Yuanshu Zhou

Multiparameter analyses of three-dimensionally cultured tumor spheroids based on respiratory activity and comprehensive gene expression profiles

Multicellular spheroids of human breast cancer cells (MCF-7) formed with two different three-dimensional (3D) culture methods were evaluated in detail on the basis of respiratory activity and high-throughput gene expression analysis. The spheroids formed with poly(dimethylsiloxane) (PDMS) microwell arrays indicated significant restriction of the spheroid size, whereas their respiratory activity was 2-fold greater than that formed with the hanging drop culture method. Fluidigm BioMark dynamic array was used for comprehensive and quantitative real-time polymerase chain reaction (qRT-PCR) analysis on the samples whose respiratory activity had been measured. Genes involved in cellular senescence and glucose metabolism indicated significantly higher values for the PDMS microwell culture method than for the hanging drop culture method (P<0.05). Interestingly, samples formed with the PDMS microwell culture method showed stronger responses for glycolysis than those formed with the hanging drop method. These results illustrate the power of multiparameter analysis to characterize multicellular spheroids cultured in different microenvironments even if they have the same morphology.

A Pt layer/Pt disk electrode configuration to evaluate respiration and alkaline phosphatase activities of mouse embryoid bodies.

A Pt layer/Pt disk electrode configuration was used as a scanning electrochemical microscopy (SECM) probe. The glass seal part of the insulator was covered with a Pt layer to form an exposed pseudo reference electrode. In a HEPES-based medium at pH 7.5, the half-wave potential (E(1/2)) for [Fe(CN)(6)](4-) oxidation and O(2) reduction measured versus the internal Pt pseudo reference was shifted by about -0.2V, compared with the E(1/2) measured versus the external Ag/AgCl reference electrode. The shape and the current of the cyclic voltammograms (CVs) did not change notably over time, indicating that the Pt layer is sufficiently stable to be used as an integrated pseudo reference for voltammetric measurements. To demonstrate the suitability for SECM applications, the Pt/Pt probe configuration was used for measuring the oxygen consumption and the alkaline phosphatase (ALP) activity of a single mouse embryoid body (mEB). Ten individual mEB samples were characterized to monitor the oxygen concentration profile. Oxygen reduction currents were monitored at -0.7 V versus the Pt pseudo reference and compared with those monitored at -0.5 V versus Ag/AgCl. The respiration rate of mEBs becomes greater with increasing cultivation dates. We have plotted the oxygen consumption rate (F(O(2))) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. The linearity of the plot was excellent (coefficient of determination R(2)=0.90). The slope of the least squares method was 1. In a 1.0mM p-aminophenylphospate (PAPP) HEPES buffer (pH 9.5) solution, APL activity of mEBs can be characterized, to monitor the p-aminophenol (PAP) oxidation current. ALP catalyzes the hydrolysis of PAPP to PAP. The E(1/2) for PAP oxidation measured versus the Pt layer was not shifted, compared with the E(1/2) versus Ag/AgCl. The mEB samples were characterized to monitor the PAP concentration profile. PAP oxidation currents were monitored at +0.3 V versus the Pt layer and compared with those monitored at +0.3 V versus Ag/AgCl. We have plotted the PAP production rate (F(PAP)) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. In this case, the linearity of the plot became slightly scattered, but it was found to be possible to evaluate ALP activities of mEB samples utilizing the Pt/Pt probe configuration. This type of probe is very useful because it is not necessary to insert a reference electrode into the measuring solution to obtain an electrical connection, and thus electrochemical measurement in a small volume becomes much easier.

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.

Noninvasive Measurement of Alkaline Phosphatase Activity in Embryoid Bodies and Coculture Spheroids with Scanning Electrochemical Microscopy

Alkaline phosphatase (ALP) is an enzyme commonly used as an undifferentiated marker of embryonic stem cells (ESCs). Although noninvasive ALP detection has long been desired for stem cell research and in cell transplantation therapy, little progress has been made in developing such techniques. In this study, we propose a noninvasive evaluation method for detecting ALP activity in mouse embryoid bodies (mEBs) using scanning electrochemical microscopy (SECM). SECM has several advantages, including being noninvasive, nonlabeled, quantitative, and highly sensitive. First, we found that SECM-based ALP evaluation permits the comparison of ALP activity among mEBs of different sizes by monitoring the p-aminophenol (PAP) production rate in aqueous solution containing p-aminophenylphosphate (PAPP) normal to the surface area of each sample. Second, coculture spheroids, consisting of mEB and MCF-7 cells for the core and the concentric outer layer, respectively, were prepared as model samples showing heterogeneous ALP activities. The overall PAP production rate dramatically declined in the presence of the MCF-7 cell outer layer, which blocked the mass transfer of PAPP to inner mEB. This result indicated that the SECM response mainly originated from ALP located at the surface of the cellular aggregate, including mEBs and coculture spheroids. Third, taking advantage of the noninvasive nature of SECM, we examined the relevance of ALP activity and cardiomyocyte differentiation. Collectively, these results suggested that noninvasive SECM-based ALP activity normalized by the sample surface enables the selection of EBs with a higher potential to differentiate into cardiomyocytes, which can contribute toward various types of stem cell research.

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.

Noninvasively measuring respiratory activity of rat primary hepatocyte spheroids by scanning electrochemical microscopy

Construction of an in vitro drug screening method for evaluating drug metabolism and toxicity by using cells is required instead of the conventional in vivo one that uses animals. In order to realize the in vitro study, analyzing the cellular activity or viability noninvasively in advance of the screening is essential. The aim of the current study is to establish a method that can evaluate the cellular activity depending on spheroid sizes by means of oxygen consumption and to determine the valid diameter of hepatocyte spheroids. To measure the respiratory activity of the spheroids, which were formed on a nanopillar sheet, we applied scanning electrochemical microscopy (SECM). From the viewpoint of high respiratory activity and its small variation, we determined that spheroids with 70 μm in diameter were adequate. We then performed a gene expression analysis by using a real-time PCR to evaluate the correlation with respiratory activity. As a result, a higher expression level of Hnf4α, which is essential for hepatocytes to fulfill many liver functions and is the indicator of well-differentiated hepatocytes, showed relatively higher respiratory activity. We concluded that the noninvasive SECM technique could evaluate the cellular activity of a single spheroid. Noninvasively measuring cellular activity by SECM makes it possible to evaluate the cellular activity prior to a nonclinical test and enables the continued monitoring of the drug response by using single spheroid. SECM becomes a powerful tool to satisfying the increasing demand for an in vitro system in the course of new drug development.

Nanoscale imaging of primary cilia with scanning ion conductance microscopy

Primary cilia are hair-like sensory organelles whose dimensions and location vary with cell type and culture condition. Herein, we employed scanning ion conductance microscopy (SICM) to visualize the topography of primary cilia from different cell types. By combining SICM with fluorescence imaging, we successfully distinguished between surface cilia that project outward from the cell surface and subsurface cilia that are trapped below it. The nanoscale structure of the ciliary pocket, which cannot be easily identified using a confocal fluorescence microscope, was observed in SICM images. Furthermore, we developed a topographic reconstruction method using current-distance profiles to evaluate the relationship between set point and topographic image and found that a low set point is important for detecting the true topography of a primary cilium using hopping mode SICM.