Developing a scalable-coculturing system for gut anaerobes and intestinal epithelium

• Project/Area Number: 19K22544
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 49:Pathology, infection/immunology, and related fields
• Research Institution: Keio University
• Principal Investigator: SASAKI Nobuo 慶應義塾大学, 医学部(信濃町), 特任助教 (30777769)
• Project Period (FY): 2019-06-28 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000); Fiscal Year 2020: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2019: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000)
• Keywords: オルガノイド / 腸内細菌 / 嫌気性細菌 / 共培養システム / プロバイオティクス / 嫌気性腸内細菌 / 共培養システム開発 / 嫌気性培養

Outline of Research at the Start

本課題研究は，正常腸管上皮細胞培養法のオルガノイドを腸内細菌研究分野へ応用させることを目的としているが，腸管上皮を含む生体の上皮細胞の生存には酸素は不可欠であり，反対に腸内細菌のほとんどは酸素存在下では生育できない．したがって，このような相反する酸素要求性を示す腸管上皮細胞と腸内細菌の共培養は原理的に不可能である．そこで我々は，生体内の腸管の生理条件をシャーレ上で再現することで，消化管感染を中心とした様々な疾患の発症メカニズムの理解につながる感染学や腸内細菌学の発展に貢献できる，新規の低酸素環境におけるオルガノイド培養法の確立を目指す．

Outline of Final Research Achievements

The aim of this study is to develop a novel culturing system of healthy human intestinal epithelium together with anaerobic gut bacteria. We modified the classical culture method for 3D adult-tissue stem cell, organoid firstly. Using the Boyden chamber, we succeeded in culturing human normal colon epithelial cells as the monolayer condition for long periods. Next, we also figure out the suitable conditioned medium to enable to generate anaerobic environments at apical surface of their epithelium. Actually, the obligate anaerobic bacteria can grow to form their colonies on the human colonic epithelial cells in our culture condition. Here, we demonstrated a novel coculturing system in modeling competitive and mutually beneficial relationship between human epithelium and gut microbes in vitro.

Academic Significance and Societal Importance of the Research Achievements

最近では企業における機能細菌（プロバイオティクス）研究開発現場において，動物愛護の観点から動物試験が禁止されるようになってきたため，モデル動物に変わる細菌の機能を試験する新規モデルの開発が急務であった．我々が開発した新規ヒト腸管上皮-腸内細菌共培養システムは，動物試験を不要とするだけではなく，細菌が与えるヒト細胞への影響を直接検証できるといった特徴も有している．そのため本研究成果は，製薬や食品メーカーが直面している商品開発の“死の谷”を克服するための一翼を担うことが期待されているので，今後積極的に本技術を産業界へ導出したいと考えている．

Research Products

• [Journal Article] 腸内細菌－宿主細胞の相互作用メカニズムを紐解く新規共培養系の開発 (2021)
  • Author(s): 佐々木 伸雄
  • Journal Title: 臨床免疫・アレルギー科 Volume: 75 Pages: 77-81
  • NAID: 40022452487
• [Journal Article] Development of a scalable co-culture system for gut anaerobes and human colon epithelium (2020)
  • Author(s): Sasaki Nobuo、Miyamoto Kentaro、Maslowski Kendle M.、Ohno Hiroshi、Kanai Takanori、Sato Toshiro
  • Journal Title: Gastroenterology Volume: in Press Issue: 1 Pages: 388-390
  • DOI: 10.1053/j.gastro.2020.03.021
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Somatic inflammatory gene mutations in human ulcerative colitis epithelium. (2020)
  • Author(s): Nanki K, Fujii M, Shimokawa M, Matano M, Nishikori S, Date S, Takano A, Toshimitsu K, Ohta Y, Takahashi S, Sugimoto S, Ishimaru K, Kawasaki K, Nagai Y, Ishii R, Yoshida K, Sasaki N, Hibi T, Ishihara S, Kanai T, Sato T.
  • Journal Title: Nature Volume: 7789 Issue: 7789 Pages: 254-259
  • DOI: 10.1038/s41586-019-1844-5
  • Peer Reviewed / Open Access
• [Journal Article] Gut pathobionts underlie intestinal barrier dysfunction and liver T helper 17 cell immune response in primary sclerosing cholangitis. (2019)
  • Author(s): Nakamoto N, Sasaki N, Aoki R, Miyamoto K, Suda W, Teratani T, Suzuki T, Koda Y, Chu PS, Taniki N, Yamaguchi A, Kanamori M, Kamada N, Hattori M, Ashida H, Sakamoto M, Atarashi K, Narushima S, Yoshimura A, Honda K, Sato T, Kanai T.
  • Journal Title: Nat Microbiol. Volume: 4 Issue: 3 Pages: 492-503
  • DOI: 10.1038/s41564-018-0333-1
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Ongoing chromosomal instability and karyotype evolution in human colorectal cancer organoids (2019)
  • Author(s): Bolhaqueiro A.C.F., Ponsioen B., Bakker B., Klaasen S., Kucukkose E., van Jaarsveld R.H., Vivie J., Verlaan-Klink I., Hami N., Spierings D.C.J., Sasaki N., Dutta D., Boj S.F., Vries R.G.J., Lansdorp P.M., van de Wetering M. van Oudenaarden A., Clevers H., Kranenburg O., Foijer F., Snippert H.J.G., Kops G.J.P.
  • Journal Title: Nature Genetics Volume: 51 Issue: 5 Pages: 824-834
  • DOI: 10.1038/s41588-019-0399-6
  • Peer Reviewed
• [Journal Article] Defining the Identity and Dynamics of Adult Gastric Isthmus Stem Cells (2019)
  • Author(s): Han S, Fink J, Jorg DJ, Lee E, Yum MK, Chatzeli L, Merker SR, Josserand M, Trendafilova T, Andersson-Rolf A, Dabrowska C, Kim H, Naumann R, Lee JH, Sasaki N, Mort RL, Basak O, Clevers H, Stange DE, Philpott A, Kim JK, Simons BD, Koo BK
  • Journal Title: Cell Stem Cell Volume: 25 Issue: 3 Pages: 342-356.e7
  • DOI: 10.1016/j.stem.2019.07.008
  • Peer Reviewed
• [Journal Article] Enteroendocrine and tuft cells support Lgr5 stem cells on Paneth cell depletion (2019)
  • Author(s): van Es Johan H.、Wiebrands Kay、L?pez-Iglesias Carmen、van de Wetering Marc、Zeinstra Laura、van den Born Maaike、Korving Jeroen、Sasaki Nobuo、Peters Peter J.、van Oudenaarden Alexander、Clevers Hans
  • Journal Title: Proceedings of the National Academy of Sciences Volume: 116 Issue: 52 Pages: 26599-26605
  • DOI: 10.1073/pnas.1801888117
  • Peer Reviewed
• [Presentation] The study of epithelial crosstalk at the host-microbes interface using organoid (2020)
  • Author(s): Nobuo Sasaki
  • Organizer: 8th Global Network Forum on Infection and Immunity: Microbiome
  • Int'l Joint Research / Invited
• [Presentation] 嫌気性腸内細菌とヒト正常大腸上皮細胞の共培養を可能にする新規培養系の開発 (2020)
  • Author(s): 佐々木 伸雄
  • Organizer: 第43回日本分子生物学会年会
  • Invited
• [Presentation] Organoid unravels the interaction mechanism between mucosal barrier and bacteria (2019)
  • Author(s): Nobuo Sasaki
  • Organizer: Keio International Symposium, The Crossroads of Intestinal Microbiology, Mucosal Immunology and Epithelial Biology
  • Int'l Joint Research / Invited
• [Presentation] 大腸がんにおいて1細胞レベルで明らかにされた単一腫瘍内における不均一性の網羅的解析 (2019)
  • Author(s): 佐々木 伸雄
  • Organizer: 第46回日本毒性学会学術年会
  • Invited
• [Presentation] オルガノイドが切り拓く腸内細菌研究の新時代 (2019)
  • Author(s): 佐々木 伸雄
  • Organizer: 日本乳酸菌学会2019年度秋季セミナー
  • Invited
• [Presentation] オルガノイドが切り拓く次世代の腸内細菌研究 (2019)
  • Author(s): 佐々木 伸雄，佐藤 俊朗
  • Organizer: 第42回日本分子生物学会年会
  • Invited
• [Book] 決定版 オルガノイド実験スタンダード (佐藤，武部，永楽/編) (2019)
  • Author(s): 佐々木 伸雄，金井 隆典，佐藤 俊朗
  • Total Pages: 372
  • Publisher: 羊土社
  • ISBN: 9784758122399
• [Patent(Industrial Property Rights)] 上皮細胞培養用培養容器及びその使用 (2020)
  • Inventor(s): 佐藤俊朗，佐々木伸雄
  • Industrial Property Rights Holder: 佐藤俊朗，佐々木伸雄
  • Industrial Property Rights Type: 特許
  • Filing Date: 2020