血中循環腫瘍細胞（CTC）を無染色で識別し、分取する装置の技術開発

• Project/Area Number: 17K18180
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: General medical chemistry; Medical Physics and Radiological Technology
• Research Institution: Waseda University
• Principal Investigator: 尾高 正朗 早稲田大学, 重点領域研究機構, 研究助手 (90748948)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Project Status: Discontinued (Fiscal Year 2019)
• Budget Amount: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2020: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000); Fiscal Year 2019: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000); Fiscal Year 2018: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2017: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
• Keywords: 血中循環腫瘍細胞 / イメージング・セルソーター / アルギン酸ナトリウムマイクロカプセル / 血中循環腫瘍細胞（CTC） / 微細流路 / 単一細胞 / 細胞塊 / 画像認識 / 細胞ゲル包埋 / 細胞分取装置 / 微細加工流路

Outline of Annual Research Achievements

血中循環腫瘍細胞（CTCs）を無染色で識別し、分取する装置の技術研究開発を行った。従来のセルソーターの光散乱強度計測や蛍光強度計測などでは取得できない、画像情報から得られる「かたち」情報に基づいて細胞を選別する「マルチイメージング・セルソーター装置の技術の開発」によってリアルタイムで細胞・細胞塊を認識し、分取する技術の研究開発を試みた。
2019年度までに、装置の主要構成パーツをあらためて環境整備し、最適化に成功し、さらに血液検体には標的の細胞より小さいサイズの赤血球が大量に含まれるのでこれを取り除くことを目的とした前処理技術として微細加工技術を駆使し、網目のある階段状に柱状構造を施した流路を使って大きさの異なる粒子を分離する手法に成功した。
また、細胞の分取過程において、緩衝液条件や直接的な電気信号による細胞傷害性が高く、回収できた細胞の生存率や活性度が低かったが、これを改善するためアルギン酸ナトリウムマイクロカプセル包埋技術を確立し、検体の細胞をカプセル化して回収する技術を確立した。
これまでに成功していたマルチイメージング・セルソーターに組み合わせ、血液検体に含まれる細胞・細胞塊の画像情報をリアルタイム処理し、その画像情報を利用して、より低傷害で標的とした細胞・細胞塊を回収する技術を確立できた。
最終年度、がん細胞転移モデル動物の血液を使って、野生型血液で存在しない末梢血中を流れるCTCsを「かたち」情報に基づいて回収し、これまで困難であったCTCsのリアルタイム解析の技術の有効性を検証し、特徴づけし、回収できた細胞や細胞塊の特性評価、既知バイオマーカーとの比較評価、従来の上皮細胞分子マーカーでは判定不能であったCTCsの上皮間葉転換(EMT)仮説についての生物学的観点からの機構の解明を進める予定していたが、所属組織の研究プロジェクトの早期終了に伴い、研究課題は廃止となった。

Research Products

• [Int'l Joint Research] 民間企業(シンガポール)
• [Journal Article] Size Distribution Analysis with On-Chip Multi-Imaging Cell Sorter for Unlabeled Identification of Circulating Tumor Cells in Blood (2019)
  • Author(s): Odaka Masao、Kim Hyonchol、Nakamura Yoshiyasu、Hattori Akihiro、Matsuura Kenji、Iwamura Moe、Miyagi Yohei、Yasuda Kenji
  • Journal Title: Micromachines Volume: 10 Issue: 2 Pages: 154-154
  • DOI: 10.3390/mi10020154
  • Peer Reviewed / Open Access
• [Journal Article] On-chip spatiotemporal electrophysiological analysis of human stem cell derived cardiomyocytes enables quantitative assessment of proarrhythmia in drug development (2018)
  • Author(s): Asahi Yumiko、Hamada Tomoyo、Hattori Akihiro、Matsuura Kenji、Odaka Masao、Nomura Fumimasa、Kaneko Tomoyuki、Abe Yasuyuki、Takasuna Kiyoshi、Sanbuissho Atsushi、Yasuda Kenji
  • Journal Title: Scientific Reports Volume: 8 Issue: 1 Pages: 14536-14536
  • DOI: 10.1038/s41598-018-32921-1
  • Peer Reviewed / Open Access
• [Journal Article] Selective digestion of Ba2+/Ca2+ alginate gel microdroplets for single-cell handling (2018)
  • Author(s): Odaka Masao、Hattori Akihiro、Matsuura Kenji、Yasuda Kenji
  • Journal Title: Japanese Journal of Applied Physics Volume: 57 Issue: 6S1 Pages: 06HH02-06HH02
  • DOI: 10.7567/jjap.57.06hh02
  • Peer Reviewed / Open Access
• [Presentation] Geometrical understanding of the local fluctuation in propagation of excitation conduction in cardiomyocyte network (2019)
  • Author(s): 青木 肖太, 坂本 一史, 本堂 義常, 服部 明弘, 尾高 正朗, 安田 賢二
  • Organizer: 第57回日本生物物理学会年会
• [Presentation] Observation of direction-dependent asymmetrical propagation velocities in excitation conduction in a same cardiomyocyte networks on a chip (2019)
  • Author(s): 坂本 一史, 青木 肖太, 本堂 義常, 尾高 正朗, 服部 明弘, 安田 賢二
  • Organizer: 第57回日本生物物理学会年会
• [Presentation] On-chip differential analysis of sequential phagocytosis on identical position of single macrophages (2019)
  • Author(s): 古本 悠也, 吾妻 利紀, 吉田 周, 北原 貴太, 坂口 友康, 尾高 正朗, 服部 明弘, 安田 賢二
  • Organizer: 第57回日本生物物理学会年会
• [Presentation] Topology of three-dimensional capillary structure determines blood vein sheet extension behavior (2019)
  • Author(s): 飯田 健斗, 橋本 広道, 尾高 正朗, 服部 明弘, 安田 賢二
  • Organizer: 第57回日本生物物理学会年会
• [Presentation] Behavior of single vascular endothelial cells in 2D structures (2019)
  • Author(s): 橋本 広道, 山中 悠希, 高野 隆治, 飯田 健斗, 尾高 正朗, 松浦 賢志, 服部 明弘, 安田 賢二
  • Organizer: 第57回日本生物物理学会年会
• [Presentation] Interactions of neurites elongated from isolated hippocampal cells in agarose width-length-controlled microchannels. (2019)
  • Author(s): 田中 悠平, 青木 肖太, 渡部 治樹, 下田 賢司, 服部 明弘, 尾高 正朗, 安田 賢二
  • Organizer: 第57回日本生物物理学会年会
• [Presentation] Precise μm agarose microfabrication technology with spot absorption microneedle for single neurite elongation property measurement (2019)
  • Author(s): 渡部 治樹, 田中 悠平, 青木 肖太, 下田 賢司, 菊池 隆仁, 服部 明弘, 尾高 正朗, 安田 賢二
  • Organizer: 第57回日本生物物理学会年会
• [Presentation] Examination of backtracking engulfment mechainism in macrophages using on-chip single cell observation assay (2019)
  • Author(s): 吉田 周, 古本 悠也, 吾妻 利紀, 北原 貴太, 坂口 友康, 尾高 正朗, 服部 明弘, 安田 賢二
  • Organizer: 第57回日本生物物理学会年会
• [Presentation] Extra volume increase of single macrophage during sequential phagocytosis occurred by using micropipette aspiration measurement assay (2019)
  • Author(s): 吾妻 利紀, 古本 悠也, 吉田 周, 尾高 正朗, 服部 明弘, 坂口 友康, 北原 貴太, 安田 賢二
  • Organizer: 第57回日本生物物理学会年会
• [Presentation] Size distribution analysis of circulating tumor cell clusters in blood using on-chip multi-imaging flow cytometry (2019)
  • Author(s): 尾高 正朗, 服部 明弘, 安田 賢二
  • Organizer: 第57回日本生物物理学会年会
• [Presentation] Direct observation of blood vein formation dynamics exploiting flexible three-dimensional gelatin-gel microfabrication technology (2019)
  • Author(s): 飯田健斗、山中悠希、橋本広道、高野隆治、尾高正朗、服部明弘、松浦賢志、安田賢二
  • Organizer: CREST「生命動態の理解と制御のための基盤技術の創出」研究領域 第7回領域会議
• [Presentation] Development of sprouting vascular endothelial cell collection method using flexible design of Matrigel for expression analysis (2019)
  • Author(s): 山中悠希、飯田健斗、高野隆治、橋本広道、尾高正朗、服部明弘、松浦賢志、安田賢二
  • Organizer: CREST「生命動態の理解と制御のための基盤技術の創出」研究領域 第7回領域会議
• [Presentation] Community effect of cardiomyocytes in synchronous behavior of beating by constructing cell cluster (1): Experimental approach (2018)
  • Author(s): 高橋直樹、山下晶洸、坂本一史、尾高正朗、服部明弘、松浦賢志、安田賢二
  • Organizer: 第56回日本生物物理学会年会
• [Presentation] A 1064/1480-nm photo-thermal etching system with fiber optics for an accurate and non-invasive micropatterning of an agarose thin layer (2018)
  • Author(s): 菊池隆仁、青木肖太、田中悠平、尾高正朗、服部明弘、松浦賢志、安田賢二?
  • Organizer: 第56回日本生物物理学会年会
• [Presentation] Extracellular field potential change analysis of spontaneous firing of an isolated neuron by an on-chip multi-electrode array system. (2018)
  • Author(s): 青木肖太、菊池隆仁、田中悠平、松浦賢志、服部明弘、尾高正朗、安田賢二
  • Organizer: 第56回日本生物物理学会年会
• [Presentation] Repulsive interactions of two neurites elongated from two isolated hippocampal cells in agarose width-length-controlled microchannels. (2018)
  • Author(s): 田中悠平、菊池隆仁、青木肖太、服部明弘、松浦賢志、尾高正朗、安田賢二
  • Organizer: 第56回日本生物物理学会年会
• [Presentation] Development of free-flow assay for precise evaluation of phagocytosis efficiency of macrophages. (2018)
  • Author(s): 古本悠也、中田吉紀、吾妻利紀、吉田周、尾高正朗、服部明弘、松浦賢志、安田賢二
  • Organizer: 第56回日本生物物理学会年会
• [Presentation] Hysteresis of single point sequential phagocytoses in macrophages using on-chip single cell measurement assay (2018)
  • Author(s): 吾妻利紀、中田吉紀、古本悠也、吉田周、服部明弘、松浦賢志、尾高正朗、安田賢二
  • Organizer: 第56回日本生物物理学会年会
• [Presentation] Identifying the maximum size of phagocytosis in macrophages using on-chip single cell measurement assay (2018)
  • Author(s): 吉田周、中田吉紀、古本悠也、吾妻利紀、服部明弘、松浦賢志、尾高正朗、安田賢二
  • Organizer: 第56回日本生物物理学会年会
• [Presentation] Evaluation of photo-thermal three-dimensional gelatin-gel microfabrication technology for clarification of endothelial cells’ dynamics (2018)
  • Author(s): 橋本広道、飯田健斗、山中悠希、高野隆治、尾高正朗、松浦賢志、服部明弘、安田賢二
  • Organizer: 第56回日本生物物理学会年会
• [Presentation] Analysis of neglecting phase in phagocytosis of macrophages using on-chip sequential single-point phagocytoses measurement assay (2018)
  • Author(s): 中田吉紀、古本悠也、吾妻利紀、吉田周、尾高正朗、松浦賢志、服部明弘、安田賢二
  • Organizer: 第56回日本生物物理学会年会
• [Presentation] Direct observation of blood vein formation dynamics exploiting flexible three-dimensional gelatin-gel microfabrication technology (2018)
  • Author(s): 飯田健斗、山中悠希、橋本広道、高野隆治、尾高正朗、服部明弘、松浦賢志、安田賢二
  • Organizer: 第56回日本生物物理学会年会
• [Presentation] Development of a method to track conductions in cardiomyocyte network with a multi-electrode system (2018)
  • Author(s): 坂本一史、関菜月、青木肖太、高橋直樹、尾高正朗、松浦賢志、服部明弘、安田賢二
  • Organizer: 第56回日本生物物理学会年会
• [Presentation] Quantitative evaluation of preciseness in design copy in microfabrication procedures of circulating tumor cell cluster size-filtration (2018)
  • Author(s): 川合絢子、岩村萌絵、松浦賢志、服部明弘、尾高正朗、安田賢二
  • Organizer: 第56回日本生物物理学会年会
• [Presentation] Development of size filtration-imaging cell sorter for real time selective collection of circulating tumor cells (CTCs) in blood (2018)
  • Author(s): 岩村萌絵、松浦賢志、川合絢子、尾高正朗、服部明弘、安田賢二
  • Organizer: 第56回日本生物物理学会年会
• [Presentation] Selective digestion of Ba2+/Ca2+ alginate microdroplets for single-cell-analysis (2018)
  • Author(s): 尾高正朗、岩村萌絵、川合絢子、服部明弘、松浦賢志、安田賢二
  • Organizer: 第56回日本生物物理学会年会
• [Presentation] Development of sprouting vascular endothelial cell collection method using flexible design of Matrigel for expression analysis (2018)
  • Author(s): 山中悠希、飯田健斗、高野隆治、橋本広道、尾高正朗、服部明弘、松浦賢志、安田賢二
  • Organizer: 第56回日本生物物理学会年会
• [Presentation] Study of Seamless Combination of Imaging Cell Sorting and Gene Analysis Technology Utilizing Characteristics of Alginate Microdroplets (2018)
  • Author(s): 尾高正朗、服部明弘、松浦賢志、安田賢二
  • Organizer: The 31th International Microprocesses and Nanotechnology Conference (MNC 2018)
  • Int'l Joint Research
• [Presentation] Time Course Change of Clustered Circulating Tumor Cell in Cancer Metastasis Blood Investigated with Imaging Biomarkers by on-Chip Multi-Imaging Cell Sorter. (2017)
  • Author(s): Masao Odaka, Akihiro Hattori, Kenji Matsuura, Moe Iwamura, Yuki Yamanaka, Kento Iida, Kenji Yasuda
  • Organizer: The 30th International Microprocesses and Nanotechnology Conference (MNC 2017)
  • Int'l Joint Research
• [Presentation] Development of real time microfabrication technology of gelatin with focused photo-thermal etching. (2017)
  • Author(s): Kento Iida, Yuki Yamanaka, Moe Iwamura, Masao Odaka, Kenji Matsuura, Akihiro Hattori, Kenji Yasuda
  • Organizer: 第55回日本生物物理学会年会
• [Presentation] A simple method for encapsulating single cells in alginate microspheres. (2017)
  • Author(s): Masao Odaka, Akihiro Hattori, Kenji Matsuura, Moe Iwamura, Yuki Yamanaka, Kento Iida, Ronald W. Davis, Laurel D. Crosby, Kenji Yasuda
  • Organizer: 第55回日本生物物理学会年会
• [Presentation] Monitoring of circulating tumor cell clusters in blood using size classifying imaging cell sorter. (2017)
  • Author(s): Moe Iwamura, Masao Odaka, Yuki Yamanaka, Kento Iida, Kenji Matsuura, Akihiro Hattori, Kenji Yasuda
  • Organizer: 第55回日本生物物理学会年会
• [Presentation] Origin of cardiomyocyte cluster beating intervals: Elucidation of selection rule of interbeat intervals of cardiomyocyte network. (2017)
  • Author(s): Naoki Takahashi, Natuki Seki, Masao Odaka, Hideyuki Terazono, Kenji Matsuura, Akihiro Hattori, Kenji Yasuda
  • Organizer: 第55回日本生物物理学会年会
• [Presentation] Adaptation of field potential duration in cardiomyocyte clusters under forced electrical stimulation intervals. (2017)
  • Author(s): Natsuki Seki, Naoki Takahashi, Masao Odaka, Kenji Matsuura, Akihiro Hattori, Kenji Yasuda
  • Organizer: 第55回日本生物物理学会年会
• [Presentation] Extracellular electrophysiological measurement of spontaneous firing of single neurons in neuronal circuit using expandable on-chip assay. (2017)
  • Author(s): Shota Aoki, Takahito Kikuchi, Kenji Matsuura, Akihiro Hattori, Masao Odaka, Kenji Yasuda
  • Organizer: 第55回日本生物物理学会年会
• [Presentation] Neurite elongation characteristics in the width-controlled channels using an in situ on-chip photothermal microfabrication assay. (2017)
  • Author(s): Takahito Kikuchi, Shota Aoki, Hideyuki Terazono, Kenji Matsuura, Akihiro Hattori, Masao Odaka, Kenji Yasuda
  • Organizer: 第55回日本生物物理学会年会
• [Presentation] Analysis of sequential single point phagocytosises in macrophages using on-chip single cell measurement assay. (2017)
  • Author(s): Yoshiki Nakata, Yuya Furumoto, Masao Odaka, Kenji Matsuura, Akihiro Hattori, Hideyuki Terazono, Kenji Yasuda
  • Organizer: 第55回日本生物物理学会年会
• [Presentation] Development of real time microfabrication technology of nano-particle suspended agarose microstructures with focused photo-thermaletching. (2017)
  • Author(s): Yuki Yamanaka, Kento Iida, Moe Iwamura, Masao Odaka, Kenji Matuura, Akihiro Hattori, Kenji Yasuda
  • Organizer: 第55回日本生物物理学会年会
• [Presentation] Optimization of antigen of macrophage phagocytosis using on-chip single cell measurement assay. (2017)
  • Author(s): Yuya Furumoto, Yoshiki Nakata, Masao Odaka, Kenji Matsuura, Akihiro Hattori, Kenji Yasuda
  • Organizer: 第55回日本生物物理学会年会
• [Presentation] Development of on-chip dual measurement system for quasi-in vivo screening of cardiotoxicity using extracellular field potential recording and optical displacement analysis of single cells. (2017)
  • Author(s): Akihiro Hattori, Naoki Takahashi, Masao Odaka, Kenji Matsuura, Hideyuki Terazono, Natsuki Seki, Kenji Yasuda
  • Organizer: The 5th International symposium for Bioimaging Joint symposium on Bioimaging between Singapore and Japan.
  • Int'l Joint Research
• [Presentation] Evaluation of cell-to-cell conduction on one-dimensional line of cardiac muscle cells network for in vitro predictive cardiotoxicity measurement beyond cell-based drug discovery electrophysiology. (2017)
  • Author(s): Kenji Matsuura, Moe Iwamura, Naoki Takahashi, Yuki Yamanaka, Natsuki Seki, Masao Odaka, Akihiro Hattori, Hideyuki Terazono, Kenji Yasuda
  • Organizer: The 5th International symposium for Bioimaging Joint symposium on Bioimaging between Singapore and Japan.
  • Int'l Joint Research
• [Presentation] Time course change of circulating tumor cell clusters in blood evaluated by imaging biomarkers of on-chip multi-imaging cell sorter (2017)
  • Author(s): Masao Odaka, Moe Iwamura, Akihiro Hattori, Kenji Matsuura, Hideyuki Terazono,Yuki Yamanaka, Kento Iida, Kenji Yasuda
  • Organizer: The 5th International symposium for Bioimaging Joint symposium on Bioimaging between Singapore and Japan.
  • Int'l Joint Research
• [Presentation] Real time on-chip imaging cell sorter for shape recognition of target cells in droplets. (2017)
  • Author(s): Mathias Girault, Hyonchol Kim, Kenji Matsuura, Masao Odaka, Akihiro Hattori, Hideyuki Terazono, Kenji Yasuda
  • Organizer: The 5th International symposium for Bioimaging Joint symposium on Bioimaging between Singapore and Japan.
  • Int'l Joint Research
• [Presentation] On-chip multi-imaging flowcytometry for non-labeled detection of cell clusters in blood with imaging biomarkers. (2017)
  • Author(s): Moe Iwamura, Yuki Yamanaka, Kento Iida, Masao Odaka, Akihiro Hattori, Kenji Matsuura, Hideyuki Terazono, Kenji Yasuda
  • Organizer: The 5th International symposium for Bioimaging Joint symposium on Bioimaging between Singapore and Japan.
  • Int'l Joint Research
• [Presentation] Origin of cardiomyocyte cluster beating rythms: Elucidation of selection rule of interbeat intervals of cardiomyocyte network from single cells. (2017)
  • Author(s): Naoki Takahashi, Natsuki Seki, Hideyuki Terazono, Masao Odaka, Akihiro Hattori, Kenji Matsuura, Kenji Yasuda
  • Organizer: The 5th International symposium for Bioimaging Joint symposium on Bioimaging between Singapore and Japan.
  • Int'l Joint Research
• [Presentation] Minimum width of artificial neuronal circuit patterns for stable elongation of neurites. (2017)
  • Author(s): Takahito Kikuchi, Shota Aoki, Masao Odaka, Akihiro Hattori, Kenji Matsuura, Hideyuki Terazono, Kenji Yasuda
  • Organizer: The 5th International symposium for Bioimaging Joint symposium on Bioimaging between Singapore and Japan.
  • Int'l Joint Research
• [Presentation] Studies on spatiotemporal adaptive regulation mechanism of macrophage phagocytosis: response of multiple stimulations due to physical contacts with multiway optical tweezers. (2017)
  • Author(s): Yoshiki Nakata, Yuya Furumoto, Masao Odaka, Akihiro Hattori, Kenji Matsuura, Hideyuki Terazono, Kenji Yasuda
  • Organizer: The 5th International symposium for Bioimaging Joint symposium on Bioimaging between Singapore and Japan.
  • Int'l Joint Research