JP2020535850A - 生体模倣オルガノイド培養物 - Google Patents
生体模倣オルガノイド培養物 Download PDFInfo
- Publication number
- JP2020535850A JP2020535850A JP2020540673A JP2020540673A JP2020535850A JP 2020535850 A JP2020535850 A JP 2020535850A JP 2020540673 A JP2020540673 A JP 2020540673A JP 2020540673 A JP2020540673 A JP 2020540673A JP 2020535850 A JP2020535850 A JP 2020535850A
- Authority
- JP
- Japan
- Prior art keywords
- cells
- organoid
- cell layer
- cell
- organoids
- Prior art date
- Legal status (The legal status 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 status listed.)
- Granted
Links
- 210000002220 organoid Anatomy 0.000 title claims abstract description 89
- 230000003592 biomimetic effect Effects 0.000 title claims abstract description 8
- 210000001519 tissue Anatomy 0.000 claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 19
- 238000012258 culturing Methods 0.000 claims abstract description 13
- 210000004027 cell Anatomy 0.000 claims description 126
- 239000010410 layer Substances 0.000 claims description 74
- 210000004379 membrane Anatomy 0.000 claims description 41
- 239000012528 membrane Substances 0.000 claims description 41
- 239000000017 hydrogel Substances 0.000 claims description 33
- 238000000338 in vitro Methods 0.000 claims description 23
- 230000002207 retinal effect Effects 0.000 claims description 18
- 210000002889 endothelial cell Anatomy 0.000 claims description 14
- 210000002919 epithelial cell Anatomy 0.000 claims description 7
- 239000002356 single layer Substances 0.000 claims description 7
- 238000003501 co-culture Methods 0.000 claims description 6
- 210000002950 fibroblast Anatomy 0.000 claims description 4
- 210000000663 muscle cell Anatomy 0.000 claims description 4
- 210000000608 photoreceptor cell Anatomy 0.000 claims description 4
- 210000000844 retinal pigment epithelial cell Anatomy 0.000 claims description 4
- 230000000968 intestinal effect Effects 0.000 claims description 3
- 241000251539 Vertebrata <Metazoa> Species 0.000 claims description 2
- 210000004556 brain Anatomy 0.000 claims description 2
- 210000001087 myotubule Anatomy 0.000 claims description 2
- 238000010899 nucleation Methods 0.000 claims description 2
- 210000003098 myoblast Anatomy 0.000 claims 1
- 230000006870 function Effects 0.000 abstract description 3
- 238000010586 diagram Methods 0.000 abstract 1
- 210000003583 retinal pigment epithelium Anatomy 0.000 description 20
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 15
- 230000010412 perfusion Effects 0.000 description 11
- 230000003993 interaction Effects 0.000 description 10
- 239000003814 drug Substances 0.000 description 8
- 230000003511 endothelial effect Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 238000011161 development Methods 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000002523 lectin Substances 0.000 description 5
- 108091008695 photoreceptors Proteins 0.000 description 5
- 208000017442 Retinal disease Diseases 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 239000000090 biomarker Substances 0.000 description 4
- 230000030833 cell death Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000004205 dimethyl polysiloxane Substances 0.000 description 4
- 230000035800 maturation Effects 0.000 description 4
- 210000004498 neuroglial cell Anatomy 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 210000004789 organ system Anatomy 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 4
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 3
- 206010038923 Retinopathy Diseases 0.000 description 3
- 229910052791 calcium Inorganic materials 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 238000005266 casting Methods 0.000 description 3
- 210000002308 embryonic cell Anatomy 0.000 description 3
- 210000001671 embryonic stem cell Anatomy 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- 238000010859 live-cell imaging Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000035790 physiological processes and functions Effects 0.000 description 3
- 239000000049 pigment Substances 0.000 description 3
- 230000000069 prophylactic effect Effects 0.000 description 3
- 210000001525 retina Anatomy 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- WHTVZRBIWZFKQO-AWEZNQCLSA-N (S)-chloroquine Chemical compound ClC1=CC=C2C(N[C@@H](C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-AWEZNQCLSA-N 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010046016 Peanut Agglutinin Proteins 0.000 description 2
- 208000007014 Retinitis pigmentosa Diseases 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 206010064930 age-related macular degeneration Diseases 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 229960003677 chloroquine Drugs 0.000 description 2
- WHTVZRBIWZFKQO-UHFFFAOYSA-N chloroquine Natural products ClC1=CC=C2C(NC(C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-UHFFFAOYSA-N 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 210000003038 endothelium Anatomy 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 230000002055 immunohistochemical effect Effects 0.000 description 2
- 238000011532 immunohistochemical staining Methods 0.000 description 2
- 230000002427 irreversible effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 208000002780 macular degeneration Diseases 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 238000011338 personalized therapy Methods 0.000 description 2
- 230000004962 physiological condition Effects 0.000 description 2
- 239000008279 sol Substances 0.000 description 2
- 230000002269 spontaneous effect Effects 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- NCYCYZXNIZJOKI-IOUUIBBYSA-N 11-cis-retinal Chemical compound O=C/C=C(\C)/C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-IOUUIBBYSA-N 0.000 description 1
- QURLONWWPWCPIC-UHFFFAOYSA-N 2-(2-aminoethoxy)ethanol;3,6-dichloro-2-methoxybenzoic acid Chemical compound NCCOCCO.COC1=C(Cl)C=CC(Cl)=C1C(O)=O QURLONWWPWCPIC-UHFFFAOYSA-N 0.000 description 1
- 239000002999 4 aminobutyrate aminotransferase inhibitor Substances 0.000 description 1
- 206010054881 Acquired pigmented retinopathy Diseases 0.000 description 1
- 102100026440 Arrestin-C Human genes 0.000 description 1
- 201000004569 Blindness Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 208000003098 Ganglion Cysts Diseases 0.000 description 1
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 description 1
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 1
- 101000785755 Homo sapiens Arrestin-C Proteins 0.000 description 1
- 101000729271 Homo sapiens Retinoid isomerohydrolase Proteins 0.000 description 1
- 101000581122 Homo sapiens Rho-related GTP-binding protein RhoD Proteins 0.000 description 1
- 101000854931 Homo sapiens Visual system homeobox 2 Proteins 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 101150117895 LAMP2 gene Proteins 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 102100031176 Retinoid isomerohydrolase Human genes 0.000 description 1
- 102100027609 Rho-related GTP-binding protein RhoD Human genes 0.000 description 1
- 102100040756 Rhodopsin Human genes 0.000 description 1
- 108090000820 Rhodopsin Proteins 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 208000005400 Synovial Cyst Diseases 0.000 description 1
- 102100020676 Visual system homeobox 2 Human genes 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000001961 anticonvulsive agent Substances 0.000 description 1
- 229960003965 antiepileptics Drugs 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000003986 cell retinal photoreceptor Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008614 cellular interaction Effects 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 210000005081 epithelial layer Anatomy 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000007277 glial cell activation Effects 0.000 description 1
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 208000036260 idiopathic disease Diseases 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 238000012625 in-situ measurement Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000008611 intercellular interaction Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000001459 lithography Methods 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 230000002132 lysosomal effect Effects 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 101150087532 mitF gene Proteins 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 230000008450 motivation Effects 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 210000000107 myocyte Anatomy 0.000 description 1
- 210000005157 neural retina Anatomy 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 229920002120 photoresistant polymer Polymers 0.000 description 1
- 230000008560 physiological behavior Effects 0.000 description 1
- 238000009832 plasma treatment Methods 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- -1 polydimethylsiloxane Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 210000000768 retinal photoreceptor cell outer segment Anatomy 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
Images
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/12—Well or multiwell plates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/02—Membranes; Filters
-
- 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
- C12N5/0618—Cells of the nervous system
- C12N5/0621—Eye cells, e.g. cornea, iris pigmented 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
- C12N2502/085—Coculture with; Conditioned medium produced by cells of the nervous system eye 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1323—Adult fibroblasts
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1335—Skeletal muscle cells, myocytes, myoblasts, myotubes
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/28—Vascular endothelial 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
- 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
- C12N2533/50—Proteins
- C12N2533/52—Fibronectin; Laminin
-
- 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
- C12N2533/70—Polysaccharides
- C12N2533/80—Hyaluronan
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Sustainable Development (AREA)
- Cell Biology (AREA)
- Ophthalmology & Optometry (AREA)
- Immunology (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Molecular Biology (AREA)
- Clinical Laboratory Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
Description
1.網膜MPSの製造/集成
バイオリアクターを製造するために、MPSの複数の層を、微細構造化されたシリコンウエハー上にポリジメチルシロキサン(PDMS)をキャスティングすることにより製造する。しかしながら、MPSの製造はこの材料に限定されず、ガラス、PC並びにPET及びそれらの組み合わせ物等の他の材料も可能である。それぞれの流し込み型(マスター)の微細構造化はフォトレジスト(SU-8、MicroChem社)のUVリソグラフィーによって実現される。
初めに、アセンブルされたMPSを50ワットの出力及び0.1Nml/分〜0.3Nml/分の酸素ガス流量並びに5分〜15分の処理期間で酸素プラズマによって滅菌する。MPSの滅菌は、同様にオートクレーブ処理又はガンマ線照射によって行うこともできる。プラズマ処理の実施後に、RPE細胞の後の接着を可能にするために半透膜の被覆を行う。このために、DMEM/F12とラミニンとの1:10〜1:25希釈を適用し、MPSを37℃及び5%のCO2で1時間〜4時間の期間にわたりインキュベートする(図3A)。iPS-RPE細胞をプレーティングする前に、余分なラミニン混合物を除去し、MPS全体を培地ですすぐ。既に事前に引き離されたiPS-RPE細胞を、5μl〜10μlの容量で直接的に上からRO及びRPE組織チャンバーを通じて膜上に加える。iPS RPE細胞を膜上に接着させることを可能にするために、iPS RPE細胞を37℃及び5%のCO2で30分間〜60分間の期間にわたりインキュベートする(図3B)。引き続き、MPS内のiPS RPE細胞を、1日間〜3日間の期間にわたり外部のシリンジポンプを介して10μl/時間〜20μl/時間の一定の培地流量で培養した。
MPSにおけるiPS-RO及びiPS-RPEの共培養の生理的機能性及び活力は、以下のように検出することができた:
Claims (15)
- 底部に半透膜(33)を備えたバイオリアクター容器(30)内でオルガノイド組織(10)の生体模倣共培養をする方法であって、
(a)少なくとも1種の第1の細胞種の細胞を膜(33)上に播種するステップと、
(b)前記播種細胞を培養して、前記膜(33)に支持された少なくとも1層の2D細胞層(20)を形成させるステップと、
(c)バイオリアクター容器(30)内の前記支持2D細胞層(20)上へ、
- 互いに対して規定の3D構造で配置されている少なくとも2種の更なる細胞種の細胞を含むオルガノイド(10)、及び
- ヒドロゲル(15)
を導入するステップであって、バイオリアクター容器(30)内のオルガノイド(10)が、導入されたヒドロゲル(15)を介して、前記支持2D細胞層(20)から離れて位置することを条件とするステップと
を含む、方法。 - ステップ(c)において、バイオリアクター容器(30)内のオルガノイド(10)が、導入されたヒドロゲル(15)を介して、バイオリアクター容器(30)の壁部(38)からも離れていることを更なる条件とする、請求項1に記載の方法。
- ステップ(c)において、オルガノイド(10)がヒドロゲル(15)と共にバイオリアクター容器(30)内に導入される、請求項1又は2に記載の方法。
- ステップ(c)が、
(c1)ヒドロゲルの一部を導入して、2D細胞層(20)に対して、及び場合によりバイオリアクター容器(30)の壁部(38)に対して規定の間隔層(18)を形成させるサブステップと、次いで
(c2)形成されたヒドロゲル間隔層(18)上にオルガノイド(10)を導入するサブステップと
を含む、請求項1又は2に記載の方法。 - 底部の半透膜(33)上の2D細胞層(20)の培養において、その基底極(24)がその頂端極(22)と別に潅流される、請求項1〜4のいずれか一項に記載の方法。
- 底部に半透膜(33)を備えたバイオリアクター容器(30)内のin vitro組織培養物であって、
- 半透膜(33)上の少なくとも第1の細胞種を含む2D細胞層(20)と、
- 互いに対して規定の3D構造で配置されている少なくとも2種の更なる細胞種の細胞を含むオルガノイド(10)と、
- オルガノイド(10)がバイオリアクター容器(30)内で埋設されており、底部の2D細胞層(20)から規定の距離離れて位置している、ヒドロゲル(15)と
を含む、in vitro組織培養物。 - オルガノイド(10)の2D細胞層(20)に対する規定の距離が、1μm〜100μm、好ましくは2μm〜20μmである、請求項6に記載のin vitro組織培養物。
- オルガノイド(10)が、バイオリアクター容器(30)の壁部(38)からも離れているように、ヒドロゲル(15)中に埋設されている、請求項6又は7に記載のin vitro組織培養物。
- 第1の2D細胞層(20)が、膜(33)の、オルガノイド(10)に向いた上面に配置されている、請求項6〜8のいずれか一項に記載のin vitro組織培養物。
- 更なる2D細胞層(25)が、膜(33)の、オルガノイド(10)と反対側の下面に配置されている、請求項9に記載のin vitro組織培養物。
- 2D細胞層(20、25)の細胞種が、
- 上皮細胞、
- 上皮様細胞、
- 内皮細胞、
- 線維細胞及び/又は線維芽細胞を含む間質細胞、並びに
- 筋芽細胞、筋細胞、及び/又は筋線維を含む筋肉細胞
から選択される、請求項6〜10のいずれか一項に記載のin vitro組織培養物。 - 膜(33)の、オルガノイド(10)に向いた上面にある第1の2D細胞層(20)が、上皮細胞を含む又は上皮細胞からなる、請求項9〜11のいずれか一項に記載のin vitro組織培養物。
- 膜(33)の、オルガノイド(10)と反対側の下面にある更なる2D細胞層(25)が、内皮細胞を含む又は内皮細胞からなる、請求項12に記載のin vitro組織培養物。
- オルガノイド(10)が、網膜オルガノイド、脳オルガノイド、膵臓オルガノイド、及び腸オルガノイドを含む、自己組織化多細胞種組織、又は細胞プリントによって製造可能な規定の3D構造を有する多細胞種組織の群から選択される、請求項6〜13のいずれか一項に記載のin vitro組織培養物。
- オルガノイド(10)が、少なくとも光受容体細胞、及び脊椎動物の神経網膜の少なくとも1種の更なる細胞種の細胞を含む網膜オルガノイドであり、かつ2D細胞層(20)が、網膜色素上皮細胞由来のコンフルエントな単層である、請求項6〜14のいずれか一項に記載のin vitro組織培養物。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE102017217738.1A DE102017217738B3 (de) | 2017-10-05 | 2017-10-05 | Mikrophysiologische Organoidkultur |
DE102017217738.1 | 2017-10-05 | ||
PCT/EP2018/076645 WO2019068640A1 (de) | 2017-10-05 | 2018-10-01 | Mikrophysiologische organoidkulur |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2020535850A true JP2020535850A (ja) | 2020-12-10 |
JP7422665B2 JP7422665B2 (ja) | 2024-01-26 |
Family
ID=63525901
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2020540673A Active JP7422665B2 (ja) | 2017-10-05 | 2018-10-01 | 生体模倣オルガノイド培養物 |
Country Status (6)
Country | Link |
---|---|
US (1) | US11459542B2 (ja) |
EP (1) | EP3692138B1 (ja) |
JP (1) | JP7422665B2 (ja) |
CA (1) | CA3078426A1 (ja) |
DE (1) | DE102017217738B3 (ja) |
WO (1) | WO2019068640A1 (ja) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE102018221838B3 (de) | 2018-12-14 | 2020-01-09 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Mikrophysiologisches Choroidea-Modell |
KR20230019453A (ko) * | 2020-05-29 | 2023-02-08 | 후지필름 셀룰러 다이내믹스, 인코포레이티드 | 망막 색소 상피 세포 및 광수용체의 이중 세포 응집체 및 이의 사용 방법 |
CN114774278B (zh) * | 2022-06-07 | 2023-06-23 | 华中科技大学同济医学院附属协和医院 | 一种3d心脏瓣膜类器官培育器的使用方法 |
WO2023247007A1 (de) | 2022-06-20 | 2023-12-28 | Technische Universität Ilmenau | Vorrichtung und verfahren zur erzeugung von organoiden sowie zellkultur |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007501633A (ja) * | 2003-05-06 | 2007-02-01 | ベル ブルック ラブズ リミテッド ライアビリティ カンパニー | 微小規模の流体ハンドリングシステムにおける三次元細胞培養 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA3000718A1 (en) * | 2015-10-02 | 2017-04-06 | Wake Forest University Health Sciences | Methods and apparatus for modeling cancer metastasis in vitro |
CA3001342A1 (en) * | 2015-10-16 | 2017-04-20 | Wake Forest University Health Sciences | Multi-layer airway organoids and methods of making and using the same |
-
2017
- 2017-10-05 DE DE102017217738.1A patent/DE102017217738B3/de active Active
-
2018
- 2018-10-01 WO PCT/EP2018/076645 patent/WO2019068640A1/de unknown
- 2018-10-01 CA CA3078426A patent/CA3078426A1/en active Pending
- 2018-10-01 EP EP18782698.7A patent/EP3692138B1/de active Active
- 2018-10-01 JP JP2020540673A patent/JP7422665B2/ja active Active
- 2018-10-01 US US16/753,047 patent/US11459542B2/en active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007501633A (ja) * | 2003-05-06 | 2007-02-01 | ベル ブルック ラブズ リミテッド ライアビリティ カンパニー | 微小規模の流体ハンドリングシステムにおける三次元細胞培養 |
Also Published As
Publication number | Publication date |
---|---|
DE102017217738B3 (de) | 2018-10-04 |
EP3692138A1 (de) | 2020-08-12 |
WO2019068640A1 (de) | 2019-04-11 |
US11459542B2 (en) | 2022-10-04 |
EP3692138B1 (de) | 2021-12-15 |
JP7422665B2 (ja) | 2024-01-26 |
CA3078426A1 (en) | 2019-04-11 |
US20200239843A1 (en) | 2020-07-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Ao et al. | One-stop microfluidic assembly of human brain organoids to model prenatal cannabis exposure | |
JP7422665B2 (ja) | 生体模倣オルガノイド培養物 | |
Manafi et al. | Organoids and organ chips in ophthalmology | |
Kim et al. | Controlling differentiation of adipose-derived stem cells using combinatorial graphene hybrid-pattern arrays | |
Benam et al. | Engineered in vitro disease models | |
Wang et al. | Microphysiological systems: design, fabrication, and applications | |
Li et al. | Toward a neurospheroid niche model: Optimizing embedded 3D bioprinting for fabrication of neurospheroid brain-like co-culture constructs | |
Hesari et al. | A hybrid microfluidic system for regulation of neural differentiation in induced pluripotent stem cells | |
Paz et al. | Challenges and opportunities for tissue-engineering polarized epithelium | |
Wang et al. | Coculture of dorsal root ganglion neurons and differentiated human corneal stromal stem cells on silk‐based scaffolds | |
Zhou et al. | Combining PLGA scaffold and MSCs for brain tissue engineering: a potential tool for treatment of brain injury | |
WO2016140716A1 (en) | Injectable microtissue systems, devices, and methods | |
Galloway et al. | Characterization of human iPSC-RPE on a prosthetic Bruch's membrane manufactured from silk fibroin | |
Zhang et al. | Flexible Fabrication of Shape‐Controlled Collagen Building Blocks for Self‐Assembly of 3D Microtissues | |
O’Grady et al. | Development of an N-cadherin biofunctionalized hydrogel to support the formation of synaptically connected neural networks | |
Wang et al. | In vitro generation of mouse colon crypts | |
KR102075246B1 (ko) | 뇌신경계 질환 치료제의 스크리닝용 뇌기저핵 모사 칩 | |
Kashani et al. | An integrated microfluidic device for stem cell differentiation based on cell-imprinted substrate designed for cartilage regeneration in a rabbit model | |
Unagolla et al. | Recent advances in organoid engineering: A comprehensive review | |
KR102395801B1 (ko) | 뇌혈관 모사용 미세유체 디바이스 및 이를 포함하는 고효율 혈액뇌관문 모사 시스템 | |
Tong et al. | Compartmentalized microfluidic chambers enable long-term maintenance and communication between human pluripotent stem cell-derived forebrain and midbrain neurons | |
Huang et al. | Generation of interconnected neural clusters in multiscale scaffolds from human-induced pluripotent stem cells | |
Lu et al. | Tissue-engineered models for glaucoma research | |
EP3418375B1 (en) | Method for producing three-dimensional tissue having vascular system structure, and three-dimensional tissue comprising gel having vascular system structure | |
Liu et al. | Engineering Neurovascular Unit and Blood–Brain Barrier for Ischemic Stroke Modeling |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
RD01 | Notification of change of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7426 Effective date: 20210310 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20210310 |
|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20210928 |
|
A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A711 Effective date: 20210928 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20210928 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20220927 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20221220 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20230320 |
|
A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20230627 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20231012 |
|
A911 | Transfer to examiner for re-examination before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20231018 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20231219 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20240116 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 7422665 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |