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.

Molecularly Imprinted Polymer-Based Sensors for the Detection of Skeletal- and Cardiac-Muscle-Related Analytes.

Molecularly imprinted polymers (MIPs) are synthetic polymers with specific binding sites that present high affinity and spatial and chemical complementarities to a targeted analyte. They mimic the molecular recognition seen naturally in the antibody/antigen complementarity. Because of their specificity, MIPs can be included in sensors as a recognition element coupled to a transducer part that converts the interaction of MIP/analyte into a quantifiable signal. Such sensors have important applications in the biomedical field in diagnosis and drug discovery, and are a necessary complement of tissue engineering for analyzing the functionalities of the engineered tissues. Therefore, in this review, we provide an overview of MIP sensors that have been used for the detection of skeletal- and cardiac-muscle-related analytes. We organized this review by targeted analytes in alphabetical order. Thus, after an introduction to the fabrication of MIPs, we highlight different types of MIP sensors with an emphasis on recent works and show their great diversity, their fabrication, their linear range for a given analyte, their limit of detection (LOD), specificity, and reproducibility. We conclude the review with future developments and perspectives.

Micropipet-Based Navigation in a Microvascular Model for Imaging Endothelial Cell Topography Using Scanning Ion Conductance Microscopy.

Scanning ion conductance microscopy (SICM) has enabled cell surface topography at a high resolution with low invasiveness. However, SICM has not been applied to the observation of cell surfaces in hydrogels, which can serve as scaffolds for three-dimensional cell culture. In this study, we applied SICM for imaging a cell surface in a microvascular lumen reconstructed in a hydrogel. To achieve this goal, we developed a micropipet navigation technique using ionic current to detect the position of a microvascular lumen. Combining this navigation technique with SICM, endothelial cells in a microvascular model and blebs were visualized successfully at the single-cell level. To the best of our knowledge, this is the first report on visualizing cell surfaces in hydrogels using a SICM. This technique will be useful for furthering our understanding of the mechanism of intravascular diseases.

Oxygen consumption rate of tumour spheroids during necrotic-like core formation.

Hypoxia is one of the major hallmarks of solid tumours and is associated with the poor prognosis of various cancers. A multicellular aggregate, termed a spheroid, has been used as a tumour model with a necrotic-like core for more than 45 years. Oxygen metabolism in spheroids has been studied using phosphorescence quenching and oxygen-sensitive electrodes. However, these conventional methods require chemical labelling and physical insertion of the electrode into each spheroid, which may be functionally and structurally disruptive. Scanning electrochemical microscopy (SECM) can non-invasively analyse oxygen metabolism. Here, we used SECM to investigate whether the changes of the internal structure of spheroids affect the oxygen metabolism. We investigated the oxygen consumption rate (OCR) of MCF-7 breast tumour spheroids with and without a necrotic-like core. A numerical simulation was used to describe a method for estimating the OCR of spheroids that settled at the bottom of the conventional culture plates. The OCR per spheroid volume decreased with increasing spheroid radius, indicating the limitation of the oxygen supply to the core of the MCF-7 spheroid. Formation of the necrotic-like core did not affect the oxygen metabolism significantly, implying that the core had minimal contribution to the OCR even before necrosis occurred. OCR analysis using SECM non-invasively monitors the change of oxygen metabolism in tumour spheroids. The approach is promising to evaluate various three-dimensional culture models.

Site-Specific Cytosol Sampling from a Single Cell in an Intact Tumor Spheroid Using an Electrochemical Syringe.

A multicellular tumor aggregate, known as a spheroid, is an indispensable tool to study cancer biology. Owing to its three-dimensional organization, a spheroid exhibits an inherent gradient of nutrients, oxygen, and metabolites within itself. The spheroid provides culture conditions that resemble the microenvironment of certain cancer cells and causes these cells to acquire characteristics relevant to tumors in our body. However, site-specific gene expression analysis in an intact spheroid with single-cell resolution has not been explored. Recently, some types of electrochemical syringes were developed to extract cellular materials from living single cells for transcriptomic analysis. Here, we investigated whether an electrochemical syringe could be used to evaluate site-specific gene expression in a spheroid. A small amount of cytosol (roughly 540-1480 fL, less than the volume of a single cell) was successfully collected from the first, second, and third layers of the spheroid using an electrochemical syringe without causing damage to the spheroid architecture. We found that the CCNB1 and CCNA2 expression levels were different between the surface and the average of the entire spheroid, indicating that there are heterogeneous cellular functions across different regions of the spheroid. This method provides opportunities to improve our understanding of spatial gene expression of single cells in a three-dimensional environment.

Continuous collection and simultaneous detection of picoliter volume of nucleic acid samples using a mille-feuille probe.

Investigation of the positional heterogeneity of messenger RNA (mRNA) expression in tissues requires a technology that facilitates analysis of mRNA expression in the selected single cells. We developed a mille-feuille probe (MP) that allows the lamination of the aqueous and organic phases in a nanopipette under voltage control. The MP was used for continuous collection of different nucleic acid samples and sequential evaluation of gene expression with mRNA barcoding tags. First, we found that the aqueous phases could be laminated into five individual layers and separated by the plugs of the organic phases in a nanopipette when the salt (THATPBCl) concentration in the organic phase was 100 mM. Second, the aspiration rate of the MP was stabilized and the velocity of the aqueous phase in the MP was lowered at higher THATPBCl concentrations in the organic phase. This was because the force during ingression of the aqueous phase into the organic - phase-filled nanopipette induced an electro-osmotic flow between the inside wall of the nanopipette and THATPBCl in the organic phase. Third, inclusion of mRNA barcoding tags in the MP facilitated complementary DNA construction and sequential analysis of gene expression. This technique has potential to be applicable to RNA sequencing from different cell samples across the life sciences. Graphical abstract We developed a mille-feuille probe (MP) that allows the lamination of the aqueous and organic phases in a nanopipette under voltage control.

Localized Gene Expression Analysis during Sprouting Angiogenesis in Mouse Embryoid Bodies Using a Double Barrel Carbon Probe.

The mouse embryonic stem (ES) cell-derived angiogenesis model is widely used as a 3D model, reproducing cell-cell interactions in the living body. Previously, many methods to analyze localized cellular function, including in situ hybridization and laser capture microdissection, have been reported. In this study, we achieved a collection of localized cells from the angiogenesis model in hydrogel. The gene expression profiles of the endothelial cells derived from mouse ES cells were evaluated. First, we collected localized cells from the live tissue model embedded in hydrogel using the double barrel carbon probe (DBCP) and quantified mRNA expression. Second, we found that vascular marker genes were expressed at a much higher level in sprouting vessels than in the central core of the embryoid body because the cells in sprouting vessels might significantly differentiate into endothelial linages, including tip/stalk cells. Third, the gene expression levels tended to be different between the top and middle regions in the sprouting vessel due to the difference in the degree of differentiation in these regions. At the top region of the vessel, both the tip and stalk cells were present. The cells in the middle region became more mature. Collectively, these results show that DBCP is very useful for analyzing localized gene expression in cells collected from 3D live tissues embedded in hydrogel. This technique can be applied to comprehensive gene expression analyses in the medical field.

Nanoscale imaging of an unlabeled secretory protein in living cells using scanning ion conductance microscopy.

Scanning ion conductance microscopy (SICM) was applied to evaluate an unlabeled secretory protein in living cells. The target protein, von Willebrand factor (vWF), was released from human endothelial cells by adding phorbol-12-myristate-13-acetate (PMA). We confirmed that SICM could be used to clearly visualize the complex network of vWF and to detect strings with widths as low as 60 nm without any artifact. By acquiring the sequential SICM images of living cells, the protrusion and strings formation were observed. We also detected the opening and closing motions of a small pore (∼500 nm), which is difficult to visualize with fluorescence methods. The results clearly demonstrate that SICM is a powerful tool to examine the changes in living cells during exocytosis.

Isolation and quantification of messenger RNA from tissue models by using a double-barrel carbon probe.

In this study, we introduce the double-barrel carbon probe (DBCP)-a simple, affordable microring electrode-which enables the collection and analysis of single cells independent of cellular positioning. The target cells were punctured by utilizing an electric pulse between the two electrodes in DBCP, and the cellular lysates were collected by manual aspiration using the DBCP. The mRNA in the collected lysate was evaluated quantitatively using real-time PCR. The histograms of single-cell relative gene expression normalized to GAPDH were fit to a theoretical lognormal distribution. In the tissue culture model, we focused on angiogenesis to prove that multiple gene expression analysis was available. Finally, we applied DBCP for the embryonic stem (ES) cell-derived cardiomyocytes to substantiate the capability of the probe to collect cells, even from high-volume samples such as spheroids. This method achieves high sensitivity for mRNA at the single-cell level and is applicable in the analysis of various biological samples independent of cellular positioning.

Modeling embryo-endometrial interface recapitulating human embryo implantation.

The initiation of human pregnancy is marked by the implantation of an embryo into the uterine environment; however, the underlying mechanisms remain largely elusive. To address this knowledge gap, we developed hormone-responsive endometrial organoids (EMO), termed apical-out (AO)-EMO, which emulate the in vivo architecture of endometrial tissue. The AO-EMO comprise an exposed apical epithelium surface, dense stromal cells, and a self-formed endothelial network. When cocultured with human embryonic stem cell-derived blastoids, the three-dimensional feto-maternal assembloid system recapitulates critical implantation stages, including apposition, adhesion, and invasion. Endometrial epithelial cells were subsequently disrupted by syncytial cells, which invade and fuse with endometrial stromal cells. We validated this fusion of syncytiotrophoblasts and stromal cells using human blastocysts. Our model provides a foundation for investigating embryo implantation and feto-maternal interactions, offering valuable insights for advancing reproductive medicine.

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.

Minimally Invasive Sub-Retinal Transplantation of RPE-J Cells on a Biodegradable Composite PCL/Collagen Nanosheet.

Retinal cells are irreparably damaged by diseases such as age-related macular degeneration (AMD). A promising method to restore partial or whole vision is through cell-based transplantation to the damaged location. However, cell transplantation using conventional vitreous surgery is an invasive procedure that may induce infections and has a high failure rate of cell engraftment. In this study, we describe the fabrication of a biodegradable composite nanosheet used as a substrate to support retinal pigment epithelial (RPE-J) cells, which can be grafted to the sub-retinal space using a minimally invasive approach. The nanosheet was fabricated using polycaprolactone (PCL) and collagen in 80:20 weight ratio, and had size of 200 µm in diameter and 300 nm in thickness. These PCL/collagen nanosheets showed excellent biocompatibility and mechanical strength in vitro. Using a custom designed 27-gauge glass needle, we successfully transplanted an RPE-J cell loaded nanosheet into the sub-retinal space of a rat model with damaged photoreceptors. The cell loaded nanosheet did not trigger immunological reaction within 2 weeks of implantation and restored the retinal environment. Thus, this composite PCL/collagen nanosheet holds great promise for organized cell transplantation, and the treatment of retinal diseases.

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.

Pericytes and shear stress each alter the shape of a self-assembled vascular network.

Blood vessel morphology is dictated by mechanical and biochemical cues. Flow-induced shear stress and pericytes both play important roles, and they have previously been studied using on-chip vascular networks to uncover their connection to angiogenic sprouting and network stabilization. However, it is unknown which shear stress values promote angiogenesis, how pericytes are directed to sprouts, and how shear stress and pericytes affect the overall vessel morphology. Here, we employed a microfluidic device to study these phenomena in three-dimensional (3D) self-assembled vasculature. Computational fluid dynamics solver (COMSOL) simulations indicated that sprouts form most frequently at locations of relatively low shear stresses (0.5-1.5 dyn cm-2). Experimental results show that pericytes limit vascular diameter. Interestingly, when treated with imatinib or crenolanib, which are chemotherapeutic drugs and inhibitors of platelet-derived growth factor receptor ß (PDGFRß), the pericyte coverage of vessels decreased significantly but vessel diameter remained unchanged. This furthers our understanding of the mechanisms underlying vascular development and demonstrates the value of this microfluidic device in future studies on drug development and vascular biology.

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.

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.

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.

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.

Electrochemical Imaging of Endothelial Permeability Using a Large-Scale Integration-Based Device.

It is important to clarify the transport of biomolecules and chemicals to tissues. Herein, we present an electrochemical imaging method for evaluating the endothelial permeability. In this method, the diffusion of electrochemical tracers, [Fe(CN)6]4-, through a monolayer of human umbilical vein endothelial cells (HUVECs) was monitored using a large-scale integration-based device containing 400 electrodes. In conventional tracer-based assays, tracers that diffuse through an HUVEC monolayer into another channel are detected. In contrast, the present method does not employ separated channels. In detail, a HUVEC monolayer is immersed in a solution containing [Fe(CN)6]4- on the device. As [Fe(CN)6]4- is oxidized and consumed at the packed electrodes, [Fe(CN)6]4- begins to diffuse through the monolayer from the bulk solution to the electrodes and the obtained currents depend on the endothelial permeability. As a proof-of-concept, the effects of histamine on the monolayer were monitored. Also, an HUVEC monolayer was cocultured with cancer spheroids, and the endothelial permeability was monitored to evaluate the metastasis of the cancer spheroids. Unlike conventional methods, the device can provide spatial information, allowing the interaction between the monolayer and the spheroids to be monitored. The developed method is a promising tool for organs-on-a-chip and drug screening in vitro.