Yuji Nashimoto

A microfluidic dual capillary probe to collect messenger RNA from adherent cells and spheroids

Collection of bioanalytes from single cells is still a challenging technology despite the recent progress in many integrated microfluidic devices. A microfluidic dual capillary probe was prepared from a theta (theta)-shaped glass capillary to analyze messenger RNA (mRNA) from adherent cells and spheroids. The cell lysis buffer solution was introduced from the injection aperture, and the cell-lysed solution from the aspiration aperture was collected for further mRNA analysis based on reverse transcription real-time PCR. The cell lysis buffer can be introduced at any targeted cells and never spilled out of the targeted area by using the microfluidic dual capillary probe because laminar flow was locally formed near the probe under the optimized injection/aspiration flow rates. This method realizes the sensitivity of mRNA at the single cell level and the identification of the cell types on the basis of the relative gene expression profiles.

Evaluation of mRNA Localization Using Double Barrel Scanning Ion Conductance Microscopy.

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 collect cytosol from living cells, which is a minute but sufficient amount to assess cellular status using qPCR analysis. The water phase barrel could be used for SICM to image topography with subcellular resolution, which could be used to determine positions for analyzing mRNA expression. This system was able to evaluate mRNA localization in single cells. After puncturing the cellular membrane in a minimally invasive manner, using SICM imaging as a guide, we collected a small amount cytosol from different positions within a single cell and showed that mRNA expression depends on cellular position. In this study, we show that SICM imaging can be utilized for the analysis of mRNA localization in single cells. In addition, we fully automated the pipet movement in the XYZ-directions during the puncturing processes, making it applicable as a high-throughput system for collecting cytosol and analyzing mRNA localization.

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.

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.

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

AbstractIn 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. Figureᅟ

Engineering of vascularized 3D cell constructs to model cellular interactions through a vascular network

Current in vitro 3D culture models lack a vascular system to transport oxygen and nutrients, as well as cells, which is essential to maintain cellular viability and functions. Here, we describe a microfluidic method to generate a perfusable vascular network that can form inside 3D multicellular spheroids and functionally connect to microchannels. Multicellular spheroids containing endothelial cells and lung fibroblasts were embedded within a hydrogel inside a microchannel, and then, endothelial cells were seeded into both sides of the hydrogel so that angiogenic sprouts from the cell spheroids and the microchannels were anastomosed to form a 3D vascular network. Solution containing cells and reagents can be perfused inside the cell spheroids through the vascular network by injecting it into a microchannel. This method can be used to study cancer cell migration towards 3D co-culture spheroids through a vascular network. We recapitulated a bone-like microenvironment by culturing multicellular spheroids containing osteo-differentiated mesenchymal stem cells (MSCs), as well as endothelial cells, and fibroblasts in the device. After the formation of vascularized spheroids, breast cancer cells were injected into a microchannel connected to a vascular network and cultured for 7 days on-chip to monitor cellular migration. We demonstrated that migration rates of the breast cancer cells towards multicellular spheroids via blood vessels were significantly higher in the bone-like microenvironment compared with the microenvironment formed by undifferentiated MSCs. These findings demonstrate the potential value of the 3D vascularized spheroids-on-a-chip for modeling in vivo-like cellular microenvironments, drug delivery through blood vessels, and cellular interactions through a vascular network.

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.

Engineering a three-dimensional tissue model with a perfusable vasculature in a microfluidic device

In this study, we developed a microfluidic platform for a three-dimensional tissue model with a perfusable capillary network, which will allow, for the first time, a perfusion-culture in a tissue model with a high cell density. Our group previously reported that a spheroid of lung fibroblasts induced angiogenic sprouts from microchannels [1]. In this study, we successfully connected angiogenic sprouts to the vessel-like hollow structure in a spheroid and perfused the formed vascular network through microfluidic channels to the spheroid. This model opens up new techniques for tissue-culture for long-term.

Development of three-dimensional tumor model with a perfusable vasculature using a microfluidic device

disorders. Characterising the changes in mouse embryos that result from ablation of lethal genes is a necessary first step towards uncovering their role in normal embryonic development and establishing any correlates amongst human congenital abnormalities. Here we present results gathered to date in the Deciphering the Mechanisms of Developmental Disorders (DMDD) programme, cataloguing the morphological defects identified from comprehensive imaging of 220 homozygous mutant embryos from 42 lethal and subviable lines, analysed at E14.5. Virtually all embryos show multiple abnormal phenotypes and amongst the 42 lines these affect most organ systems. Within each mutant line, the phenotypes of individual embryos form distinct but overlapping sets. Subcutaneous edema, malformations of the heart or great vessels, abnormalities in forebrain morphology and the musculature of the eyes are all prevalent phenotypes, as is loss or abnormal size of the hypoglossal nerve. The most striking finding is that no matter how profound the malformation, each phenotype shows highly variable penetrance within a mutant line. These findings have challenging implications for efforts to identify human disease correlates.

Remodeling in synthetic vascular network - experiment and modeling

Vascular network is first generated by vasculogenesis and angiogenesis. The vessel diameters are later modified by vascular flow, which is called remodeling process. Various theoretical models were proposed for this remodeling process to explain vascular patterns in vivo. However, if we fully understand the mechanism of pattern formation, we should be able to generate the vascular pattern from scratch. In the present study, we utilized a microdevice to generate perfusable vascular network (Kim et al., 2013), and established a long-term perfusion method using braille display. Synthetic vasculature first starts as an irregular network. Then the endothelial cells in the microdevice exhibits remodelling process. Unused part of the vascular bed gradually disappears, resulting in one thick blood vessel in the devise. First, we formulated a model to explain the early phase of network formation. Time-lapse observation of first 12 hours of culture revealed the endothelial cell dynamics. The cultured cells seldom moved but extended long protrusions, and stochastic collisions of the protrusion and cell body established the connections between cells. Effect of neighbouring cell on formation and elongation of protrusion is not observed. Based on these findings, we use stochastic model with experimentally measured parameter to reproduce the initial phase of meshwork formation in this culture system. Next, we established amodel to reproduce the remodelling process in a microdevice. We utilized a simple model in which vessel wall change its position for optimized share stress value. We implemented the vessel wall change by phase field method, and the Stokes flow by MAC method. The numerical simulation reproduces pruning process observed in the microdevice, and final thickness of remaining vasculature can be obtained analytically.