMWs) were employed to enhance the conductivity and mechanical strength of gelatin methacryloyl (GelMA) gels. The values of electrical resistivity and stiffness of hybrid GelMA-PdMGSMW hydrogels were varied by the concentration of the sub-micron wires in the gels. Compared with pristine GelMA gels, hybrid GelMA-PdMGSMW gels were more efficient in regulating adhesion and spreading of C2C12 myoblasts. Formation, contractility, and metabolic activity of C2C12 myotubes in GelMA hydrogels also increased upon inclusion of the PdMGSMWs and applying electrical stimulation. The latter phenomenon is likely because of the electrical conductivity of hybrid GelMA gels.

Ultralow-voltage field-ionization discharge on whiskered silicon nanowires for gas-sensing applications.

Several hundred million volts per centimetre of electric-field strength are required to field-ionize gas species. Such fields are produced on sharp metallic tips under a bias of a few kilovolts. Here, we show that field ionization is possible at dramatically lower fields on semiconductor nanomaterials containing surface states, particularly with metal-catalysed whiskers grown on silicon nanowires. The low-voltage field-ionization phenomena observed here cannot be explained solely on the basis of the large field-amplification effect of suspended gold nanoparticles present on the whisker tips. We postulate that field penetration causes upward band-bending at the surface of exposed silicon containing surface states in the vicinity of the catalyst. Band-bending enables the valence electron to tunnel into the surface states at reduced fields. This work provides a basis for development of low-voltage ionization sensors. Although demonstrated on silicon, low-voltage field ionization can be detected on any sharp semiconductor tip containing proper surface states.