マイクロメッシュを用いた層状細胞構造の構築と機能発現およびその計測

• Project Area: Hyper Bio Assembler for 3D Celluler Innovation
• Project/Area Number: 26106710
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Science and Engineering
• Research Institution: The University of Tokyo
• Principal Investigator: 鷲津 正夫 東京大学, 工学(系)研究科(研究院), 教授 (10201162)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥18,330,000 (Direct Cost: ¥14,100,000、Indirect Cost: ¥4,230,000); Fiscal Year 2015: ¥9,490,000 (Direct Cost: ¥7,300,000、Indirect Cost: ¥2,190,000); Fiscal Year 2014: ¥8,840,000 (Direct Cost: ¥6,800,000、Indirect Cost: ¥2,040,000)
• Keywords: マイクロ・ナノデバイス / 移植・再生医療 / バイオリアクター / 細胞組織 / 細胞シート / 微細加工

Outline of Annual Research Achievements

チップ上でエレクトロポレーションを行うと同時に電気泳動により外来DNA（プラスミド）を核に送達するシステムを開発した。細胞は，孔径2μm・ピッチ15μm程度のオリフィスを持つシート上で付着培養される。このオリフィスシートを両側から電極ではさみ，4V-200msec程度の電圧を印加すると，膜の可逆的破壊が生じ，そこを通して流れる導電電流下の電気泳動によりプラスミドが核に移動する。このように，プラスミドは核に直接送達されるため，パルス印加2時間後くらいからプラスミド遺伝子の発現が見られる。オリフィスを十分に密に作り，細胞核の直下に確率的に1個以上のオリフィスが来るように作れば，すべての核にプラスミドを送達することが可能になる。この手法を用いて，体細胞に4つの山中因子を導入したところ，Oct4とNanogの発現，すなわち細胞の初期化が起きた。この遺伝子直接送達法は，タイミングを正確に制御して即時に遺伝子を導入できるという特徴があるので，今後の細胞のリプログラミングや分化制御の研究に役立つ物と期待される。

Research Progress Status

27年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

27年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] Cell Adhesion Minimization by a Novel Mesh Culture Method Mechanically Directs Trophoblast Differentiation and Self-Assembly Organization of Human Pluripotent Stem Cells (2015)
  • Author(s): Okeyo O. Kennedy, Osamu Kurosawa, Satoshi Yamazaki, Hidehiro Oana, Hidetoshi Kotera, Hiromitsu Nakauchi, Masao Washizu
  • Journal Title: Tissue Engineering Part C Volume: なし Issue: 10 Pages: 1105-15
  • DOI: 10.1089/ten.tec.2015.0038
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] 電場を用いた細胞マ ニピュレーションとその新展開 (2014)
  • Author(s): 阪本祥太・オケヨケネディ・鷲津正夫
  • Journal Title: 電気化学会誌 Volume: 82 (11) Pages: 1012-1016
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] Cell Adhesion Control using Microstructured Meshes Induces Self-Assembly-Mediated Organoid Formation by Human iPS Cells (2015)
  • Author(s): Kennedy O. Okeyo, Osamu Kurosawa, Satoshi Yamazaki, Hidehiro Oana, Hidetoshi Kotera, and Masao Washizu
  • Organizer: 2015 IEEE/ International Symposium on Micro-NanoMechatronics and Human Science
  • Place of Presentation: 名古屋大学, 愛知県 名古屋市
  • Year and Date: 2015-11-23
  • Int'l Joint Research
• [Presentation] Microstructured Meshes as Platforms for Cell Culture and Induction of Self-Assembly-Mediated Organoid Formation (2015)
  • Author(s): Kennedy Omondi Okeyo, Osamu Kurosawa, Satoshi Yamazaki, Hidehiro Oana, Hidetoshi Kotera, and Masao Washizu
  • Organizer: The 28th International Microprocesses and Nanotechnology Conference
  • Place of Presentation: 富山国際会議場,富山県 富山市
  • Year and Date: 2015-11-10
  • Int'l Joint Research / Invited
• [Presentation] Engineering Free Standing 2D and 3D Biostructures by Induction of Self-Assembly Organization of Cells in a Suspension (2015)
  • Author(s): Kennedy Okeyo and Masao Washizu
  • Organizer: Lab-on-a-Chip, Microfluidics & Microarrays World Congress
  • Place of Presentation: San Diego USA
  • Year and Date: 2015-10-28
  • Int'l Joint Research / Invited
• [Presentation] Lab-on-a CD Device for Rapid Sorting of Blood Cells and Real Time Analysis : Toward Early Detection of Maralia (2015)
  • Author(s): Kennedy O. Okeyo, Osamu Kurosawa, Hitoshi Suzuki, Yoshihide Saito and Masao Washizu
  • Organizer: 2nd Africa international biotechnology and biomedical conference
  • Place of Presentation: Nairobi Keniya
  • Year and Date: 2015-09-17
  • Int'l Joint Research / Invited
• [Presentation] Control of Cell Orientation in Mesenchymal Cell Sheets Fabricated Using Microstructured Mesh Sheet (2014)
  • Author(s): Rina Yanaru, Kennedy Omondi Okeyo, Osamu Kurosawa, Hidehiro Oana, Hidetoshi Kotera, Masao Washizu
  • Organizer: IEEE MHS2014
  • Place of Presentation: Nagoya University,Aichi Nagoya City
  • Year and Date: 2014-11-09
  • Int'l Joint Research
• [Presentation] Generation of epithelial cell sheets with defined cell orientation using microstructured mesh sheets as a substrate for cell culture (2014)
  • Author(s): K.O. Okeyo, R. Yanaru, O. Kurosawa, H. Oana, H. Kotera, and M. Washizu
  • Organizer: μTAS2014
  • Place of Presentation: San Antonio, USA
  • Year and Date: 2014-10-26
  • Int'l Joint Research
• [Presentation] Stable and long term culture of stem cells under shear flow on a microstructured mesh sheet embedded in a fluidic chamber (2014)
  • Author(s): K.O. Okeyo, T. Isozaki, O. Kurosawa, H. Oana, H. Kotera, and M. Washizu
  • Organizer: μTAS2014
  • Place of Presentation: San Antonio, USA
  • Year and Date: 2014-10-26
  • Int'l Joint Research
• [Presentation] Biomanipulation, cell fusion, single-cell surgery (2014)
  • Author(s): Masao Washizu
  • Organizer: Joint Meeting of the 1st Africa International Biotechnology & Biomedical Conference and the 8th International Workshop on Approaches to Single-Cell Analysis
  • Place of Presentation: Nairobi, Kenya
  • Year and Date: 2014-09-09
  • Int'l Joint Research / Invited
• [Presentation] A New Approach to Cell Culture and Cell Sheet Fabrication by the “Shoji” Process (2014)
  • Author(s): Kennedy O. Okeyo, R. Yanaru, O. Kurosawa, H. Oana, and M. Washizu
  • Organizer: RSC Tokyo International Conference 2014
  • Place of Presentation: Makuhari Messe, Chiba City Mihamaku
  • Year and Date: 2014-09-04
  • Int'l Joint Research / Invited
• [Presentation] Cell culture on “Shoji” for Self-Assembly Driven Fabrication of 2D and 3D Cellular Structure (2014)
  • Author(s): Kennedy O. Okeyo, R. Yanaru, O. Kurosawa, H. Oana, and M. Washizu
  • Organizer: International Symposium on 3D Tissue Fabrications
  • Place of Presentation: Komaba Campus, Tokyo University, Tokyo Meguroku
  • Year and Date: 2014-05-20
  • Int'l Joint Research / Invited
• [Remarks] 東京大学大学院工学系研究科 鷲津・小穴研究室
  • URL: http://www.washizu.t.u-tokyo.ac.jp/