研究者業績

山田 真澄

ヤマダ マスミ  (Masumi Yamada)

基本情報

所属
千葉大学 大学院工学研究院 共生応用化学コース 教授
学位
博士(工学)(2006年3月 東京大学)

J-GLOBAL ID
200901066152886720
researchmap会員ID
6000014797

論文

 90
  • Ayumi Hayashi, Runa Hemmi, Yuhei Saito, Rie Utoh, Tatsuo Taniguchi, Masumi Yamada
    Analytical Chemistry 96(17) 6764-6773 2024年4月30日  査読有り最終著者責任著者
    Tremendous efforts have been made to develop practical and efficient microfluidic cell and particle sorting systems; however, there are technological limitations in terms of system complexity and low operability. Here, we propose a sheath flow generator that can dramatically simplify operational procedures and enhance the usability of microfluidic cell sorters. The device utilizes an embedded polydimethylsiloxane (PDMS) sponge with interconnected micropores, which is in direct contact with microchannels and seamlessly integrated into the microfluidic platform. The high-density micropores on the sponge surface facilitated fluid drainage, and the drained fluid was used as the sheath flow for downstream cell sorting processes. To fabricate the integrated device, a new process for sponge-embedded substrates was developed through the accumulation, incorporation, and dissolution of PMMA microparticles as sacrificial porogens. The effects of the microchannel geometry and flow velocity on the sheath flow generation were investigated. Furthermore, an asymmetric lattice-shaped microchannel network for cell/particle sorting was connected to the sheath flow generator in series, and the sorting performances of model particles, blood cells, and spiked tumor cells were investigated. The sheath flow generation technique developed in this study is expected to streamline conventional microfluidic cell-sorting systems as it dramatically improves versatility and operability.
  • Shota Mashiyama, Runa Hemmi, Takeru Sato, Atsuya Kato, Tatsuo Taniguchi, Masumi Yamada
    Lab on a Chip 24(2) 171-181 2024年1月17日  査読有り最終著者責任著者
    Although droplet microfluidics has been studied for the past two decades, its applications are still limited due to the low productivity of microdroplets resulting from the low integration of planar microchannel structures. In this study, a microfluidic system implementing inverse colloidal crystals (ICCs), a spongious matrix with regularly and densely formed three-dimensional (3D) interconnected micropores, was developed to significantly increase the throughput of microdroplet generation. A new bottom-up microfabrication technique was developed to seamlessly integrate the ICCs into planar microchannels by accumulating non-crosslinked spherical PMMA microparticles as sacrificial porogens in a selective area of a mold and later dissolving them. We have demonstrated that the densely arranged micropores on the spongious ICC of the microchannel function as massively parallel micronozzles, enabling droplet formation on the order of >10 kHz. Droplet size could be adjusted by flow conditions, fluid properties, and micropore size, and biopolymer particles composed of polysaccharides and proteins were produced. By further parallelization of the unit structures, droplet formation on the order of >100 kHz was achieved. The presented approach is an upgrade of the existing droplet microfluidics concept, not only in terms of its high throughput, but also in terms of ease of fabrication and operation.
  • Yuken Hasebe, Masumi Yamada, Rie Utoh, Minoru Seki
    Journal of Bioscience and Bioengineering 135(5) 417-422 2023年5月  責任著者
    Technologies for efficiently expanding Chinese hamster ovary (CHO) cells, the primary host cells for antibody production, are of growing industrial importance. Various processes for the use of microcarriers in CHO suspension cultures have been developed, but there have been very few studies on cell-adhesive microcarriers that are similar in size to cells. In this study, we proposed a new approach to suspension cultures of CHO cells using cell-sized condensed and crosslinked gelatin microparticles (GMPs) as carriers. Unlike commercially available carriers with sizes typically greater than 100 μm, each cell can adhere to the surface of multiple particles and form loose clusters with voids. We prepared GMPs of different average diameters (27 and 48 μm) and investigated their effects on cell adhesion and cluster formation. In particular, small GMPs promoted cell proliferation and increased IgG4 production by the antibody-producing CHO cell line. The data obtained in this study suggest that cell-sized particles, rather than larger ones, enhance cell proliferation and function, providing useful insights for improving suspension-culture-based cell expansion and cell-based biologics production for a wide range of applications.
  • Mai Takagi, Masumi Yamada, Rie Utoh, Minoru Seki
    Lab on a Chip 23(9) 2257-2267 2023年5月  責任著者
    Spheroid formation assisted by microengineered chambers is a versatile approach for morphology-controlled three-dimensional (3D) cell cultivation with physiological relevance to human tissues. However, the limitation in diffusion-based oxygen/nutrient transport has been a critical issue for the densely packed cells in spheroids, preventing maximization of cellular functions and thus limiting their biomedical applications. Here, we have developed a multiscale microfluidic system for the perfusion culture of spheroids, in which porous microchambers, connected with microfluidic channels, were engineered. A newly developed process of centrifugation-assisted replica molding and salt-leaching enabled the formation of single micrometer-sized pores on the chamber surface and in the substrate. The porous configuration generates a vertical flow to directly supply the medium to the spheroids, while avoiding the formation of stagnant flow regions. We created seamlessly integrated, all PDMS/silicone-based microfluidic devices with an array of microchambers. Spheroids of human liver cells (HepG2 cells) were formed and cultured under vertical-flow perfusion, and the proliferation ability and liver cell-specific functions were compared with those of cells cultured in non-porous chambers with a horizontal flow. The presented system realizes both size-controlled formation of spheroids and direct medium supply, making it suitable as a precision cell culture platform for drug development, disease modelling, and regenerative medicine.
  • Natsumi Shimmyo, Makoto Furuhata, Masumi Yamada, Rie Utoh, Minoru Seki
    Analyst 147(8) 1622-1630 2022年4月  責任著者
    Numerous attempts have been made to develop efficient systems to purify trace amounts of circulating tumor cells (CTCs) from blood samples. However, current technologies are limited by complexities in device fabrication, system design, and process operability. Here we describe a facile, scalable, and highly efficient approach to physically capturing CTCs using a rationally designed microfluidic isolator with an array of microslit channels. The wide but thin microslit channels with a depth of several micrometers selectively capture CTCs, which are larger and less deformable than other blood cells, while allowing other blood cells to just flow through. We investigated in detail the effects of the microchannel geometry and operating parameters on the capture efficiency and selectivity of several types of cultured tumor cells spiked in blood samples as the CTC model. Additionally, in situ post-capture staining of the captured cells was demonstrated to investigate the system's applicability to clinical cancer diagnosis. The presented approach is simple in operation but significantly effective in capturing specific cells and hence it may have great potential in implementating cell physics-based CTC isolation techniques for cancer liquid biopsy.
  • Akihiro Morita, Masumi Yamada, Rie Utoh, Kanta Momiyama, Hideki Iwadate, Minoru Seki
    Journal of Bioscience and Bioengineering 133(3) 265-272 2022年3月  責任著者
    Numerous attempts have been made to organize isolated primary hepatocytes into functional three-dimensional (3D) constructs, but technologies to introduce extracellular matrix (ECM) components into such assemblies have not been fully developed. Here we report a new approach to forming hepatocyte-based 3D tissues using fibrillized collagen microparticles (F-CMPs) as intercellular binders. We created thick tissues with a thickness of w200 mm simply by mixing FCMPs with isolated primary rat hepatocytes and culturing them in cell culture inserts. Owing to the incorporated FCMPs, the circular morphology of the formed tissues was stabilized, which was strong enough to be manually manipulated and retrieved from the chamber of the insert. We confirmed that the F-CMPs dramatically improved the cell viability and hepatocyte-specific functions such as albumin production and urea synthesis in the formed tissues. The presented approach provides a versatile strategy for hepatocyte-based tissue engineering, and will have a significant impact on biomedical applications and pharmaceutical research. (c) 2021, The Society for Biotechnology, Japan. All rights reserved.
  • Rie Utoh, Sakiko Enomoto, Masumi Yamada, Keigo Yamanaka, Yuya Yajima, Kazuya Furusawa, Minoru Seki
    Materials Science and Engineering: C 129 112417-112417 2021年10月  責任著者
  • Kotone Saeki, Hisataka Hiramatsu, Ayaka Hori, Yu Hirai, Masumi Yamada, Rie Utoh, Minoru Seki
    ACS Omega 5(34) 21641-21650 2020年9月1日  責任著者
    Although many types of technologies for hydrogel-based cell cultivation have recently been developed, strategies to integrate cell-adhesive micrometer-sized supports with bulk-scale hydrogel platforms have not been fully established. Here, we present a highly unique approach to produce cell-adhesive, protein-based microfibers assisted by the sacrificial template of alginate; we applied these fibers as microengineered scaffolds for hydrogel-based cell encapsulation. Two types of microfluidic devices were designed and fabricated: a single-layered device for producing relatively thick (φ of 10-60 μm) alginate-protein composite fibers with a uniform cross-sectional morphology and a four-layered device for preparing thinner (φ of ∼4 μm) ones through the formation of patterned microfibers with eight distinct alginate-protein composite regions. Following chemical cross-linking of protein molecules and the subsequent removal of the sacrificial alginate from the double-network matrices, microfibers composed only of cross-linked proteins were obtained. We used gelatin, albumin, and hemoglobin as the protein material, and the gelatin-based cell-adhesive fibers were further encapsulated in hydrogels together with the mammalian cells. We clarified that the thinner fibers were especially effective in promoting cell proliferation, and the shape of the constructs was maintained even after removing the hydrogel matrices. The presented approach offers cells with biocompatible solid supports that enhance cell adhesion and proliferation, paving the way for the next generation of techniques for tissue engineering and multicellular organoid formation.
  • Tinglin Mu, Hajime Toyoda, Yuki Kimura, Masumi Yamada, Rie Utoh, Daisuke Umeno, Minoru Seki
    Analytical Chemistry 92(3) 2580-2588 2020年2月4日  責任著者
    Visualization and quantification of intracellular molecules of mammalian cells are crucial steps in clinical diagnosis, drug development, and basic biological research. However, conventional methods rely mostly on labor-intensive, centrifugation-based manual operations for exchanging the cell carrier medium and have limited reproducibility and recovery efficiency. Here we present a microfluidic cell processor that can perform four-step exchange of carrier medium, simply by introducing a cell suspension and fluid reagents into the device. The reaction time period for each reaction step, including fixation, membrane permeabilization, and staining, was tunable in the range of 2 to 15 min by adjusting the volume of the reaction tube connecting the neighboring exchanger modules. We double-stained the cell nucleus and cytoskeleton (F-actin) using the presented device with an overall reaction period of ∼30 min, achieving a high recovery ratio and high staining efficiency. Additionally, intracellular cytokine (IL-2) was visualized for T cells to demonstrate the feasibility of the device as a pretreatment system for downstream flow-cytometric analysis. The presented approach would facilitate the development of laborless, automated microfluidic systems that integrate cell processing and analysis operations and would pave a new path to high-throughput biological experiments.
  • Shuhei Aoyama, Kenji Monden, Yuto Akiyama, Masumi Yamada, Minoru Seki
    Analytical Chemistry 91(21) 13377-13382 2019年11月5日  
    Lateral flow immunoassay devices have revolutionized the style of on-site disease detection and point-of-care testing in the past few decades. The surface nanotopography of a solid substrate is a dominant parameter in the efficiency of antibody immobilization, but precise control over surface roughness has not been fully investigated. Here we presented lateral flow immunoassay platforms with nanometer-scale surface roughness, reproducibly engineered using thermal nanoimprinting lithography, and investigated the effects of surface nanotopography on immunoadsorption and immunoassay performance. We fabricated three types of imprinted polycarbonate sheets with microcone array structures having different degrees of surface roughness using three types of molds fabricated by micromachining or laser ablation. The structures fabricated by laser-ablated nickel mold exhibited numerous bumps measuring several tens of nanometers, which enhanced antibody adsorption. We performed sandwich immunoassays of C-reactive protein in serum samples and achieved highly sensitive detection with a detection limit of ∼0.01 μg mL and a broad dynamic range. The present results provide useful information on the remarkable effect of nanoengineered surfaces on biomolecule adsorption, and the platforms presented here will widen the applicability and versatility of lateral flow immunoassay devices. -1
  • Takuma Yanai, Takatomo Ouchi, Masumi Yamada, Minoru Seki
    Micromachines 10(6) 2019年6月1日  責任著者
    We herein propose a new hydrodynamic mechanism of particle separation using dual-depth, lattice-patterned asymmetric microchannel networks. This mechanism utilizes three-dimensional (3D) laminar flow profiles formed at intersections of lattice channels. Large particles, primarily flowing near the bottom surface, frequently enter the shallower channels (separation channels), whereas smaller particles flowing near the microchannel ceiling primarily flow along the deeper channels (main channels). Consequently, size-based continuous particle separation was achieved in the lateral direction in the lattice area. We confirmed that the depth of the main channel was a critical factor dominating the particle separation efficiencies, and the combination of 15-μm-deep separation channels and 40-μm-deep main channels demonstrated the good separation ability for 3-10-μm particles. We prepared several types of microchannels and successfully tuned the particle separation size. Furthermore, the input position of the particle suspension was controlled by adjusting the input flow rates and/or using a Y-shaped inlet connector that resulted in a significant improvement in the separation precision. The presented concept is a good example of a new type of microfluidic particle separation mechanism using 3D flows and may potentially be applicable to the sorting of various types of micrometer-sized objects, including living cells and synthetic microparticles.
  • Ryoken Ozawa, Hideki Iwadate, Hajime Toyoda, Masumi Yamada, Minoru Seki
    Lab on a Chip 19(10) 1828-1837 2019年5月21日  責任著者
    Even though a number of microfluidic systems for particle/cell sorting have been proposed, facile and versatile platforms that provide sufficient sorting throughput and good operability are still under development. Here we present a simple but effective numbering-up strategy to dramatically increase the throughput of a continuous-flow particle/cell sorting scheme based on hydrodynamic filtration (HDF). A microfluidic channel equipped with multiple branches has been employed as a unit structure for size-based filtration, which realizes precise sorting without necessitating sheath flows. According to the concept of resistive circuit models, we designed and fabricated microdevices incorporating 64 or 128 closely assembled, multiplied units with a separation size of 5.0/7.0 μm. In proof-of-concept experiments, we successfully separated single micrometer-sized model particles and directly separated blood cells (erythrocytes and leukocytes) from blood samples. Additionally, we further increased the unit numbers by laminating multiple layers at a processing speed of up to 15 mL min . The presented numbering-up strategy would provide a valuable insight that is universally applicable to general microfluidic particle/cell sorters and may facilitate the actual use of microfluidic systems in biological studies and clinical diagnosis. -1
  • Aruto Hori, Yuki Watabe, Masumi Yamada, Yuya Yajima, Rie Utoh, Minoru Seki
    ACS Applied Bio Materials 2(5) 2237-2245 2019年5月20日  責任著者
    With the recent progress in three-dimensional (3D) cell culture techniques for regenerative medicine and drug development, hydrogel-based tissue engineering approaches that can precisely organize cells into functional formats have attracted increasing attention. However, challenges remain in creating continuous microconduits within hydrogels to effectively deliver oxygen and nutrients to the embedded cells. Here we propose a one-step, fully liquid state, and all-aqueous process to create porous hydrogels that can encapsulate living cells without the need for extensive processing protocols, including the incorporation and removal of sacrificial materials. An unusual bicontinuous state of aqueous two-phase dispersion was utilized, and one of the two phases, encapsulating living cells, was rapidly photo-cross-linked to form hydrogel sponges. We optimized the volumetric mixing ratio of gelatin methacrylate (GelMA)-rich and polyethylene glycol (PEG)-rich solutions and investigated the effects of the formed continuous microconduits on the cell functions by creating liver-tissue mimetic 3D constructs. The presented technology provides a facile and versatile strategy for fabricating microstructured hydrogels for cell culture and would bring new insights for the development of porous materials by fully aqueous bicontinuous dispersions.
  • Hideki Iwadate, Masumi Yamada, Naoki Kimura, Rina Hashimoto, Yuya Yajima, Rie Utoh, Minoru Seki
    Sensors and Actuators, B: Chemical 287 486-495 2019年5月15日  責任著者
    Thin membrane-based cell culture platforms have recently gained much attention for facilitating separated but adjacent cocultures of heterotypic cells in in vivo tissue-mimetic microenvironments. Here we propose a new approach to preparing significantly thin but highly stable membranes composed of extracellular matrix (ECM) components, mainly type I collagen, that are supported by PDMS microstencil plates. A collagen solution was introduced into the through holes of a microstencil plate and dried to form condensed membranes with a thickness of less than 1 μm. A coculture of HepG2 and Swiss-3T3 cells was performed on both membrane surfaces, and gene expression assays revealed the superiority of the presented thin membranes. Release of the membranes from the plate was possible, as was the integration of the membranes into microfluidic systems to perform perfusion cultures of cells. We were also able to prepare Matrigel membranes by the same procedure. The presented thin membrane-based cell culture platforms would provide physiologically suitable conditions for cells and thus would be widely applicable to a variety of in vitro cell culture systems.
  • Shuhei Aoyama, Yuto Akiyama, Kenji Monden, Masumi Yamada, Minoru Seki
    Analyst 144(5) 1519-1526 2019年3月7日  
    Although various types of on-site immunoassay platforms have been developed, facile and reliable sample-to-answer immunoassay systems are still under development. In this study, we proposed a lateral-flow immunoassay system utilizing a polymer sheet with microcone array structures fabricated by thermal imprinting. By adding a surfactant to the running/washing buffer, we were able to dispense with complicated chemical modification protocols, which are usually necessary to enhance antibody adsorption. We investigated three types of polymeric materials and confirmed that polycarbonate is most suitable as an imprinted polymer substrate. Experiments using microcone arrays with different distances revealed that the increased surface area with nanometer-scale surface roughness was key to achieving stable immobilization of antibodies. To assess the applicability of this assay to clinical diagnosis, C-reactive protein in a pure buffer and in a serum sample was analyzed as a model, with a ∼0.1 μg mL lower limit of detection. The presented approach would open a new path to the development of immunoassay-based on-site point-of-care testing by virtue of its simplicity of operation, high sensitivity, and versatility. -1
  • Masumi Yamada
    Journal of the Society of Powder Technology, Japan 56(7) 398-402 2019年  責任著者
    Microfluidic processes are capable of producing various types of microparticles with precisely controlled sizes, morphologies, and compositions. In this study we briefly introduce our recent progresses on the production of microparticles, utilizing the unique phenomena of formation and rapid dissolution of microdroplets in microfluidic channels. Water and a polar organic solvents are used either as the continuous or the dispersed phase, one of which contains the materials for particles (e.g., polymers). Because of the dissolution of the droplets, the particle materials are concentrated, forming microparticles with various morphologies and compositions. In this review, we explain the formation of such droplets in a non-equilibrium state and the production of polymeric, lipid, polysaccharide, protein, and carbon nanotube microparticles. Additionally, the application of the collagen microparticles to tissue engineering is presented.
  • Mayu Fukushi, Keita Kinoshita, Masumi Yamada, Yuya Yajima, Rie Utoh, Minoru Seki
    RSC Advances 9(16) 9136-9144 2019年  責任著者
    Vascular tissue models created in vitro are of great utility in the biomedical research field, but versatile, facile strategies are still under development. In this study, we proposed a new approach to prepare vascular tissue models in PDMS-based composite channel structures embedded with barium salt powders. When a cell-containing hydrogel precursor solution was continuously pumped in the channel, the precursor solution in the vicinity of the channel wall was selectively gelled because of the barium ions as the gelation agent supplied to the flow. Based on this concept, we were able to prepare vascular tissue models, with diameters of 1-2 mm and with tunable morphologies, composed of smooth muscle cells in the hydrogel matrix and endothelial cells on the lumen. Perfusion culture was successfully performed under a pressurized condition of ∼120 mmHg. The presented platform is potentially useful for creating vascular tissue models that reproduce the physical and morphological characteristics similar to those of vascular tissues in vivo.
  • Yuya Yajima, Chu Ning Lee, Masumi Yamada, Rie Utoh, Minoru Seki
    Journal of Bioscience and Bioengineering 126(1) 111-118 2018年7月  責任著者
    Although the reconstruction of functional 3D liver tissue models in vitro presents numerous challenges, it is in great demand for drug development, regenerative medicine, and physiological studies. Here we propose a new approach to perform perfusion cultivation of liver cells by assembling cell-laden hydrogel microfibers. HepG2 cells were densely packed into the core of sandwich-type anisotropic microfibers, which were produced using microfluidic devices. The obtained microfibers were bundled up and packed into a perfusion chamber, and perfusion cultivation was performed. We evaluated cell viability and functions, and also monitored the oxygen consumption. Furthermore, fibers covered with vascular endothelial cells were united during the perfusion culture, to form vascular network-like conduits between fibers. The presented technique can structurally mimic the hepatic lobule in vivo and could prove to be a useful model for various biomedical research applications.
  • Manami Sugimoto, Yoichi Kitagawa, Masumi Yamada, Yuya Yajima, Rie Utoh, Minoru Seki
    Lab on a Chip 18(9) 1378-1387 2018年5月7日  責任著者
    Cell migration and invasion are of significant importance in physiological phenomena, including wound healing and cancer metastasis. Here we propose a new system for quantitatively evaluating cancer cell invasion in a three-dimensional (3D), in vivo tissue-like environment. This system uses composite hydrogel microfibers whose cross section has a relatively soft micropassage region and that were prepared using a multilayered microfluidic device; cancer cells are encapsulated in the core and fibroblasts are seeded in the shell regions surrounding the core. Cancer cell proliferation is guided through the micropassage because of the physical restriction imposed by the surrounding solid shell regions. Quantitative analysis of cancer cell invasion is possible simply by counting the cancer cell colonies that form outside the fiber. This platform enables the evaluation of anticancer drug efficacy (cisplatin, paclitaxel, and 5-fluorouracil) based on the degree of invasion and the gene expression of cancer cells (A549 cells) with or without the presence of fibroblasts (NIH-3T3 cells). The presented hydrogel fiber-based migration assays could be useful for studying cell behaviors under 3D coculture conditions and for drug screening and evaluation.
  • Rie Utoh, Junji Komori, Hiroyuki Kuge, Kohei Tatsumi, Masumi Yamada, Shinji Hirohashi, Masahiro Tsutsumi, Toshihiro Amanuma, Akira Yoshioka, Yoshiyuki Nakajima, Kenjiro Wake, Teruo Okano, Eric Lagasse, Kazuo Ohashi
    Journal of Hepatology 68(4) 744-753 2018年4月  
    Background & Aims: Since the first account of the myth of Prometheus, the amazing regenerative capacity of the liver has fascinated researchers because of its enormous medical potential. Liver regeneration is promoted by multiple types of liver cells, including hepatocytes and liver non-parenchymal cells (NPCs), through complex intercellular signaling. However, the mechanism of liver organogenesis, especially the role of adult hepatocytes at ectopic sites, remains unknown. In this study, we demonstrate that hepatocytes alone spurred liver organogenesis to form an organ-sized complex 3D liver that exhibited native liver architecture and functions in the kidneys of mice. Methods: Isolated hepatocytes were transplanted under the kidney capsule of monocrotaline (MCT) and partial hepatectomy (PHx)-treated mice. To determine the origin of NPCs in neo-livers, hepatocytes were transplanted into MCT/PHx-treated green fluorescent protein transgenic mice or wild-type mice transplanted with bone marrow cells isolated from green fluorescent protein-mice. Results: Hepatocytes engrafted at the subrenal space of mice underwent continuous growth in response to a chronic hepatic injury in the native liver. More than 1.5 years later, whole organ-sized liver tissues with greater mass than those of the injured native liver had formed. Most remarkably, we revealed that at least three types of NPCs with similar phenotypic features to the liver NPCs were recruited from the host tissues including bone marrow. The neo-livers in the kidney exhibited liver-specific functions and architectures, including sinusoidal vascular systems, zonal heterogeneity, and emergence of bile duct cells. Furthermore, the neo-livers successfully rescued the mice with lethal liver injury. Conclusion: Our data clearly show that adult hepatocytes play a leading role as organizer cells in liver organogenesis at ectopic sites via NPC recruitment. Lay summary: The role of adult hepatocytes at ectopic locations has not been clarified. In this study, we demonstrated that engrafted hepatocytes in the kidney proliferated, recruited non-parenchymal cells from host tissues including bone marrow, and finally created an organ-sized, complex liver system that exhibited liver-specific architectures and functions. Our results revealed previously undescribed functions of hepatocytes to direct liver organogenesis through non-parenchymal cell recruitment and organize multiple cell types into a complex 3D liver at ectopic sites. Transcript profiling: Microarray data are deposited in GEO (GEO accession: GSE99141).
  • Shunji Homma, Shuichi Iwata, Daisuke Kobayashi, Yosuke Matsukuma, Mitsuhiro Ohta, Masumi Yamada
    Journal of Chemical Engineering of Japan 51(4) 299-299 2018年  
  • Masumi Yamada, Minoru Seki
    Journal of Chemical Engineering of Japan 51(4) 318-330 2018年  査読有り責任著者
    With the recent developments in microfluidic instruments and devices, various types of micrometer-sized materials have been produced by employing multiphase flow patterns formed in the microchannel. In particular, microparticles and microfibers, which are compatible with biomolecule incorporation or living cell encapsulations, have been gaining significant attention as new tools for biochemical analysis, cellular physiological studies, tissue engineering, cell transplantation, and controlled drug delivery. Herein, we introduce recent developments in microfluidic systems to produce alginate-based hydrogel microparticles and microfibers. By utilizing droplet dispersions either in equilibrium or nonequilibrium states, or by employing parallel laminar flows, microengineered functional materials that are difficult to generate using conventional devices and operations can be obtained. New and interesting multiphase phenomena are reviewed, together with the pros and cons of these systems and their applications. Furthermore, the fundamentals of multiphase microfluidics and the materials used to prepare particles and fibers are briefly introduced.
  • Kazuki Akamatsu, Koki Minezaki, Masumi Yamada, Minoru Seki, Shin Ichi Nakao
    Langmuir 33(49) 14087-14092 2017年12月12日  査読有り
    Direct observation of double emulsion droplet permeation through a microchannel that mimicked 100 μm membrane pores with a porosity of 66.7% provided insights regarding splitting mechanisms in porous membranes. The microchannel was fabricated by standard soft lithography, and the oil-in-water-in-oil double emulsion droplets were prepared with a glass capillary device. By changing the flow rate from 0.5 to 5.0 × 10 m s , three characteristic behaviors were observed: (a) passage into one channel without splitting; (b) division into two smaller components; and (c) stripping of the middle water phase of the double emulsion droplets into a smaller double emulsion droplet and a smaller water-in-oil single emulsion droplet. The mechanisms are discussed with respect to the balance of viscous forces and interfacial tension, the contact point with the tip of the channel, and the relative position of the innermost droplet within the middle droplet. -2 -1
  • Yuya Yajima, Masumi Yamada, Rie Utoh, Minoru Seki
    ACS Biomaterials Science and Engineering 3(9) 2144-2154 2017年9月11日  査読有り責任著者
    In closely packed artificial 3D cellular constructs, cells located near the center of the constructs are not functional because of the limited supply of oxygen and nutrition. Here we describe a simple, unique, and highly versatile approach to organizing cells into thick but porous 3D tissues, using cell-sized collagen microparticles as particulate scaffolds. When cells and particles are mixed and seeded in a noncell-adhesive planar chamber, they gather to form sheet-shaped structures with a thickness of 100-150 μm. In the construct, uniformly distributed particles work as a binder between cells and modulate the strong intercellular contraction. We confirmed that several factors, including the particle/cell ratio and particle size, critically affect the stability and shrink behaviors of porous tissues prepared using mouse embryonic fibroblasts (NIH-3T3 cells). Cross-sectional observation, together with cell proliferation and viability assays, revealed that the cells composing the tissues are functional primarily because interior pores between cells/particles worked as a path for efficient molecular transport. Furthermore, we prepared thick cell tissues of a liver model using human hepatocarcinoma cells (HepG2 cells), and confirmed that liver-specific functions were upregulated when composite tissues were formed using collagen microparticles prepared with several different stabilization protocols by glutaraldehyde, genipin, and methyl acetate). The process presented would be highly useful in enabling one-step production of thick cellular constructs in which porosity and morphology are tunable.
  • Masumi Yamada, Wataru Seko, Takuma Yanai, Kasumi Ninomiya, Minoru Seki
    Lab on a Chip 17(2) 304-314 2017年1月21日  査読有り筆頭著者責任著者
    Hydrodynamic microfluidic platforms have been proven to be useful and versatile for precisely sorting particles/cells based on their physicochemical properties. In this study, we demonstrate that a simple lattice-shaped microfluidic pattern can work as a virtual sieve for size-dependent continuous particle sorting. The lattice is composed of two types of microchannels (“main channels” and “separation channels”). These channels cross each other in a perpendicular fashion, and are slanted against the macroscopic flow direction. The difference in the densities of these channels generates an asymmetric flow distribution at each intersection. Smaller particles flow along the streamline, whereas larger particles are filtered and gradually separated from the stream, resulting in continuous particle sorting. We successfully sorted microparticles based on size with high accuracy, and clearly showed that geometric parameters, including the channel density and the slant angle, critically affect the sorting behaviors of particles. Leukocyte sorting and monocyte purification directly from diluted blood samples have been demonstrated as biomedical applications. The presented system for particle/cell sorting would become a simple but versatile unit operation in microfluidic apparatus for chemical/biological experiments and manipulations.
  • Sakurako Tomii, Masumi Yamada, Masahiro Mizuno, Yasuhiro Yamada, Takashi Kojima, Masahito Kushida, Minoru Seki
    RSC Advances 7(29) 17773-17780 2017年  査読有り責任著者
    Carbon nanomaterials assembled into micrometer-scale configurations are highly useful because of their unique molecular transport and adsorption properties, improved operability, and versatility in industrial/research applications. Here we propose a facile approach to assemble carbon nanotubes (CNTs) into monodisperse microparticles through rapid condensation and accumulation of CNTs in aqueous droplets in a non-equilibrium state. The droplets were generated by means of a microfluidic process or membrane emulsification, using a water-soluble polar organic solvent as the continuous phase. Because water molecules in the droplets were rapidly dissolved into the continuous phase, the CNTs were dramatically condensed and stable microparticles were finally formed. We prepared microparticles using both multi-walled and single-walled CNTs, and the size was controllable in the range of 10-40 μm simply by changing the initial CNT concentration. Interestingly, the morphologies of the particles were not spherical in many cases, and they were controllable by changing the type of the organic solvent and/or using additives in the dispersed phase. Physicochemical characterization suggested good compatibility of the CNT microparticles when used as supports for catalysts, adsorbents, and electrodes.
  • Keita Kinoshita, Masaki Iwase, Masumi Yamada, Yuya Yajima, Minoru Seki
    Biotechnology Journal 11(11) 1415-1423 2016年11月1日  査読有り責任著者
    Vascular tissues fabricated in vitro are useful tools for studying blood vessel-related cellular physiologies and for constructing relatively large 3D tissues. An efficient strategy for fabricating vascular tissue models with multilayered, branched, and thick structures through the in situ hydrogel formation in fluidic channels is proposed. First, an aqueous solution of RGD-alginate containing smooth muscle cells (SMCs) is introduced into channel structures made of agarose hydrogel, forming a cell-embedding Ca-alginate hydrogel layer with a thickness of several hundred micrometers on the channel surface because of the Ca ions diffused from the agarose hydrogel matrix. Next, endothelial cells (ECs) are introduced and cultured for up to seven days to form hierarchically organized, multilayered vascular tissues. The factors affecting the thickness of the Ca-alginate hydrogel layer, and prepared several types of microchannels with different morphologies are examined. The fabricated vascular tissue models are easily recovered from the channel by simply detaching the agarose hydrogel plates. In addition, the effect of O tension (20 or 80%) on the viability and elastin production of SMCs during the perfusion culture is evaluated. This technique would pave a new way for vascular tissue engineering because it enables the facile production of morphologically in vivo vascular tissue-like structures that can be employed for various biomedical applications. 2+ 2
  • Natsuki Nakajima, Masumi Yamada, Shunta Kakegawa, Minoru Seki
    Analytical Chemistry 88(11) 5637-5643 2016年6月7日  査読有り責任著者
    When analyzing the kinetics of liquid-liquid extraction (LLE), the change in the concentration of extracted target molecules over time should be monitored for a known interfacial area. Herein, we developed a microfluidic system for precisely analyzing the kinetics of LLE using droplets of a constant size even in the presence of additives. Extraction is initiated by exchanging the carrier fluid for the droplets with a target solution and then terminated by phase separation, based on the principle of hydrodynamic filtration. By using one out of several pairs of outlet/buffer inlet at a given time, the extraction time period is tuned stepwise without changing the flow rate condition. We successfully demonstrated droplet-based LLE by controlling the extraction period from ∼0.03 to ∼1.2 s and evaluated the extraction kinetics of rhodamine B from the continuous aqueous phase to droplets of 1-octanol with a diameter of ∼40 μm. In addition, the effect of additives on extraction efficiency was evaluated. The system presented in this study would be useful for determining rate constants for interfacial mass transfer in general LLE kinetic studies as well as for developing new extraction chemistries and optimizing microfluidic chemical/biochemical analysis systems that involve an LLE process.
  • Yumie Ichihara, Rie Utoh, Masumi Yamada, Tatsuya Shimizu, Yasuko Uchigata
    Heliyon 2(6) e00129 2016年6月1日  
    Pancreatic islets are heterogeneous clusters mainly composed of α and β cells, and these clusters range in diameter from 50 to several hundred micrometers. Native small islets are known to have a higher insulin secretion ability in vitro and to provide better transplantation outcomes when compared with large islets. In this study, we prepared microengineered pseudo-islets from dispersed rat islet cells using precisely-fabricated agarose gel-based microwells with different diameters (100, 300, or 500 μm) to investigate the function and survival of islet cell aggregates with well-controlled sizes. We observed that dead cells were rarely present in the small pseudo-islets with an average diameter of ∼60 μm prepared using 100 μm microwells. In contrast, we observed more dead cells in the larger pseudo-islets prepared using 300 and 500 μm microwells. The relative amount of hypoxic cells was significantly low in the small pseudo-islets whereas a hypoxic condition was present in the core region of the larger pseudo-islets. In addition, we found that the small-sized pseudo-islets reconstituted the in vivo-tissue like arrangement of the α and β cells, and restored the high insulin secretory capacity in response to high glucose. These results clearly suggest that precise size control of pseudo-islets is essential for maintaining islet cell function and survival in vitro. The small-sized pseudo-islets may be advantageous for providing a better therapeutic approach for treating type 1 diabetes mellitus via islet reorganization and transplantation.
  • Kosei Endo, Takahisa Anada, Masumi Yamada, Minoru Seki, Keiichi Sasaki, Osamu Suzuki
    Biomedical Materials (Bristol) 10(6) 065019-065019 2015年12月14日  
    The present study investigated whether alginate (Alg) hydrogel microbeads have a role in maintaining mouse bone marrow stromal ST-2 cells and release the cells after being stimulated by synthetic octacalcium phosphate (OCP), which is a mineral crystal capable of stimulating osteoblastic differentiation during a conversion process to hydroxyapatite (HA). The ST-2 cell suspension in the alginate solution, which contained various concentrations of OCP granules with diameters less than 53 μm, was extruded drop-wise into a stirred gelation solution containing BaCl using an encapsulator with nitrogen gas stream. The Alg-microbeads (Alg/OCP • ST-2 microbeads) that were generated, which had a diameter of approximately 400 μm, were incubated for up to 14 d and then assessed for osteoblastic differentiation. Alg-microbeads with cells were also incubated to identify the possible conversion from OCP to HA. Osteoblast differentiation markers in ST-2 cells, alkaline phosphatase (ALP) and collagen type I, were up-regulated in the presence of higher amounts of OCP. X-ray diffraction analysis and Fourier transform infrared spectroscopy confirmed that the OCP tended to convert to HA over time, suggesting that the OCP in Alg-microbeads interacts three-dimensionally with ST-2 cells and stimulates its osteoblastic differentiation. The release of ST-2 cells from the microbeads was also estimated. ST-2 cells were identified outside of the microbeads, although the cell number tended to decrease with increasing OCP. These results suggest that Alg/OCP microbeads could be used as a vehicle to activate osteoblastic cells and deliver them to sites where bone regeneration is needed. 2
  • Masumi Yamada, Ayaka Hori, Sari Sugaya, Yuya Yajima, Rie Utoh, Masayuki Yamato, Minoru Seki
    Lab on a Chip 15(19) 3941-3951 2015年8月12日  査読有り筆頭著者責任著者
    The reconstitution of extracellular matrix (ECM) components in three-dimensional (3D) cell culture environments with microscale precision is a challenging issue. ECM microparticles would potentially be useful as solid particulate scaffolds that can be incorporated into 3D cellular constructs, but technologies for transforming ECM proteins into cell-sized stable particles are currently lacking. Here, we describe new processes to produce highly condensed collagen microparticles by means of droplet microfluidics or membrane emulsification. Droplets of an aqueous solution of type I collagen were formed in a continuous phase of polar organic solvent followed by rapid dissolution of water molecules into the continuous phase because the droplets were in a non-equilibrium state. We obtained highly unique, disc-shaped condensed collagen microparticles with a final collagen concentration above 10% and examined factors affecting particle size and morphology. After testing the cell-adhesion properties on the collagen microparticles, composite multicellular spheroids comprising the particles and primary rat hepatocytes were formed using microfabricated hydrogel chambers. We found that the ratio of the cells and particles is critical in terms of improvement of hepatic functions in the composite spheroids. The presented methodology for incorporating particulate-form ECM components in multicellular spheroids would be advantageous because of the biochemical similarity with the microenvironments in vivo.
  • Masahiro Mizuno, Taro Toyota, Miki Konishi, Yoshiyuki Kageyama, Masumi Yamada, Minoru Seki
    Langmuir 31(8) 2334-2341 2015年3月3日  査読有り責任著者
    A new microfluidic process was used to generate unique micrometer-sized hierarchical lipid particles having spherical lipid-core and multilamellar-shell structures. The process includes three steps: (1) formation of monodisperse droplets in a nonequilibrium state at a microchannel confluence, using a phospholipid-containing water-soluble organic solvent as the dispersed phase and water as the continuous phase; (2) dissolution of the organic solvent of the droplet into the continuous phase and concentration of the lipid molecules; and (3) reconstitution of multilamellar lipid membranes and simultaneous formation of a lipid core. We demonstrated control of the lipid particle size by the process conditions and characterized the obtained particles by transmission electron microscopy and microbeam small-angle X-ray scattering analysis. In addition, we prepared various types of core-shell and core-core-shell particles incorporating hydrophobic/hydrophilic compounds, showing the applicability of the presented process to the production of drug-encapsulating lipid particles.
  • 山田 真澄, 関 実
    膜 40(3) 137-142 2015年  
    Microfluidic technologies are capable of producing small materials such as particles, fibers, and sheets with sizes of several hundred micrometers. In this paper, we briefly introduce our recent progresses on the fabrication of hydrogel materials for tissue engineering using microfluidic devices. The first topic is the production of thin, stripepatterned sheets made of alginate hydrogel, having soft and solid regions. These functional hydrogel sheets were used to generate heterotypic cellular constructs composed of hepatocytes and non-parenchymal cells, for the purpose of upregulating hepatic functions. The second topic is the formation of vascular–tissue like tubular structures composed of alginate hydrogel and two types of cells, within hydrogel microchannels made of agarose hydrogel. These studies are good examples of the application of microfluidic devices to the production of thin hydrogel materials that are potentially useful for various biological research and tissue engineering.
  • Yuya Yajima, Masumi Yamada, Emi Yamada, Masaki Iwase, Minoru Seki
    Biomicrofluidics 8(2) 024115-024115 2014年3月1日  査読有り責任著者
    We present facile strategies for the fabrication of two types of microfluidic devices made of hydrogels using the natural biopolymers, alginate, and gelatin as substrates. The processes presented include the molding-based preparation of hydrogel plates and their chemical bonding. To prepare calcium-alginate hydrogel microdevices, we suppressed the volume shrinkage of the alginate solution during gelation using propylene glycol alginate in the precursor solution along with sodium alginate. In addition, a chemical bonding method was developed using a polyelectrolyte membrane of poly-L-lysine as the electrostatic glue. To prepare gelatin-based microdevices, we used microbial transglutaminase to bond hydrogel plates chemically and to cross-link and stabilize the hydrogel matrix. As an application, mammalian cells (fibroblasts and vascular endothelial cells) were cultivated on the microchannel surface to form three-dimensional capillary-embedding tissue models for biological research and tissue engineering.
  • Tsubasa Ono, Masumi Yamada, Yusuke Suzuki, Tatsuo Taniguchi, Minoru Seki
    RSC Advances 4(26) 13557-13564 2014年  査読有り責任著者
    A simple but versatile microfluidic process is presented for the production of monodisperse polymeric microparticles using non-equilibrium droplets. Oil-in-water (o/w) droplets were formed within microchannels by means of a water-soluble polar organic solvent containing polymer molecules as a dispersed phase. The droplets of solvent were rapidly dissolved into the continuous phase during flow through the microchannel, whereas water-insoluble polymers were precipitated to form monodisperse polymeric particles. In this way, we successfully synthesized particles with sizes significantly smaller than that of the initial droplets, using polystyrene and poly(methyl methacrylate) as the polymer molecules and typically ethyl acetate as the polar solvent. Particles obtained under several different conditions exhibited unique non-spherical morphologies, which were caused by the translocation and segregation of the precipitated polymer and diminishing solvent-rich droplet. We confirmed that various factors influence the particle morphology, including the polymer concentration, molecular weight of the polymer, type of the polar solvent, and the presence of additives in the dispersed phase. This journal is © the Partner Organisations 2014.
  • Yoichi Kitagawa, Yoji Naganuma, Yuya Yajima, Masumi Yamada, Minoru Seki
    Biofabrication 6(3) 035011-035011 2014年  査読有り責任著者
    Multilayered microfluidic devices with a micronozzle array structure have been developed to prepare unique hydrogel microfibers with highly complex cross-sectional morphologies. Hydrogel precursor solutions with different compositions are introduced through vertical micronozzles, united and focused, and continuously gelled to form hydrogel fibers with multiple regions of different physicochemical composition. We prepared alginate hydrogel microfibers with diameters of 60 ∼ 130 μm and 4/8 parallel regions in the periphery. Neuron-like PC12 cells encapsulated in the parallel region, which was made of a soft hydrogel matrix, proliferated and formed linear intercellular networks along the fiber length because of the physical restrictions imposed by the relatively rigid regions. After cultivation for 14 days, one-millimeter-long intercellular networks that structurally mimic complex nerve bundles found in vivo were formed. The proposed fibers should be useful for producing various in vivo linear tissues and should be applicable to regenerative medicine and physiological studies of cells.
  • Aoi Kobayashi, Kenta Yamakoshi, Yuya Yajima, Rie Utoh, Masumi Yamada, Minoru Seki
    Journal of Bioscience and Bioengineering 116(6) 761-767 2013年12月  査読有り責任著者
    Here we demonstrate the production of stripe-patterned heterogeneous hydrogel sheets for the high-density 3D coculture of multiple cell types, by using microchannel-combined micronozzle devices. The prepared hydrogel sheet, composed of multiple regions with varying physical stiffness, regulates the direction of proliferation of encapsulated cells and enables the formation of arrays of rod-like heterotypic organoids inside the hydrogel matrix. We successfully prepared stripe-patterned hydrogel sheets with a uniform thickness of ~100μm and a width of several millimeters. Hepatoma cells (HepG2) and fibroblasts (Swiss 3T3) were embedded inside the hydrogel matrix and cocultured, to form heterotypic micro-organoids mimicking in vivo hepatic cord structures. The upregulation of hepatic functions by the 3D coculture was confirmed by analyzing liver-specific functions. The presented heterogeneous hydrogel sheet could be useful, as it provides relatively large, but precisely-controlled, 3-dimensional microenvironments for the high-density coculture of multiple types of cells. © 2013 The Society for Biotechnology, Japan.
  • Sari Sugaya, Masumi Yamada, Ayaka Hori, Minoru Seki
    Biomicrofluidics 7(5) 54120-54120 2013年9月6日  査読有り責任著者
    In this study, a microfluidic process is proposed for preparing monodisperse micrometer-sized hydrogel beads. This process utilizes non-equilibrium aqueous droplets formed in a polar organic solvent. The water-in-oil droplets of the hydrogel precursor rapidly shrunk owing to the dissolution of water molecules into the continuous phase. The shrunken and condensed droplets were then gelled, resulting in the formation of hydrogel microbeads with sizes significantly smaller than the initial droplet size. This study employed methyl acetate as the polar organic solvent, which can dissolve water at 8%. Two types of monodisperse hydrogel beads-Ca-alginate and chitosan-with sizes of 6-10 μm (coefficient of variation < 6%) were successfully produced. In addition, we obtained hydrogel beads with non-spherical morphologies by controlling the degree of droplet shrinkage at the time of gelation and by adjusting the concentration of the gelation agent. Furthermore, the encapsulation and concentration of DNA molecules within the hydrogel beads were demonstrated. The process presented in this study has great potential to produce small and highly concentrated hydrogel beads that are difficult to obtain by using conventional microfluidic processes. © 2013 AIP Publishing LLC.
  • Masahiro Mizuno, Masumi Yamada, Ryusuke Mitamura, Kohei Ike, Kaori Toyama, Minoru Seki
    Analytical Chemistry 85(16) 7666-7673 2013年8月20日  査読有り責任著者
    A simple microfluidic system has been presented to perform continuous two-parameter cell sorting based on size and surface markers. Immunomagnetic bead-conjugated cells are initially sorted based on size by utilizing the hydrodynamic filtration (HDF) scheme, introduced into individual separation lanes, and simultaneously focused onto one sidewall by the hydrodynamic effect. Cells are then subjected to magnetophoretic separation in the lateral direction, and finally they are individually recovered through multiple outlet branches. We successfully demonstrated the continuous sorting of JM (human lymphocyte cell line) cells using anti-CD4 immunomagnetic beads and confirmed that accurate size-and surface marker-based sorting was achieved. In addition, the sorting of cell mixtures was performed at purification ratios higher than 90%. The proposed system enables two-dimensional cell sorting without necessitating complicated setups and operations, and thus, it can be a useful tool for general biological experiments including cell-based disease diagnosis, stem cell engineering, and cellular physiological studies. © 2013 American Chemical Society.
  • Ayaki Miyama, Masumi Yamada, Sari Sugaya, Minoru Seki
    RSC Advances 3(30) 12299-12306 2013年8月14日  責任著者
    A simple microfluidic process for producing yarn-ball-shaped hydrogel microbeads was devised. The mechanism employed parallel flows of aqueous solutions of a hydrogel precursor, a gelation agent, and a buffer to generate incompletely gelled alginate hydrogel microfibers. Water-in-oil droplets were generated simultaneously to fragment the fibers and fold the fragments into a yarn-ball-like shape. Alginate beads with an average outer diameter of ∼200 μm and fiber width of 10-30 μm were fabricated, which had a relatively large void volume and a high surface-to-volume ratio, enabling the efficient transport of molecules in and out of the hydrogel matrix. It was found that the concentration of the gelation agent was critical for obtaining the yarn-ball-shaped beads. In addition, to test the feasibility of the beads for use in bioencapsulation, mammalian cells were encapsulated within the hydrogel matrices of the beads at high densities, and the proliferation ability of the cells was investigated by changing the precursor concentration. The fabricated hydrogel beads should be useful as a new material for bioimmobilization and bioencapsulation and could find applications in cell transplantation therapies. © The Royal Society of Chemistry 2013.
  • Kenichi Ohara, Masaru Kojima, Akira Fukushima, Shun Onozaki, Mitsuhiro Horade, Masumi Yamada, Minoru Seki, Yasushi Mae, Tatsuo Arai
    Journal of Robotics and Mechatronics 25(4) 665-672 2013年6月  
    The construction of 3D tissue is an important issue in regenerative medicine. While small 3D tissue has been realized in vitro, large functional 3D tissue has not been achieved due to difficulties in supplying oxygen and nutrients to the tissue. In this paper, we propose automated 3D construction based on hydrogel fiber to produce active 3D tissues. Results of several preliminary experiments in generating suitable hydrogel fibers have resulted in a lattice structure as an example of 3D tissue. In experiments in 3D structure construction, we observed cell growth in the constructed lattice confirming structure functionality.
  • Masaki Iwase, Masumi Yamada, Emi Yamada, Minoru Seki
    Journal of Robotics and Mechatronics 25(4) 682-689 2013年6月  責任著者
    This paper presents a fabrication process for cell aggregates with controlled shapes that can be used as building units for constructing relatively large tissue models. Microfabricated hydrogel-based chambers with non-adhesive surface characteristics were prepared via a micromolding process. Alginate was used as the hydrogel matrix, which facilitated the efficient formation of aggregates from cells retained inside the microchamber. We employed several types of toroidal and lattice-shaped hydrogel microchambers with different geometries. We examined the effect of cell type on the aggregate formation process using NIH-3T3, C2C12, and HepG2 cells and clearly observed that aggregation behavior is highly dependent on cell type. In addition, we tried to construct 2-layered capillarylike tissues by stacking heterotypic toroidal cell aggregates, which mimic blood vessels. The presented cell aggregate-based tissue fabrication process could become a versatile approach for preparing complex and scaffold-free 3D tissue models.
  • Tomoki Morijiri, Masumi Yamada, Toshikatsu Hikida, Minoru Seki
    Microfluidics and Nanofluidics 14(6) 1049-1057 2013年6月  査読有り
    We present a centrifugal microfluidic system for precise cell/particle sorting using the concept of counterflow centrifugal elutriation (CCE). A conventional CCE system uses a rotor device incorporating a flow-through separation chamber, in which the balance of centrifugal and counterflow drag forces exerted on particles is gradually shifted by changing the flow rate and/or the rotation speed. In the present system, both the centrifugal and the fluid forces are generated through microdevice rotation in order to significantly simplify the setup of the conventional CCE. In addition, the density gradient of the medium is employed to elute particles/cells of different sedimentation velocities stepwise from the separation chamber instead of changing the rotation speed. We successfully separated polymer particles with diameters of 1.0-5.0 μm using a branched loading channel for focusing particles to the center of the separation chamber. We also demonstrated the sorting of blood cells for biological applications. This system may provide a versatile means for cell/particle sorting in a general biological laboratory and function as a unit operation in various centrifugal microfluidic platforms for biochemical experiments and clinical diagnosis. © 2012 Springer-Verlag Berlin Heidelberg.
  • Masumi Yamada, Rie Utoh, Kazuo Ohashi, Kohei Tatsumi, Masayuki Yamato, Teruo Okano, Minoru Seki
    Biomaterials 33(33) 8304-8315 2012年11月  査読有り
    We have developed a hydrogel-based cell cultivation platform for forming 3D restiform hepatic micro-organoids consisting of primary rat hepatocytes and feeder cells (Swiss 3T3 cells). Sodium alginate solutions containing hepatocytes/3T3 cells were continuously introduced into a microfluidic channel to produce cell-incorporating anisotropic Ba-alginate hydrogel microfibers, where hepatocytes at the center were closely sandwiched by 3T3 cells. Hydrogel fiber-based cultivation under high oxygen tension enabled the formation of heterotypic micro-organoids with a length of up to 1 mm and a diameter of ~50 μm, mimicking the hepatic cord structures found in the liver, while maintaining a high hepatocyte viability (~80%) over 30 days. Long-term observation of up to 90 days revealed a significant enhancement of hepatic functions because of heterotypic and homotypic cell-cell interactions, including albumin secretion and urea synthesis as well as expression of hepatocyte-specific genes, compared with conventional monolayer culture and single cultivation in the hydrogel fibers. The encapsulated hepatic constructs were recovered as scaffold-free micro-organoids by enzymatically digesting the hydrogel matrices using alginate lyase. This technique for creating heterotypic micro-organoids with precisely ordered multiple cell types will be useful for the development of a new liver tissue engineering approach and may be applicable to the fabrication of extracorporeal bioartificial liver (BAL) devices and assessment tools for drug development and testing. © 2012 Elsevier Ltd.
  • Sari Sugaya, Shunta Kakegawa, Shizuka Fukushima, Masumi Yamada, Minoru Seki
    Langmuir 28(39) 14073-14080 2012年10月2日  査読有り責任著者
    This study presents a simple but highly versatile method of fabricating picoliter-volume hydrogel patterns on poly(dimethylsiloxane) (PDMS) substrates. Hydrophilic regions were prepared on hydrophobic PDMS plates by trapping and melting functional polymer particles and performing subsequent reactions with partially oxidized dextran. Small aliquots of a gelation solution were selectively trapped on the hydrophilic areas by a simple dipping process that was utilized to make thin hydrogel patterns by the in situ gelation of a sol solution. Using this process, we successfully formed calcium alginate, collagen I, and chitosan hydrogels with a thickness of several micrometers and shapes that followed the hydrophilized regions. In addition, alginate and collagen gel patterns were used to capture cells with different adhesion properties selectively on or off the hydrogel structures. The presented strategy could be applicable to the preparation of a variety of hydrogels for the development of functional biosensors, bioreactors, and cell cultivation platforms. © 2012 American Chemical Society.
  • Kaori Toyama, Masumi Yamada, Minoru Seki
    Biomedical Microdevices 14(4) 751-757 2012年8月  査読有り責任著者
    Separation/purification of nuclei from cells is a critical process required for medical and biochemical research applications. Here, we report a flow-through microfluidic device for isolating cell nuclei by selectively digesting the cell membrane by using the concept of hydrodynamic filtration (HDF). When a cell suspension is continuously introduced into a microchannel (main channel) possessing multiple side channels, cells flow through the main channel, whereas the carrier medium of the cells is drained through the side channels. Introductions of a cell treatment solution containing a surfactant and a washing buffer enable the two-step exchange of the carrier-medium and the cell treatment by the surfactant for a short span of time. The precise control of the treatment time by changing the flow rate and/or the size of the microchannel enables the selective digestion of cell membranes, resulting in the isolation of cell nuclei after separation from membrane debris and cytoplasmic components according to size. We examined several surfactant molecules and demonstrated that Triton X-100 exhibited high efficiency regarding nucleus isolation for both adherent (HeLa) and nonadherent (JM) cells, with a recovery ratio of ~80 %. In addition, the isolation efficiency was evaluated by western blotting. The presented flow-through microfluidic cell-nucleus separator may be a useful tool for general biological applications, because of its simplicity in operation, high reproducibility, and accuracy. © 2012 Springer Science+Business Media, LLC.
  • Masumi Yamada, Sari Sugaya, Yoji Naganuma, Minoru Seki
    Soft Matter 8(11) 3122-3130 2012年3月21日  査読有り筆頭著者責任著者
    Hydrogel materials with microscale heterogeneity are of great interest in the effort to spatially control cellular microenvironments in tissue engineering applications. Here we present a microfluidic system to continuously synthesize chemically and physically anisotropic Ca-alginate hydrogel microfibers enabling the guidance of cell proliferation to form linear cell colonies and intracellular networks. The microfluidic gelation process involves 2 critical steps to obtain alginate microfibers using axisymmetric microchannels with uniform depth: introduction of a buffer solution between the sodium alginate (NaA) and CaCl solutions to modulate the gelation speed, and use of a thickener to balance the viscosities of the solutions. We synthesized hydrogel fibers with diameters of ∼7 to 200 μm, maintaining the anisotropy in the cross-section, and examined factors affecting the fiber diameter and uniformity. Moreover, parallel alginate flows with and without propylene glycol alginate (PGA) enabled the formation of sandwich-type solid-soft-solid hydrogel fibers, which were used to guide the direction of growth of cells inoculated in the soft-core, with the help of outer polycation membranes made of poly-l-lysine. We demonstrated the formation of linear colonies of 3T3 and HeLa cells inside the anisotropic fiber and observed elongated nuclei along the fiber direction. In addition, the heterogeneous morphology of the fiber was utilized to guide neurite elongation and generate cellular networks by using neuron-like PC12 cells. The hydrogel fibers reported here can be used as an innovative tool for investigating cell and tissue morphogenesis in heterogeneous microenvironments, and for creating tissue models with precise control of cellular alignment and elongation. © 2012 The Royal Society of Chemistry. 2
  • Tatsuru Moritani, Masumi Yamada, Minoru Seki
    Microfluidics and Nanofluidics 11(5) 601-610 2011年11月  査読有り
    A microfluidic system is presented to generate multiple daughter droplets from a mother droplet, by the multistep hydrodynamic division of the mother droplet at multiple branch points in a microchannel. A microchannel network designed based on the resistive circuit model enables us to control the distribution ratio of the flow rate, which dominates the division ratios of the mother droplets. We successfully generated up to 15 daughter droplets from a mother droplet with a variation in diameter of less than 2%. In addition, we examined factors affecting the division ratio, including the average fluid velocity, interfacial tension, fluid viscosity, and the distribution ratio of volumetric flow rates at a branch point. Additionally, we actively controlled the volume of the mother droplets and examined its influence on the size of the daughter droplets, demonstrating that the size of the daughter droplets was not significantly influenced by the volume of the mother droplet when the distribution ratio was properly controlled. The presented system for controlling droplet division would be available as an innovative means for preparing monodisperse emulsions from polydisperse emulsions, as well as a technique for making a microfluidic dispenser for digital microfluidics to analyze the droplet compositions. © Springer-Verlag 2011.
  • Manami Masubuchi, Taro Toyota, Masumi Yamada, Minoru Seki
    Chemical Communications 47(29) 8433-8435 2011年8月7日  査読有り
    Molecular self-assemblies exhibiting automatic motions have received much attention as potential artificial models of living organisms. We have developed a microfluidic picolitre nozzle-array device to form multilamellar lipid tubes (MLTs) under fluidic shear stress, which transformed into different two patterns (yarn-balls and double-helixes) and also exhibited unique self-actuation behaviors. © 2011 The Royal Society of Chemistry.
  • Tomoki Morijiri, Satoshi Sunahiro, Masashi Senaha, Masumi Yamada, Minoru Seki
    Microfluidics and Nanofluidics 11(1) 105-110 2011年7月  査読有り
    A simple and efficient device for density-based particle sorting is in high demand for the purification of specific cells, bacterium, or environmental particles for medical, biochemical, and industrial applications. Here we present microfluidic systems to achieve size- and densitybased particle separation by adopting the sedimentation effect for a size-based particle sorting technique utilizing microscale hydrodynamics, called "pinched-flow fractionation (PFF)." Two schemes are presented: (a) the particle inertia scheme, which utilizes the inertial force of particle movement induced by the momentum change in the curved microchannel, and (b) the device rotation scheme, in which rotation of the microdevice exerts centrifugal force on the flowing particles. In the experiments, we successfully demonstrated continuous sorting of microparticles according to size and density by using these two schemes, and showed that the observed particle movements were in good agreement with the theoretical estimations. The presented schemes could potentially become one of the functional components for integrated bioanalysis systems that can manipulate/separate small amount of precious biological samples. © Springer-Verlag 2011.

MISC

 159
  • 山田真澄
    化学とマイクロ・ナノシステム学会誌 19(2) 42-43 2020年9月  筆頭著者最終著者責任著者
  • 山田真澄
    ぶんせき 341-341 2020年9月  筆頭著者責任著者
  • Hideki Iwadate, Naoki Kimura, Rina Hashimoto, Yuya Yajima, Rie Utoh, Masumi Yamada, Minoru Seki
    21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017 177-178 2020年  
    A new process is presented to fabricate basement membrane-mimetic cell co-culture platforms using microfabricated PDMS stencil plates. An aqueous solution of extracellular matrices (ECM) was introduced into the through-holes of the microstencil plate and then dried, to form vitrified and condensed ECM films. Because the microstencil plate physically supported the films, we were able to produce significantly thin ECM films with a thickness of 0.5-2 μm, which is comparable to the basement membranes in vivo (< 1 μm). We co-cultured heterotypic cells on both sides of the film, demonstrating the usability of the presented platform in investigating cellular physiology.
  • Mayu Fukushi, Yuya Yajima, Rie Utoh, Masumi Yamada, Kazuya Furusawa, Minoru Seki
    21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017 1145-1146 2020年  
    Here we present a new approach to rapid formation of collagen gel tubes using microfluidic devices made of phosphate particle-embedding PDMS (PP-PDMS). An acidic solution of collagen, introduced into the PP-PDMS channel, was rapidly transformed into a hydrogel layer on the channel surface because it was neutralized by the phosphate ions supplied from the PP-PDMS substrate. After removing the non-gelled collagen solution at the center, collagen gel tubes were obtained. Based on this concept, we successfully fabricated vascular tissue-like multilayered structures incorporating vascular endothelial cells (ECs) and smooth muscle cells (SMCs). The presented technique is highly useful for preparing functional tubular tissues that reconstituted the in vivo tissue-like extracellular matrix environments.
  • Natsumi Shimmyo, Makoto Furuhata, Masumi Yamada, Rie Utoh, Minoru Seki
    MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences 1155-1156 2020年  
    Here we present an efficient microfluidic system for selectively capturing circulating tumor cells (CTCs) from blood samples, employing parallelized structures of cell trappers. The thin configuration of the trapper (depth of several micrometers) enables capture of CTCs based on physical characteristics; CTCs are larger and less-deformable than other blood cells. Parallelization of the cell trappers achieved high-throughput processing of the blood sample (~100 µL/min) and a high capture efficiency (~90%) of CTCs despite the simple configuration of the trapper. The presented system would be applicable to easy, low-cost, and highly sensitive cancer diagnosis for tumor patients.

書籍等出版物

 5

共同研究・競争的資金等の研究課題

 22

産業財産権

 22