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.

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.

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.

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 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.

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.

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.

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.

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.

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.

A new perfusion culture method with a self-organized capillary network.

A lack of perfusion has been one of the most significant obstacles for three-dimensional culture systems of organoids and embryonic tissues. Here, we developed a simple and reliable method to implement a perfusable capillary network in vitro. The method employed the self-organization of endothelial cells to generate a capillary network and a static pressure difference for culture medium circulation, which can be easily introduced to standard biological laboratories and enables long-term cultivation of vascular structures. Using this culture system, we perfused the lumen of the self-organized capillary network and observed a flow-induced vascular remodeling process, cell shape changes, and collective cell migration. We also observed an increase in cell proliferation around the self-organized vasculature induced by flow, indicating functional perfusion of the culture medium. We also reconstructed extravasation of tumor and inflammatory cells, and circulation inside spheroids including endothelial cells and human lung fibroblasts. In conclusion, this system is a promising tool to elucidate the mechanisms of various biological processes related to vascular flow.

Fabrication of three-dimensional calcium alginate hydrogels using sacrificial templates of sugar.

Hydrogels are receiving increasing attention in bioapplications. Among hydrogels, calcium alginate (Ca-alginate) hydrogels are widely used for their biocompatibility, low toxicity, low cost, and rapid fabrication by simple mixing of Ca2+ and sodium alginate (Na-alginate). For bioapplications using hydrogels, it is necessary to construct designed hydrogel structures. Although several methods have been proposed for fabricating designed hydrogels, a simple and low-cost method is desirable. Therefore, we developed a new method using sacrificial templates of sugar structures to fabricate three-dimensional (3D) designed Ca-alginate hydrogels. In this method, Na-alginate solution is mixed with molten sugar, and the resulting highly viscous material used to mold 3D sugar structures as sacrificial templates. Since sugar constructs are easily handled compared to hydrogels, sugar templates are useful for preparing 3D constructs. Finally, the sugar and Na-alginate structure is immersed in a CaCl2 solution to simultaneously dissolve the template and form the Ca-alginate hydrogel. The resulting hydrogel takes the shape of the sugar template. By stacking and fusing various sugar structures, such as fibers and blocks, 3D designed Ca-alginate hydrogels can be successfully fabricated. This simple and low-cost method shows excellent potential for application to a variety of bioapplications.

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.

Recent Advances in Electrochemiluminescence-Based Systems for Mammalian Cell Analysis.

Mammalian cell analysis is essential in the context of both fundamental studies and clinical applications. Among the various techniques available for cell analysis, electrochemiluminescence (ECL) has attracted significant attention due to its integration of both electrochemical and spectroscopic methods. In this review, we summarize recent advances in the ECL-based systems developed for mammalian cell analysis. The review begins with a summary of the developments in luminophores that opened the door to ECL applications for biological samples. Secondly, ECL-based imaging systems are introduced as an emerging technique to visualize single-cell morphologies and intracellular molecules. In the subsequent section, the ECL sensors developed in the past decade are summarized, the use of which made the highly sensitive detection of cell-derived molecules possible. Although ECL immunoassays are well developed in terms of commercial use, the sensing of biomolecules at a single-cell level remains a challenge. Emphasis is therefore placed on ECL sensors that directly detect cellular molecules from small portions of cells or even single cells. Finally, the development of bipolar electrode devices for ECL cell assays is introduced. To conclude, the direction of research in this field and its application prospects are described.