Yuji Nashimoto

Creating vascular networks in tissues is crucial for tissue engineering. Although recent studies have demonstrated the formation of vessel-like structures in a tissue model, long-term culture is still challenging due to the lack of active perfusion in vascular networks. Here, we present a method to create a three-dimensional cellular spheroid with a perfusable vascular network in a microfluidic device. By the definition of the cellular interaction between human lung fibroblasts (hLFs) in a spher

Information regarding spatial mRNA localization in single cells is necessary for a better understanding of cellular functions in tissues. Here, we report a method for evaluating localization of mRNA in single cells using double-barrel scanning ion conductance microscopy (SICM). Two barrels in a nanopipette were filled with aqueous and organic electrolyte solutions and used for SICM and as an electrochemical syringe, respectively. We confirmed that the organic phase barrel could be used to collec

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 functi

The cytosol of a single adherent cell was collected by the electrical cell lysis method with a Pt-ring capillary probe, and the cellular messenger RNA (mRNA) was analyzed at a single-cell level. The ring electrode probe was positioned 20 microm above the cultured cells that formed a monolayer on an indium-tin oxide (ITO) electrode, and an electric pulse with a magnitude of 40 V was applied for 10 micros between the probe and the ITO electrodes in an isotonic sucrose solution. Immediately after t

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 form

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

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 i

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 p

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 p

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 n