Publications by authors named "Masaya Hagiwara"

12 Publications

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Weakening of resistance force by cell-ECM interactions regulate cell migration directionality and pattern formation.

Commun Biol 2021 06 28;4(1):808. Epub 2021 Jun 28.

Department of Micro-Nano Mechanical Science and Engineering, Nagoya University, Nagoya, Japan.

Collective migration of epithelial cells is a fundamental process in multicellular pattern formation. As they expand their territory, cells are exposed to various physical forces generated by cell-cell interactions and the surrounding microenvironment. While the physical stress applied by neighbouring cells has been well studied, little is known about how the niches that surround cells are spatio-temporally remodelled to regulate collective cell migration and pattern formation. Here, we analysed how the spatio-temporally remodelled extracellular matrix (ECM) alters the resistance force exerted on cells so that the cells can expand their territory. Multiple microfabrication techniques, optical tweezers, as well as mathematical models were employed to prove the simultaneous construction and breakage of ECM during cellular movement, and to show that this modification of the surrounding environment can guide cellular movement. Furthermore, by artificially remodelling the microenvironment, we showed that the directionality of collective cell migration, as well as the three-dimensional branch pattern formation of lung epithelial cells, can be controlled. Our results thus confirm that active remodelling of cellular microenvironment modulates the physical forces exerted on cells by the ECM, which contributes to the directionality of collective cell migration and consequently, pattern formation.
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http://dx.doi.org/10.1038/s42003-021-02350-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8239002PMC
June 2021

Engineering approaches to control and design the in vitro environment towards the reconstruction of organs.

Dev Growth Differ 2020 Apr 10;62(3):158-166. Epub 2020 Jan 10.

Cluster for Pioneering Research, RIKEN, Saitama, Japan.

In vitro experimental models pertaining to human cells are considered essential for most biological experiments, such as drug development and analysis of disease mechanisms, because of their genetic consistency and ease for detailed and long-term analysis. Recent development of organoid cultures, such as intestine, liver, and kidney cultures, greatly promotes the potential of in vitro experiments. However, conventional culture methods that use manual pipetting have limitations in regenerating complex biosystems. Our body autonomously organizes cells to form a specific tissue shape, and the self-organization process occurs in an extremely systematic manner. In order to emulate this sophisticated process in vitro; first, methodologies for cell culture and organization of in vitro systems need to be updated; second, understanding the self-organizing system is a crucial issue. In this review, recent advancements in engineering technologies to control the microenvironment during cell culture are introduced. Both static and dynamic control have been developed for decades in engineering fields, and the means by which such technologies can help to elucidate and design a biosystem is discussed.
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http://dx.doi.org/10.1111/dgd.12647DOI Listing
April 2020

Self-organized formation of developing appendages from murine pluripotent stem cells.

Nat Commun 2019 08 23;10(1):3802. Epub 2019 Aug 23.

Laboratory of Developmental Systems, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan.

Limb development starts with the formation of limb buds (LBs), which consist of tissues from two different germ layers; the lateral plate mesoderm-derived mesenchyme and ectoderm-derived surface epithelium. Here, we report means for induction of an LB-like mesenchymal/epithelial complex tissues from murine pluripotent stem cells (PSCs) in vitro. The LB-like tissues selectively differentiate into forelimb- or hindlimb-type mesenchymes, depending on a concentration of retinoic acid. Comparative transcriptome analysis reveals that the LB-like tissues show similar gene expression pattern to that seen in LBs. We also show that manipulating BMP signaling enables us to induce a thickened epithelial structure similar to the apical ectodermal ridge. Finally, we demonstrate that the induced tissues can contribute to endogenous digit tissue after transplantation. This PSC technology offers a first step for creating an artificial limb bud in culture and might open the door to inducing other mesenchymal/epithelial complex tissues from PSCs.
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http://dx.doi.org/10.1038/s41467-019-11702-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707191PMC
August 2019

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations.

J Vis Exp 2019 03 21(145). Epub 2019 Mar 21.

Department of Biological Functions Engineering, Kyushu Institute of Technology.

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.
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http://dx.doi.org/10.3791/59214DOI Listing
March 2019

Epidermal growth factor induced macropinocytosis directs branch formation of lung epithelial cells.

Biochem Biophys Res Commun 2018 12 13;507(1-4):297-303. Epub 2018 Nov 13.

Graduate School of Science, Osaka Prefecture University, 1-1 Gakuencho, Sakai-shi, Osaka, 599-8570, Japan.

Lung branching morphogenesis is a complex system involving many molecular interactions to filling the three dimensional spaces; however, the underlying developmental mechanisms are still not fully understood. In this paper, we have investigated the effect of epidermal growth factor (EGF) on normal human bronchial epithelial cells and their three-dimensional (3D) branching pattern formation by using in vitro experiments and mathematical simulation. The results show that EGF is essential for 3D branch pattern formation and its receptor is highly expressed at the tip of branches to generate the drive force for cells to migrate. Macropinocytosis induced by EGFR expression is firmly contributed to the nutrition uptake at the tip of branches. Our findings for effective branching formation of human lung cells contribute to further understanding molecular mechanisms of organogenesis, and the important mechanisms also possibly participate in related lung disease such as malformation.
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http://dx.doi.org/10.1016/j.bbrc.2018.11.028DOI Listing
December 2018

High repeatability from 3D experimental platform for quantitative analysis of cellular branch pattern formations.

Integr Biol (Camb) 2018 05;10(5):306-312

NanoSquare Research Institute, Osaka Prefecture University, Osaka, Japan.

Three-dimensional (3D) cell and tissue cultures more closely mimic biological environments than two-dimensional (2D) cultures and are therefore highly desirable in culture experiments. However, 3D cultures often fail to yield repeatable experimental results because of variation in the initial culture conditions, such as cell density and distribution in the extracellular matrix, and therefore reducing such variation is a paramount concern. Here, we present a 3D culture platform that demonstrates highly repeatable experimental results, obtained by controlling the initial cell cluster shape in the gel cube culture device. A micro-mould with the desired shape was fabricated by photolithography or machining, creating a 3D pocket in the extracellular matrix contained in the device. Highly concentrated human bronchial epithelial cells were then injected in the pocket so that the cell cluster shape matched the fabricated mould shape. Subsequently, the cubic device supplied multi-directional scanning, enabling high-resolution capture of the whole tissue structure with only a low-magnification lens. The proposed device significantly improved the repeatability of the developed branch pattern, and multi-directional scanning enabled quantitative analysis of the developed branch pattern formations. A mathematical simulation was also conducted to reveal the mechanisms of branch pattern formation. The proposed platform offers the potential to accelerate any research field that conducts 3D culture experiments, including tissue regeneration and drug development.
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http://dx.doi.org/10.1039/c8ib00032hDOI Listing
May 2018

Gefitinib Enhances Mitochondrial Biological Functions in NSCLCs with Mutations at a High Cell Density.

Anticancer Res 2017 09;37(9):4779-4788

NanoSquare Research Institution, Research Center for the 21st Century, Organization for Research Promotion, Osaka Prefecture University, Sakai, Japan

Background/aim: Gefitinib is a tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR) and has been approved for the treatment of non-small cell lung cancers (NSCLCs) with EGFR mutations. Here we demonstrated that gefitinib induced a significantly enhanced biological activity of succinate-tetrazolium reductase (STR) in mitochondria and mitochondrial membrane potential in HCC827 cells (EGFR mutation NSCLCs, sensitive to gefitinib) at a high cell density.

Materials And Methods: We assessed the biological activity (STR, mitochondrial membrane potential, expression level of Bcl-2 family proteins) of gefitinib on NSCLCs at different cell densities.

Results: The 3D cell culture experiments showed the enhanced mitochondrial biological activity in clustered cell culture treated with gefitinib. Interestingly, the expression levels of Bcl-x and Bax, were affected by the cellular number and gefitinib treatment. We also found that gefitinib prevented additive anticancer activity in the combinational treatment with doxorubicin, which induces mitochondria-dependent apoptotic cell death.

Conclusion: Our results indicate that gefitinib may work as a mitochondrial protector against combinational treatment with mitochondria-dependent anticancer agents in high-cell-density.
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http://dx.doi.org/10.21873/anticanres.11884DOI Listing
September 2017

In Vitro Experimental Model for the Long-Term Analysis of Cellular Dynamics During Bronchial Tree Development from Lung Epithelial Cells.

Tissue Eng Part C Methods 2017 06 24;23(6):323-332. Epub 2017 May 24.

2 Department of Biological Science, Osaka Prefecture University , Osaka, Japan .

Lung branching morphogenesis has been studied for decades, but the underlying developmental mechanisms are still not fully understood. Cellular movements dynamically change during the branching process, but it is difficult to observe long-term cellular dynamics by in vivo or tissue culture experiments. Therefore, developing an in vitro experimental model of bronchial tree would provide an essential tool for developmental biology, pathology, and systems biology. In this study, we succeeded in reconstructing a bronchial tree in vitro by using primary human bronchial epithelial cells. A high concentration gradient of bronchial epithelial cells was required for branching initiation, whereas homogeneously distributed endothelial cells induced the formation of successive branches. Subsequently, the branches grew in size to the order of millimeter. The developed model contains only two types of cells and it facilitates the analysis of lung branching morphogenesis. By taking advantage of our experimental model, we carried out long-term time-lapse observations, which revealed self-assembly, collective migration with leader cells, rotational motion, and spiral motion of epithelial cells in each developmental event. Mathematical simulation was also carried out to analyze the self-assembly process and it revealed simple rules that govern cellular dynamics. Our experimental model has provided many new insights into lung development and it has the potential to accelerate the study of developmental mechanisms, pattern formation, left-right asymmetry, and disease pathogenesis of the human lung.
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http://dx.doi.org/10.1089/ten.TEC.2017.0126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5510150PMC
June 2017

Tissue in Cube: In Vitro 3D Culturing Platform with Hybrid Gel Cubes for Multidirectional Observations.

Adv Healthc Mater 2016 07 29;5(13):1566-71. Epub 2016 Apr 29.

Nanoscience and Nanotechnology Research Center, Research Organization for the 21st Century, Osaka Prefecture University, 1-2 Gakuen-cho, Naka-ku Sakai-shi, Osaka, 599-8570, Japan.

An in vitro 3D culturing platform enabling multidirectional observations of 3D biosamples is presented. The 3D structure of biosamples can be recognized without fluorescence. The cubic platform employs two types of hydrogels that are compatible with conventional culture dishes or well plates, facilitating growth in culture, ease of handling, and viewing at multiple angles.
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http://dx.doi.org/10.1002/adhm.201600167DOI Listing
July 2016

In vitro reconstruction of branched tubular structures from lung epithelial cells in high cell concentration gradient environment.

Sci Rep 2015 Jan 27;5:8054. Epub 2015 Jan 27.

1] Mechanical and Aerospace Engineering Department, University of California Los Angeles, 420 Westwood Plaza, Los Angeles, CA 90095, USA [2] Bioengineering Department, University of California Los Angeles, 420 Westwood Plaza, Los Angeles, CA 90095, USA.

We have succeeded in developing hollow branching structure in vitro commonly observed in lung airway using primary lung airway epithelial cells. Cell concentration gradient is the key factor that determines production of the branching cellular structures, as optimization of this component removes the need for heterotypic culture. The higher cell concentration leads to the more production of morphogens and increases the growth rate of cells. However, homogeneous high cell concentration does not make a branching structure. Branching requires sufficient space in which cells can grow from a high concentration toward a low concentration. Simulation performed using a reaction-diffusion model revealed that long-range inhibition prevents cells from branching when they are homogeneously spread in culture environments, while short-range activation from neighboring cells leads to positive feedback. Thus, a high cell concentration gradient is required to make branching structures. Spatial distributions of morphogens, such as BMP-4, play important roles in the pattern formation. This simple yet robust system provides an optimal platform for the further study and understanding of branching mechanisms in the lung airway, and will facilitate chemical and genetic studies of lung morphogenesis programs.
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http://dx.doi.org/10.1038/srep08054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4306969PMC
January 2015

On-chip microrobot for investigating the response of aquatic microorganisms to mechanical stimulation.

Lab Chip 2013 Mar;13(6):1070-8

Department of Biological Functions and Engineering, Kyushu Institute of Technology, Kitakyushu 808-0196, Japan.

In this paper, we propose a novel, magnetically driven microrobot equipped with a frame structure to measure the effects of stimulating aquatic microorganisms. The design and fabrication of the force-sensing structure with a displacement magnification mechanism based on beam deformation are described. The microrobot is composed of a Si-Ni hybrid structure constructed using micro-electro-mechanical system (MEMS) technologies. The microrobots with 5 μm-wide force sensors are actuated in a microfluidic chip by permanent magnets so that they can locally stimulate the microorganisms with the desired force within the stable environment of the closed microchip. They afford centimetre-order mobility (untethered drive) and millinewton-order forces (high power) as well as force-sensing. Finally, we apply the developed microrobots for the quantitative evaluation of the stimuation of Pleurosira laevis (P. laevis) and determine the relationship between the applied force and the response of a single cell.
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http://dx.doi.org/10.1039/c2lc41190cDOI Listing
March 2013

On-chip magnetically actuated robot with ultrasonic vibration for single cell manipulations.

Lab Chip 2011 Jun 12;11(12):2049-54. Epub 2011 May 12.

Department of Mechanical Science and Engineering, Nagoya University, Nagoya, Japan.

This paper presents an innovative driving method for an on-chip robot actuated by permanent magnets in a microfluidic chip. A piezoelectric ceramic is applied to induce ultrasonic vibration to the microfluidic chip and the high-frequency vibration reduces the effective friction on the MMT significantly. As a result, we achieved 1.1 micrometre positioning accuracy of the microrobot, which is 100 times higher accuracy than without vibration. The response speed is also improved and the microrobot can be actuated with a speed of 5.5 mm s(-1) in 3 degrees of freedom. The novelty of the ultrasonic vibration appears in the output force as well. Contrary to the reduction of friction on the microrobot, the output force increased twice as much by the ultrasonic vibration. Using this high accuracy, high speed, and high power microrobot, swine oocyte manipulations are presented in a microfluidic chip.
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http://dx.doi.org/10.1039/c1lc20164fDOI Listing
June 2011
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