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WO2020005012A1 - Procédé de production d'organoïde tridimensionnel humain par un procédé non chirurgical - Google Patents

Procédé de production d'organoïde tridimensionnel humain par un procédé non chirurgical Download PDF

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Publication number
WO2020005012A1
WO2020005012A1 PCT/KR2019/007894 KR2019007894W WO2020005012A1 WO 2020005012 A1 WO2020005012 A1 WO 2020005012A1 KR 2019007894 W KR2019007894 W KR 2019007894W WO 2020005012 A1 WO2020005012 A1 WO 2020005012A1
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WO
WIPO (PCT)
Prior art keywords
tissue
hydrogel
vivo
biomarker
organoids
Prior art date
Application number
PCT/KR2019/007894
Other languages
English (en)
Korean (ko)
Inventor
이현숙
박지호
이수현
김성수
Original Assignee
서울대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020190076937A external-priority patent/KR102272551B1/ko
Application filed by 서울대학교 산학협력단 filed Critical 서울대학교 산학협력단
Publication of WO2020005012A1 publication Critical patent/WO2020005012A1/fr

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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C12N5/0677—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/119—Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • the separating step is a step of separating a three-dimensional organoid having an average diameter of 0.1mm or more, 0.25mm or more, 0.5mm or more, 0.75mm or more, 1mm or more, or 1.25mm or more, 1.5mm or more, 1.75mm or more or 2mm or more.
  • the upper limit of the average diameter of the organoid may vary depending on the culture period, and may be about 15 mm, 12.5 mm, 10 mm, 7.5 mm, 5 mm, or 2.5 mm, but is not limited thereto).
  • Another example provides a composition or kit for genetic information analysis or karyotype analysis comprising the three-dimensional organoid.
  • Another example provides a genetic information analysis or karyotype analysis method comprising measuring the genetic information or karyotype of the three-dimensional organoid. Determining the genetic information or karyotype of the three-dimensional organoids is extracted nucleic acid (DNA and / or RNA) from the organoids to obtain the genetic information (gene or genomic sequence, etc.) by conventional methods (e.g. WGS, etc.) Conventional genetic information analysis or karyotype analysis, such as analyzing, and / or visualizing the organoid by immunofluorescence staining or the like.
  • the hydrogel provides a method for confirming the expression of the biomarker in the ex vivo tissue structure, characterized in that the matrigel, the ex vivo tissue structure is artificial organs, bio organs, mini organs, organoids, spheroids, spheres
  • In vitro tissues characterized in that at least one selected from the group consisting of upper body, and embryonic body Provided are methods for identifying the expression of a bio
  • Figure 6a shows the result of immunostaining after 3 days of primary antibody, 3 days of secondary antibody after the step of removing the matrigel from the human organ sample-derived organoids according to an embodiment.
  • Figure 7 shows the results of karyotyping after the step of removing the matrigel from the human organ sample-derived organoid according to an embodiment and the result of karyotype without removing the matrigel.
  • the Matrigel was removed for 20 minutes to 1 hour using a recovery solution (354253, Corning, 1 ml / well). I went through the steps.
  • the matrigel-free organoids were transferred to a 15 ml conical tube and washed three times with PBS (Phosphate Buffer Saline) on ice (4 ° C).
  • the organoids retaining the washed spherical form were transferred to 2 ml vials using a pipette and filled with 4% PFA (paraformaldehyde) and fixed on ice for 1 hour. At this time, 4% PFA was changed three times in 10 minute intervals for the first 30 minutes so that the final concentration was 4%.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Evolutionary Biology (AREA)
  • Medical Informatics (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Theoretical Computer Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un organoïde tridimensionnel obtenu par la culture in vitro de tissu d'organe humain obtenu par un procédé non chirurgical, son procédé de production, son procédé de culture, et une utilisation pour une analyse d'informations génétiques, une analyse de caryotype, une validation d'effets de médicaments et/ou un criblage de médicaments de l'organoïde obtenu tel que décrit ci-dessus.
PCT/KR2019/007894 2018-06-29 2019-06-28 Procédé de production d'organoïde tridimensionnel humain par un procédé non chirurgical WO2020005012A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20180075990 2018-06-29
KR10-2018-0075990 2018-06-29
KR10-2019-0076937 2019-06-27
KR1020190076937A KR102272551B1 (ko) 2018-06-29 2019-06-27 비수술적 방법에 의한 인간유래 3차원 오거노이드의 제조 방법

Publications (1)

Publication Number Publication Date
WO2020005012A1 true WO2020005012A1 (fr) 2020-01-02

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2019/007894 WO2020005012A1 (fr) 2018-06-29 2019-06-28 Procédé de production d'organoïde tridimensionnel humain par un procédé non chirurgical

Country Status (1)

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WO (1) WO2020005012A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017048193A1 (fr) * 2015-09-15 2017-03-23 Agency For Science, Technology And Research (A*Star) Dérivation d'organoïdes hépatiques à partir de cellules souches pluripotentes humaines
US20170191030A1 (en) * 2014-05-16 2017-07-06 Koninklijke Nederlandse Akademie Van Wetenschappen Improved culture method for organoids
KR20180136410A (ko) * 2017-06-14 2018-12-24 서울대학교산학협력단 돌연변이 마우스 유래 췌장 오거노이드 및 그 용도

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170191030A1 (en) * 2014-05-16 2017-07-06 Koninklijke Nederlandse Akademie Van Wetenschappen Improved culture method for organoids
WO2017048193A1 (fr) * 2015-09-15 2017-03-23 Agency For Science, Technology And Research (A*Star) Dérivation d'organoïdes hépatiques à partir de cellules souches pluripotentes humaines
KR20180136410A (ko) * 2017-06-14 2018-12-24 서울대학교산학협력단 돌연변이 마우스 유래 췌장 오거노이드 및 그 용도

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BOJ, SYLVIA F. ET AL.: "Organoid models of human and mouse ductal pancreatic cancer", CELL, vol. 160, 2015, pages 324 - 338, XP029132656, DOI: 10.1016/j.cell.2014.12.021 *
NIKOLIC, MARKO Z ET AL.: "Human embryonic lung epithelial tips are multipotent progenitors that can be expanded in vitro as long-term self-renewing organoids", ELIFE, vol. 6, no. 2, 30 June 2017 (2017-06-30), pages 1 - 3, XP055627915 *

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