Scanning electrochemical microscopy for determining oxygen consumption rates of cells in hydrogel fibers fabricated using an extrusion 3D bioprinter.

Three-dimensional (3D)-cultured cells have attracted the attention of researchers in tissue engineering- and drug screening-related fields. Among them, 3D cellular fibers have attracted significant attention because they can be stacked to prepare more complex tissues and organs. Cellular fibers are widely fabricated using extrusion 3D bioprinters. For these applications, it is necessary to evaluate cellular activities, such as the oxygen consumption rate (OCR), which is one of the major metabolic activities. We previously reported the use of scanning electrochemical microscopy (SECM) to evaluate the OCRs of cell spheroids. However, the SECM approach has not yet been applied to hydrogel fibers prepared using the bioprinters. To the best of our knowledge, this is the first study to evaluate the OCR of cellular fibers printed by extrusion 3D bioprinters. First, the diffusion theory was discussed to address this issue. Next, diffusion models were simulated to compare realistic models with this theory. Finally, the OCRs of MCF-7 cells in the printed hydrogel fibers were evaluated as a proof of concept. Our proposed approach could potentially be used to evaluate the OCRs of tissue-engineered fibers for organ transplantation and drug screening using in-vitro models.

Microfluidic vascular formation model for assessing angiogenic capacities of single islets.

Pancreatic islet transplantation presents a promising therapy for individuals suffering from type 1 diabetes. To maintain the function of transplanted islets in vivo, it is imperative to induce angiogenesis. However, the mechanisms underlying angiogenesis triggered by islets remain unclear. In this study, we introduced a microphysiological system to study the angiogenic capacity and dynamics of individual islets. The system, which features an open-top structure, uniquely facilitates the inoculation of islets and the longitudinal observation of vascular formation in in vivo like microenvironment with islet-endothelial cell communication. By leveraging our system, we discovered notable islet-islet heterogeneity in the angiogenic capacity. Transcriptomic analysis of the vascularized islets revealed that islets with high angiogenic capacity exhibited upregulation of genes related to insulin secretion and downregulation of genes related to angiogenesis and fibroblasts. In conclusion, our microfluidic approach is effective in characterizing the vascular formation of individual islets and holds great promise for elucidating the angiogenic mechanisms that enhance islet transplantation therapy.

Vasculature-on-a-Chip with a Porous Membrane Electrode for In Situ Electrochemical Detection of Nitric Oxide Released from Endothelial Cells.

Vasculature-on-a-chip is a microfluidic cell culture device used for modeling vascular functions by culturing endothelial cells. Porous membranes are widely used to create cell culture environments. However, in situ real-time measurements of cellular metabolites in microchannels are challenging. In this study, a novel microfluidic device with a porous membrane electrode was developed for the in situ monitoring of nitric oxide (NO) released by endothelial cells in real time. In this system, a porous Au membrane electrode was placed directly beneath the cells for in situ and real-time measurements of NO, a biomarker of endothelial cells. First, the device was electrochemically characterized to construct a calibration plot for NO. Next, NO released by human umbilical vein endothelial cells under l-arginine stimulation was successfully quantified. Furthermore, the changes in NO release with culture time (in days) using the same sample were successfully recorded by exploiting minimally invasive measurements. This is the first report on the combination of a microfluidic device and porous membrane electrode for the electrochemical analysis of endothelial cells. This device will contribute to the development of organ-on-a-chip technology for real-time in situ cell analyses.

Fabrication and Cell Culture Applications of Core-Shell Hydrogel Fibers Composed of Chitosan/DNA Interfacial Polyelectrolyte Complexation and Calcium Alginate: Straight and Beaded Core Variations.

Core-shell hydrogel fibers are widely used in cell culture applications. A simple and rapid method is presented for fabricating core-shell hydrogel fibers, consisting of straight or beaded core fibers, for cell culture applications. The core fibers are prepared using interfacial polyelectrolyte complexation (IPC) with chitosan and DNA. Briefly, two droplets of chitosan and DNA are brought in contact to form an IPC film, which is dragged to prepare an IPC fiber. The incubation time and DNA concentration are adjusted to prepare straight and beaded IPC fibers. The fibers with Ca2+ are immersed in an alginate solution to form calcium alginate shell hydrogels around the core IPC fibers. To the best of the knowledge, this is the first report of core-shell hydrogel fibers with IPC fiber cores. To demonstrate cell culture, straight hydrogel fibers are applied to fabricate hepatic models consisting of HepG2 and 3T3 fibroblasts, and vascular models consisting of human umbilical vein endothelial cells and 3T3 fibroblasts. To evaluate the effect of co-culture, albumin secretion, and angiogenesis are evaluated. Beaded hydrogel fibers are used to fabricate many size-controlled spheroids for fiber and cloning applications. This method can be widely applied in tissue engineering and cell analysis.

Microarray-Based Electrochemical Biosensing.

Microarrays are widely utilized in bioanalysis. Electrochemical biosensing techniques are often applied in microarray-based assays because of their simplicity, low cost, and high sensitivity. In such systems, the electrodes and sensing elements are arranged in arrays, and the target analytes are detected electrochemically. These sensors can be utilized for high-throughput bioanalysis and the electrochemical imaging of biosamples, including proteins, oligonucleotides, and cells. In this chapter, we summarize recent progress on these topics. We categorize electrochemical biosensing techniques for array detection into four groups: scanning electrochemical microscopy, electrode arrays, electrochemiluminescence, and bipolar electrodes. For each technique, we summarize the key principles and discuss the advantages, disadvantages, and bioanalysis applications. Finally, we present conclusions and perspectives about future directions in this field.

Oxygen metabolism analysis of a single organoid for non-invasive discrimination of cancer subpopulations with different growth capabilities.

Heterogeneous nature is a pivotal aspect of cancer, rendering treatment problematic and frequently resulting in recurrence. Therefore, advanced techniques for identifying subpopulations of a tumour in an intact state are essential to develop novel screening platforms that can reveal differences in treatment response among subpopulations. Herein, we conducted a non-invasive analysis of oxygen metabolism on multiple subpopulations of patient-derived organoids, examining its potential utility for non-destructive identification of subpopulations. We utilised scanning electrochemical microscopy (SECM) for non-invasive analysis of oxygen metabolism. As models of tumours with heterogeneous subpopulations, we used patient-derived cancer organoids with a distinct growth potential established using the cancer tissue-originated spheroid methodology. Scanning electrochemical microscopy measurements enabled the analysis of the oxygen consumption rate (OCR) for individual organoids as small as 100 µm in diameter and could detect the heterogeneity amongst studied subpopulations, which was not observed in conventional colorectal cancer cell lines. Furthermore, our oxygen metabolism analysis of pre-isolated subpopulations with a slow growth potential revealed that oxygen consumption rate may reflect differences in the growth rate of organoids. Although the proposed technique currently lacks single-cell level sensitivity, the variability of oxygen metabolism across tumour subpopulations is expected to serve as an important indicator for the discrimination of tumour subpopulations and construction of novel drug screening platforms in the future.

Simple, Rapid, and Large-Scale Fabrication of Multi-Branched Hydrogels Based on Viscous Fingering for Cell Culture Applications.

Hydrogels are widely used in cell culture applications. For fabricating tissues and organs, it is essential to produce hydrogels with specific structures. For instance, multiple-branched hydrogels are desirable for the development of network architectures that resemble the biological vascular network. However, existing techniques are inefficient and time-consuming for this application. To address this issue, a simple, rapid, and large-scale fabrication method based on viscous fingering is proposed. This approach utilizes only two plates. To produce a thin solution, a high-viscosity solution is introduced into the space between the plates, and one of the plates is peeled off. During this procedure, the solution's high viscosity results in the formation of multi-branched structures. Using this strategy, 180 mm × 200 mm multi-branched Pluronic F-127 hydrogels are successfully fabricated within 1 min. These structures are used as sacrificial layers for the fabrication of polydimethylsiloxane channels for culturing human umbilical vein endothelial cells (HUVECs). Similarly, multi-branched Matrigel and calcium (Ca)-alginate hydrogel structures are fabricated, and HUVECs are successfully cultured inside the hydrogels. Also, the hydrogels are collected from the plate, while maintaining their structures. The proposed fabrication technique will contribute to the development of network architectures such as vascular structures in tissue engineering.

Highly Sensitive Electrochemical Endotoxin Sensor Based on Redox Cycling Using an Interdigitated Array Electrode Device.

The Limulus amebocyte lysate (LAL) reaction-based assay, the most commonly used endotoxin detection method, requires a skilled technician. In this study, to develop an easy-to-use and highly sensitive endotoxin sensor, we created an electrochemical endotoxin sensor by using an interdigitated array electrode (IDAE) device with advantages of amplifiable signals via redox cycling and portability. We added Boc-Leu-Gly-Arg-p-aminophenol (LGR-pAP) as an electrochemical substrate for an LAL reaction and detected p-aminophenol (pAP) released from LGR-pAP as a product of an endotoxin-induced LAL reaction via an IDAE device. The IDAE device showed a great redox cycling efficiency of 79.8%, and a 4.79-fold signal amplification rate. Then, we confirmed that pAP was detectable in the presence of LGR-pAP through chronoamperometry with the potential of the anode stepped from -0.3 to 0.5 V vs. Ag/AgCl while the cathode was biased at -0.3 V vs. Ag/AgCl. Then, we performed an endotoxin assay by using the IDAE device. Our endotoxin sensor detected as low as 0.7 and 1.0 endotoxin unit/L after the LAL reaction for 1 h and 45 min, respectively, and these data were within the cut-off value for ultrapure dialysis fluid. Therefore, our highly sensitive endotoxin sensor is useful for ensuring medical safety.

Batteryless wireless magnetostrictive Fe30Co70/Ni clad plate for human coronavirus 229E detection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been garnered increasing for its rapid worldwide spread. Each country had implemented city-wide lockdowns and immigration regulations to prevent the spread of the infection, resulting in severe economic consequences. Materials and technologies that monitor environmental conditions and wirelessly communicate such information to people are thus gaining considerable attention as a countermeasure. This study investigated the dynamic characteristics of batteryless magnetostrictive alloys for energy harvesting to detect human coronavirus 229E (HCoV-229E). Light and thin magnetostrictive Fe-Co/Ni clad plate with rectification, direct current (DC) voltage storage capacitor, and wireless information transmission circuits were developed for this purpose. The power consumption was reduced by improving the energy storage circuit, and the magnetostrictive clad plate under bending vibration stored a DC voltage of 1.9 V and wirelessly transmitted a signal to a personal computer once every 5 min and 10 s under bias magnetic fields of 0 and 10 mT, respectively. Then, on the clad plate surface, a novel CD13 biorecognition layer was immobilized using a self-assembled monolayer of -COOH groups, thus forming an amide bond with -NH2 groups for the detection of HCoV-229E. A bending vibration test demonstrated the resonance frequency changes because of HCoV-229E binding. The fluorescence signal demonstrated that HCoV-229E could be successfully detected. Thus, because HCoV-229E changed the dynamic characteristics of this plate, the CD13-modified magnetostrictive clad plate could detect HCoV-229E from the interval of wireless communication time. Therefore, a monitoring system that transmits/detects the presence of human coronavirus without batteries will be realized soon.

Electrochemical sensing of oxygen metabolism for a three-dimensional cultured model with biomimetic vascular flow.

Microphysiological systems (MPSs) with three-dimensional (3D) cultured models have attracted considerable interest because of their potential to mimic human health and disease conditions. Recent MPSs have shown significant advancements in engineering perfusable vascular networks integrated with 3D culture models, realizing a more physiological environment in vitro; however, a sensing system that can monitor their activity under biomimetic vascular flow is lacking. We designed an open-top microfluidic device with sensor capabilities and demonstrated its application in analyzing oxygen metabolism in vascularized 3D tissue models. We first validated the platform by using human lung fibroblast (hLF) spheroids. Then, we applied the platform to a patient-derived cancer organoid and evaluated the changes in oxygen metabolism during drug administration through the vascular network. We found that the platform could integrate a perfusable vascular network with 3D cultured cells, and the electrochemical sensor could detect the change in oxygen metabolism in a quantitative, non-invasive, and real-time manner. This platform would become a monitoring system for 3D cultured cells integrated with a perfusable vascular network.

High-Sensitivity Amperometric Dual Immunoassay Using Two Cascade Reactions with Signal Amplification of Redox Cycling in Nanoscale Gap.

Here, we report a high-sensitivity dual immunoassay using Lumulus amebocyte lysate (LAL) and blood coagulation cascade reactions with redox cycling in a nanoscale-gap electrode. Endotoxin and factor XIa were used as the label molecules for the immunoassay of two types of analytes to induce the LAL and coagulation cascade reactions, respectively, when each corresponding analyte existed in the sample solution. In addition to the signal amplification by the cascade reactions, we employed redox cycling in a nanoscale gap to achieve a highly sensitive assay. The nanoscale-gap electrode amplifies the amperometric signals from p-aminophenol liberated from artificial substrates in the final steps of the cascade reactions. First, the cross reaction between the LAL and coagulation cascade reactions was investigated. The results indicated that these cascade reactions did not efficiently proceed in a single solution owing to the cross reaction. Therefore, we selected to induce two cascade reactions in different solutions by bisecting the beads after the immunocomplex formation on the beads. The cross reactions of factor XIa with the LAL cascade reaction and of endotoxin with the coagulation cascade reaction were investigated. The effects of these cross reactions were revealed to be negligible by bisecting the beads before inducing the cascade reactions. Finally, a dual immunoassay for goat and human immunoglobulin G was performed, for which the limits of detection were 70 pg/mL (470 fmol/L) and 1.0 ng/mL (6.6 pmol/L), respectively. Thus, our dual immunoassay provides a sensitive platform for clinical diagnosis requiring detection of multiple analytes.

Highly Sensitive Electrochemical Immunoassay Using Signal Amplification of the Coagulation Cascade.

Here, we report a highly sensitive immunoassay for human immunoglobulin G (IgG) that uses signal amplification of the coagulation cascade. Z-Phe-Pro-Lys-p-nitroaniline (FPK-pNA) was used as a substrate for thrombin activation in the last step of the coagulation cascade. During the coagulation cascade, pNA is liberated from FPK-pNA and can be detected electrochemically. Using square wave voltammetry with a glassy carbon electrode, we demonstrated that pNA can be quantified in a solution modeling the coagulation cascade prepared by mixing FPK-pNA and pNA. Characterization of the reactivity of thrombin toward FPK-pNA revealed that thrombin efficiently reacted with FPK-pNA. Subsequent characterization of factor XIa activity of factor XIa-labeled antibody revealed that factor XIa was not inactivated during labeling. Finally, a coagulation cascade-based immunoassay for human IgG was performed using a factor XIa-labeled antibody on magnetic beads. The limit of detection for human IgG was 5.0 pg/mL (33 fM) indicating that the coagulation cascade can amplify the immunoassay sensitivity compared to immunoassay using a thrombin-labeled antibody as a condition without a coagulation cascade. Coagulation cascade-based immunoassay was also highly selective. In the near future, we will report a highly sensitive immunoassay for the simultaneous detection of multiple analytes using a coagulation cascade-based immunoassay and Limulus amebocyte lysate reaction-based immunoassay we previously reported.

Electrochemical microwell sensor with Fe-N co-doped carbon catalyst to monitor nitric oxide release from endothelial cell spheroids.

Endothelial cells have been widely used for vascular biology studies; recent progress in tissue engineering have offered three-dimensional (3D) culture systems for vascular endothelial cells which can be considered as physiologically relevant models. To facilitate the studies, we developed an electrochemical device to detect nitric oxide (NO), a key molecule in the vasculature, for the evaluation of 3D cultured endothelial cells. Using an NO-sensitive catalyst composed of Fe-N co-doped reduced graphene oxide, the real-time monitoring of NO release from the endothelial cell spheroids was demonstrated.

Dissolved Oxygen-Sensing Chip Integrating an Open Container Connected with a Position-Raised Channel for Estimation of Cellular Mitochondrial Activity.

The measurement of oxygen consumption of adherent cells is a profoundly important issue for estimating the bioenergetic health and metabolism activity of cells. The study describes the construction of a microfluidic chip consisting of an open container connected with a position-raised channel and dissolved oxygen (DO)-sensing gold ultramicroelectrodes for quantifying the oxygen consumption rate (OCR) of adherent cells. The microfluidic chip design can reduce the action of shear force on the adherent cells during medium replacement. The residual concentration of analytes in the open container was only 4.4% after solution replacement via the position-raised channel. The DO reduction current measured by ultramicroelectrodes averaged in the range of 40-60 s presented high reproducibility with a 1.1% relative standard deviation suitable for OCR calculation. After short-term (90 min) cultivation, the microfluidic chip can monitor the time-dependent change in the OCR of 3T3-L1 cells for several hours by repeatedly replacing the culture medium or with the stimulation of different mitochondrial inhibitors. The presented microfluidic cell-based chip has great promise for drug screening and chemosensitivity testing by measuring OCR to evaluate the mitochondrial function of adherent cells.

Fabrication of High-Density Vertical Closed Bipolar Electrode Arrays by Carbon Paste Filling Method for Two-Dimensional Chemical Imaging.

In this study, a carbon paste filling method was proposed as a simple strategy for fabricating high-density bipolar electrode (BPE) arrays for bipolar electrochemical microscopy (BEM). High spatiotemporal resolution imaging was achieved using the fabricated BPE array. BEM, which is an emerging microscopic system in recent years, achieves label-free and high spatiotemporal resolution imaging of molecular distributions using high-density BPE arrays and electrochemiluminescence (ECL) signals. We devised a simple method to fabricate a BPE array by filling a porous plate with carbon paste and succeeded in fabricating a high-density BPE array (15 µm pitch). After a detailed observation of the surface of the BPE array using a scanning electron microscope, the basic electrochemical and ECL emission characteristics were evaluated using potassium ferricyanide solution as a sample solution. Moreover, inflow imaging of the sample molecules was conducted to evaluate the imaging ability of the prepared BPE array. In addition, Prussian Blue containing carbon ink was applied to the sample solution side of the BPE array to provide catalytic activity to hydrogen peroxide, and the quantification and inflow imaging of hydrogen peroxide by ECL signals was achieved. This simple fabrication method of the BPE array can accelerate the research and development of BEM. Furthermore, hydrogen peroxide imaging by BEM is an important milestone for achieving bioimaging with high spatiotemporal resolution such as biomolecule imaging using enzymes.

Electrochemical Glue for Binding Chitosan-Alginate Hydrogel Fibers for Cell Culture.

Three-dimensional organs and tissues can be constructed using hydrogels as support matrices for cells. For the assembly of these gels, chemical and physical reactions that induce gluing should be induced locally in target areas without causing cell damage. Herein, we present a novel electrochemical strategy for gluing hydrogel fibers. In this strategy, a microelectrode electrochemically generated HClO or Ca2+, and these chemicals were used to crosslink chitosan-alginate fibers fabricated using interfacial polyelectrolyte complexation. Further, human umbilical vein endothelial cells were incorporated into the fibers, and two such fibers were glued together to construct "+"-shaped hydrogels. After gluing, the hydrogels were embedded in Matrigel and cultured for several days. The cells spread and proliferated along the fibers, indicating that the electrochemical glue was not toxic toward the cells. This is the first report on the use of electrochemical glue for the assembly of hydrogel pieces containing cells. Based on our results, the electrochemical gluing method has promising applications in tissue engineering and the development of organs on a chip.

Ion Conductance-Based Perfusability Assay of Vascular Vessel Models in Microfluidic Devices.

We present a novel methodology based on ion conductance to evaluate the perfusability of vascular vessels in microfluidic devices without microscopic imaging. The devices consisted of five channels, with the center channel filled with fibrin/collagen gel containing human umbilical vein endothelial cells (HUVECs). Fibroblasts were cultured in the other channels to improve the vascular network formation. To form vessel structures bridging the center channel, HUVEC monolayers were prepared on both side walls of the gel. During the culture, the HUVECs migrated from the monolayer and connected to the HUVECs in the gel, and vascular vessels formed, resulting in successful perfusion between the channels after culturing for 3-5 d. To evaluate perfusion without microscopic imaging, Ag/AgCl wires were inserted into the channels, and ion currents were obtained to measure the ion conductance between the channels separated by the HUVEC monolayers. As the HUVEC monolayers blocked the ion current flow, the ion currents were low before vessel formation. In contrast, ion currents increased after vessel formation because of creation of ion current paths. Thus, the observed ion currents were correlated with the perfusability of the vessels, indicating that they can be used as indicators of perfusion during vessel formation in microfluidic devices. The developed methodology will be used for drug screening using organs-on-a-chip containing vascular vessels.

Topography and Permeability Analyses of Vasculature-on-a-Chip Using Scanning Probe Microscopies.

Microphysiological systems (MPS) or organs-on-chips (OoC) can emulate the physiological functions of organs in vitro and are effective tools for determining human drug responses in preclinical studies. However, the analysis of MPS has relied heavily on optical tools, resulting in difficulties in real-time and high spatial resolution imaging of the target cell functions. In this study, the role of scanning probe microscopy (SPM) as an analytical tool for MPS is evaluated. An access hole is made in a typical MPS system with stacked microchannels to insert SPM probes into the system. For the first study, a simple vascular model composed of only endothelial cells is prepared for SPM analysis. Changes in permeability and local chemical flux are quantitatively evaluated during the construction of the vascular system. The morphological changes in the endothelial cells after flow stimulation are imaged at the single-cell level for topographical analysis. Finally, the possibility of adapting the permeability and topographical analysis using SPM for the intestinal vascular system is further evaluated. It is believed that this study will pave the way for an in situ permeability assay and structural analysis of MPS using SPM.

Nanoscale Visualization of Morphological Alteration of Live-Cell Membranes by the Interaction with Oligoarginine Cell-Penetrating Peptides.

The interactions between the cell membrane and biomolecules remain poorly understood. For example, arginine-rich cell-penetrating peptides (CPPs), including octaarginines (R8), are internalized by interactions with cell membranes. However, during the internalization process, the exact membrane dynamics introduced by these CPPs are still unknown. Here, we visualize arginine-rich CPPs and cell-membrane interaction-induced morphological changes using a system that combines scanning ion-conductance microscopy and spinning-disk confocal microscopy, using fluorescently labeled R8. This system allows time-dependent, nanoscale visualization of structural dynamics in live-cell membranes. Various types of membrane remodeling caused by arginine-rich CPPs are thus observed. The induction of membrane ruffling and the cup closure are observed as a process of endocytic uptake of the peptide. Alternatively suggested is the concave structural formation accompanied by direct peptide translocation through cell membranes. Studies using R8 without fluorescent labeling also demonstrate a non-negligible effect of the fluorescent moiety on membrane structural alteration.

Electrochemiluminescence imaging of respiratory activity of cellular spheroids using sequential potential steps.

The respiratory activity of cultured cells can be electrochemically monitored using scanning electrochemical microscopy (SECM) with high spatial resolution. However, in SECM, the electrode takes a long time to scan, limiting simultaneous measurements with large biological samples such as cell spheroids. Therefore, for rapid electrochemical imaging, a novel strategy is needed. Herein, we report electrochemiluminescence (ECL) imaging of spheroid respiratory activity for the first time using sequential potential steps. L-012, a luminol analog, was used as an ECL luminophore, and H2O2, a sensitizer for ECL of L-012, was generated by the electrochemical reduction of dissolved O2. The ECL imaging visualized spheroid respiratory activity-evidenced by ECL suppression-corresponding to O2 distribution around the spheroids. This method enabled the time-lapse imaging of respiratory activity in multiple spheroids with good spatial resolution comparable to that of SECM. Our work provides a promising high-throughput imaging strategy for elucidating spheroid cellular dynamics.