CN117247905A - Construction method and application of hydrothorax and ascites-derived digestive tract tumor cell organoid model - Google Patents
Construction method and application of hydrothorax and ascites-derived digestive tract tumor cell organoid model Download PDFInfo
- Publication number
- CN117247905A CN117247905A CN202311515256.1A CN202311515256A CN117247905A CN 117247905 A CN117247905 A CN 117247905A CN 202311515256 A CN202311515256 A CN 202311515256A CN 117247905 A CN117247905 A CN 117247905A
- Authority
- CN
- China
- Prior art keywords
- organoid
- primary
- cell
- culture
- ascites
- 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.)
- Pending
Links
- 210000002220 organoid Anatomy 0.000 title claims abstract description 88
- 206010003445 Ascites Diseases 0.000 title claims abstract description 30
- 238000010276 construction Methods 0.000 title claims abstract description 21
- 206010048612 Hydrothorax Diseases 0.000 title claims abstract description 20
- 210000001035 gastrointestinal tract Anatomy 0.000 title claims abstract description 16
- 210000004881 tumor cell Anatomy 0.000 title description 5
- 210000004027 cell Anatomy 0.000 claims abstract description 56
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 32
- 108010082117 matrigel Proteins 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 9
- 239000006285 cell suspension Substances 0.000 claims abstract description 8
- 230000003211 malignant effect Effects 0.000 claims abstract description 8
- 210000004748 cultured cell Anatomy 0.000 claims abstract description 7
- 238000007877 drug screening Methods 0.000 claims abstract description 7
- 239000013049 sediment Substances 0.000 claims abstract description 7
- 238000012258 culturing Methods 0.000 claims abstract description 3
- 238000002156 mixing Methods 0.000 claims abstract description 3
- 239000003814 drug Substances 0.000 claims description 19
- 239000001963 growth medium Substances 0.000 claims description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 6
- 239000007640 basal medium Substances 0.000 claims description 6
- 239000012091 fetal bovine serum Substances 0.000 claims description 6
- 229960005322 streptomycin Drugs 0.000 claims description 6
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 6
- 239000002246 antineoplastic agent Substances 0.000 claims description 5
- 210000003743 erythrocyte Anatomy 0.000 claims description 5
- 229930012538 Paclitaxel Natural products 0.000 claims description 4
- 150000001413 amino acids Chemical class 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 4
- 229960001592 paclitaxel Drugs 0.000 claims description 4
- 210000002966 serum Anatomy 0.000 claims description 4
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 claims description 4
- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical compound C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 claims description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 3
- 102100028412 Fibroblast growth factor 10 Human genes 0.000 claims description 3
- 108090001047 Fibroblast growth factor 10 Proteins 0.000 claims description 3
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 claims description 3
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 claims description 3
- 229960004316 cisplatin Drugs 0.000 claims description 3
- 229960002949 fluorouracil Drugs 0.000 claims description 3
- 229960004768 irinotecan Drugs 0.000 claims description 3
- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 claims description 3
- 102000045246 noggin Human genes 0.000 claims description 3
- 108700007229 noggin Proteins 0.000 claims description 3
- DWAFYCQODLXJNR-BNTLRKBRSA-L oxaliplatin Chemical compound O1C(=O)C(=O)O[Pt]11N[C@@H]2CCCC[C@H]2N1 DWAFYCQODLXJNR-BNTLRKBRSA-L 0.000 claims description 3
- 229960001756 oxaliplatin Drugs 0.000 claims description 3
- GAGWJHPBXLXJQN-UORFTKCHSA-N Capecitabine Chemical compound C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](C)O1 GAGWJHPBXLXJQN-UORFTKCHSA-N 0.000 claims description 2
- GAGWJHPBXLXJQN-UHFFFAOYSA-N Capecitabine Natural products C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1C1C(O)C(O)C(C)O1 GAGWJHPBXLXJQN-UHFFFAOYSA-N 0.000 claims description 2
- 101100373143 Drosophila melanogaster Wnt5 gene Proteins 0.000 claims description 2
- ZDZOTLJHXYCWBA-VCVYQWHSSA-N N-debenzoyl-N-(tert-butoxycarbonyl)-10-deacetyltaxol Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=4C=CC=CC=4)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 ZDZOTLJHXYCWBA-VCVYQWHSSA-N 0.000 claims description 2
- 101710110302 R-spondin-1 Proteins 0.000 claims description 2
- 102100022762 R-spondin-1 Human genes 0.000 claims description 2
- 102000052549 Wnt-3 Human genes 0.000 claims description 2
- 108700020985 Wnt-3 Proteins 0.000 claims description 2
- 229960004117 capecitabine Drugs 0.000 claims description 2
- 229960003668 docetaxel Drugs 0.000 claims description 2
- 230000002934 lysing effect Effects 0.000 claims description 2
- 239000008188 pellet Substances 0.000 claims description 2
- 229940127089 cytotoxic agent Drugs 0.000 claims 2
- 238000004113 cell culture Methods 0.000 description 19
- 206010027476 Metastases Diseases 0.000 description 18
- 230000009401 metastasis Effects 0.000 description 18
- 239000000523 sample Substances 0.000 description 18
- 241000282326 Felis catus Species 0.000 description 17
- 208000005718 Stomach Neoplasms Diseases 0.000 description 12
- 229940079593 drug Drugs 0.000 description 12
- 206010017758 gastric cancer Diseases 0.000 description 12
- 201000011549 stomach cancer Diseases 0.000 description 12
- 230000000694 effects Effects 0.000 description 11
- 238000011282 treatment Methods 0.000 description 11
- 206010025538 Malignant ascites Diseases 0.000 description 6
- 238000001556 precipitation Methods 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 206010009944 Colon cancer Diseases 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000002512 chemotherapy Methods 0.000 description 3
- 229940044683 chemotherapy drug Drugs 0.000 description 3
- 239000000306 component Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 201000002528 pancreatic cancer Diseases 0.000 description 3
- 208000008443 pancreatic carcinoma Diseases 0.000 description 3
- 239000013610 patient sample Substances 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 2
- 206010026673 Malignant Pleural Effusion Diseases 0.000 description 2
- 208000015634 Rectal Neoplasms Diseases 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000002038 chemiluminescence detection Methods 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
- 238000005138 cryopreservation Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 235000020776 essential amino acid Nutrition 0.000 description 2
- 239000003797 essential amino acid Substances 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 238000011463 hyperthermic intraperitoneal chemotherapy Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 238000007422 luminescence assay Methods 0.000 description 2
- 230000003278 mimic effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 206010038038 rectal cancer Diseases 0.000 description 2
- 201000001275 rectum cancer Diseases 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 206010006895 Cachexia Diseases 0.000 description 1
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 101000825954 Homo sapiens R-spondin-1 Proteins 0.000 description 1
- 208000005016 Intestinal Neoplasms Diseases 0.000 description 1
- 102000006833 Multifunctional Enzymes Human genes 0.000 description 1
- 108010047290 Multifunctional Enzymes Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000003445 biliary tract Anatomy 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 238000003570 cell viability assay Methods 0.000 description 1
- 238000012200 cell viability kit Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- 238000011281 clinical therapy Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 102000050526 human RSPO1 Human genes 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 201000002313 intestinal cancer Diseases 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000011462 intraperitoneal chemotherapy Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 229920000117 poly(dioxanone) Polymers 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
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
- C12N5/0693—Tumour cells; Cancer cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/44—Thiols, e.g. mercaptoethanol
-
- 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
- C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
-
- 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
- C12N2501/40—Regulators of development
- C12N2501/415—Wnt; Frizzeled
-
- 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
- C12N2501/70—Enzymes
- C12N2501/72—Transferases [EC 2.]
- C12N2501/727—Kinases (EC 2.7.)
-
- 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
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Oncology (AREA)
- General Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a construction method of a primary cell organoid model and application thereof in drug screening, wherein the construction method comprises the following steps: (1) Centrifuging malignant hydrothorax and ascites samples from digestive tract tumor patients, collecting cell sediment, and preparing single-cell suspension; (2) Primary culturing the single cell suspension to obtain primary cultured cells; (3) And mixing the primary cultured cells with matrigel for organoid culture to obtain a primary cell organoid model. By adopting the method, the organoids cultivated by using the primary cells have higher uniformity and more quantity of organoids cultivated successfully, and meanwhile, the problems of passage and limited library establishment of the organoids in the organoid cultivation field are solved.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a construction method of a hydrothorax and ascites source digestive tract tumor cell organoid model and application thereof in medicine screening.
Background
Digestive tract tumor peritoneal metastasis (Gastrointestinal cancer peritoneal metastasis, GICPM) is a major bottleneck problem in current clinical therapies, and the cause of death in patients with advanced digestive tract tumors is mostly related to peritoneal metastasis cachexia. Peritoneal metastasis is a common metastasis site of digestive tract tumors including gastric cancer, colorectal cancer, biliary tract tumors, pancreatic cancer and the like, and patients with tumors that develop peritoneal metastasis often have poor prognosis and short total Survival (OS). Peritoneal metastasis is one of the common distant sites of gastric cancer, and studies have shown that peritoneal metastasis accounts for 50% of distant metastasis of gastric cancer.
In recent years, with the popularization of clinical diagnosis and treatment technologies such as laparoscopic technology, intraperitoneal chemotherapy and intraperitoneal hot infusion chemotherapy (hyperthermic intraperitoneal chemotherapy, HIPEC), the promotion of multidisciplinary treatment modes, and the deep clinical and basic research in the fields related to peritoneal transfer treatment, the overall prognosis of patients with peritoneal transfer is improved to a certain extent than in the past. Although attempts at peritoneal transfer reduction in combination with thermal infusion chemotherapy suggest that overall survival may be prolonged in some studies, most of the studies are retrospective analyses and preliminary conclusions show that patients receiving peritoneal reduction need to be strictly screened, still few patients who can benefit from this approach. Although the present second generation high throughput genome sequencing (NGS) can guide clinical medication selection to a certain extent, the characteristics of strong tumor heterogeneity, large individual differences of patients and the like exist, so that the curative effect of the medicine cannot be rapidly and effectively evaluated in an individuation mode, and the selection of clinical medication can be guided accurately.
Organoids (Organoids) refer to tissue analogs having a spatial structure formed by in vitro three-dimensional (3D) culture using adult stem cells or pluripotent stem cells. Although organoids are not truly human organs, they mimic real organs in structure and function, maximally mimic in vivo tissue structure and function, and enable subculture. At present, although the construction of a Patient tumor-derived organoid model (PDOs) can be used for screening clinical drugs to guide the individual accurate treatment of patients, there are still some limitations in the construction and use of organoid models: (1) passage limitation: in order to avoid the difference between the phenotype after the passage of the tumor organoid and the in-vivo situation, the general passage of the organoid from the tumor needs to be controlled within 2 to 3 generations, and the maximum time is not more than 5 generations; (2) promiscuous cell effects: some of the impurity cells such as fibroblasts and the like are mixed in the tumor tissue, and the growth of the impurity cells in a large amount influences the growth state of the tumor organoids; (3) The organoid biological sample bank construction requires a large sample size: most of the organoids have a success rate of about 63-70%, and organoids are limited in subculture, so that a larger sample size is required for library construction.
In addition, organoid culture based on malignant hydrothorax and ascites sources still has no mature standard culture system at present, because hydrothorax and ascites are obviously different from the tumor microenvironment of solid tumor tissues, the body fluid components in hydrothorax and ascites are very complex, and the reactivity of different patients to drug treatment is also greatly different. Patients with advanced digestive tract tumors mostly suffer from malignant hydrothorax and ascites which are difficult to control due to distant metastasis, and the survival prognosis of the patients is seriously affected. Therefore, the construction of the tumor organoid culture system derived from malignant hydrothorax and ascites of the digestive tract tumor has extremely important application value for researching the digestive tract tumor and guiding the individual and accurate treatment of patients.
Disclosure of Invention
In order to solve at least part of the technical problems in the prior art, the invention provides a construction method of a primary cell organoid model and application thereof in drug screening. In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a method for constructing a primary cell organoid model, comprising the steps of:
(1) Centrifuging the hydrothorax and ascites sample, collecting cell sediment, and preparing single-cell suspension;
(2) Primary culturing the single cell suspension to obtain primary cultured cells;
(3) And mixing the primary cultured cells with matrigel for organoid culture to obtain a primary cell organoid model.
In a preferred embodiment, the medium of primary culture comprises the following components:
IMDM basal medium; 10% fetal bovine serum; 8-12 mu M Y-27632;0.05-0.15w/w% penicillin-streptomycin; 0.05-0.15w/w% beta-mercaptoethanol and 0.5-1.5w/w% MEM optional amino acid solution.
In another preferred embodiment, the organoid culture medium comprises:
DMEM/F12 basal medium; 3% -5% of fetal bovine serum; 50-100 ng/mL EGF;50-200 ng/mL FGF-10;50-200 ng/mL R-Spondin-1;50-150 ng/mL Noggin;50-100 ng/mL Wnt-3 a, 3-7w/w% KSR serum replacement; 8-12 mu M Y-27632;0.05-0.15w/w% penicillin-streptomycin; 0.05-0.15w/w% beta-mercaptoethanol; 5-1.5w/w% MEM optional amino acid solution.
The invention also relates to application of the organoid model constructed by the construction method in drug screening.
In a preferred embodiment of the present invention, the drug is a chemotherapeutic drug including, but not limited to, oxaliplatin, cisplatin, fluorouracil, capecitabine, paclitaxel, docetaxel, and irinotecan.
The invention has the following beneficial technical effects：
1. In the technical scheme of the invention, the method comprises the step of collecting digestive tract tumor cell precipitation, and the step of primary cell culture is also added. This step allows for rapid expansion of the more dry tumor cells from the mixed cells in the pellet in a short period of time, helping the growth of the tumor organoid and retaining a large number of "seeds" of the cultured organoid.
2. The culture medium component for primary cell culture provided by the invention is beneficial to improving the efficiency and the cell activity of primary culture.
3. The organoid medium composition provided by the invention helps to improve organoid uniformity and number.
4. The organoid model based on the 3D culture technology solves the defect that the traditional 2D culture primary cells lack structural characteristics of tumor histology, so that the tumor cannot fully respond to the reactivity of the tumor to the medicine in vivo.
5. The invention solves the technical problem that the current organoid culture system based on hydrothorax and ascites sources is not mature.
6. The culture system provided by the invention can rapidly amplify and purify primary cells and organoids from hydrothorax and ascites in a short period, a large number of primary cells ensure sufficient seeds for the construction of organoids, the defects of restriction of organoids passage and change of growth state after passage can be well overcome, and the organoid model constructed by using the primary cells has higher growth uniformity, more organoids and better growth state, thereby being beneficial to the construction of a organoid biological sample library.
Drawings
Fig. 1a: a primary cell culture organoid effect diagram of gastric cancer ascites sample sources;
fig. 1b: a gastric cancer ascites sample source cell precipitation culture organoid effect diagram;
fig. 1c: in the organoid culture stage, the quantity of organoids cultured by directly using an ascites precipitation cell sample is compared with the quantity of organoids cultured by constructing a primary cell sample from an ascites source;
fig. 2: primary cell culture, primary cell organoid culture and organoid pathology HE staining identification charts of ascites samples derived from different digestive tract tumors (gastric cancer, intestinal cancer and pancreatic cancer) show that organoids cultured by a primary cell organoid model construction system have typical cancer structures; wherein F3P1 refers to the P1 generation organoids of the F3 primary cells in culture;
fig. 3: based on the case display of common chemotherapeutic drug screening results of primary cell organoid models derived from ascites of different gastric cancer patients (the relative percentage quantification of live cells for drug sensitivity is detected by treating different drugs for 96 hours), the figure shows that the sensitivity of different patients to different chemotherapeutic drugs is different.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present invention, it is understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.
Example 1: cultivation and construction of digestive tract tumor organoid model
1.1 Reagent consumable preparation
(1) Consumable: 50 A mL centrifuge tube, a 70 mu m sterile filter, a 1.5 mL EP tube, a 5 mL rubber head dropper, a 10 mu L-1000 mu L pipette tip, a 24-hole cell culture plate (Corning), and a 10 cm cell culture dish (Corning);
(2) Reagent: HBSS buffer (Gibco); DPBS balanced salt buffer (Gibco); 0.25% EDTA-trypsin (Gibco, cat 25200072); type II collagenase (Gibco, cat: 17101-015); matrigel (R & D systems, cat: 3536-005-02); DMSO (Gibco) (3) primary culture medium: IMDM basal Medium (Iscove's Modified Dulbeccos's Medium, gibco, cat 12440053); 10% fetal bovine serum (Gibco,); 10. m Y-27632 (PeproTech, cat: 1293823); 0.1% penicillin-streptomycin (Gibco); 0.1% beta-mercaptoethanol (Gibco, cat: 21985023); 1% MEM non-essential amino acid solution (Gibco, cat: 11140050);
(4) Organoid medium: DMEM/F12 basal medium (Gibco, cat: 11330032); 3% -5% fetal bovine serum (Gibco); 50-100 ng/mL EGF (PeproTech, cat: AF-100-15); 50-200 ng/mL FGF-10 (PeproTech, cat: 100-26); 50-200 ng/mL Human R-Spondin-1 (PeproTech, cat: 120-38); 50-150 ng/mL Noggin (PeproTech, cat: 120-10C); 50-100 ng/mL Wnt-3α (PeproTech, cat: 315-20); 5% Gibco ™ Knock Out ™ serum replacement (KSR, gibco, cat: 10828028); 10. m Y-27632 (PeproTech, cat: 1293823); 0.1% penicillin-streptomycin (Gibco); 0.1% beta-mercaptoethanol (Gibco, cat: 21985023); 1% MEM non-essential amino acid solution (Gibco, cat: 11140050);
(5) Frozen stock solution: 10% dmso+90% foetal calf serum.
1.2 Hydrothorax and ascites sample pretreatment
(1) Treatment of hydrothorax and ascites: 100-300 mL from digestive tract tumor patients (27 malignant hydrothorax and ascites samples from Beijing tumor hospitals including 9 gastric cancer patient samples, 9 colorectal cancer patient samples and 9 pancreatic cancer patient samples are collected, informed consent of the patients is obtained and approval of the ethical committee of the hospital is obtained), and fresh and isolated malignant hydrothorax and ascites samples are transferred into a 50 mL centrifuge tube under aseptic conditions, wherein the temperature is 4 ℃, the speed is 2000rpm, and the time is 5min, and cell sediment is collected;
(2) Lysing erythrocytes (whether or not to operate according to actual need): observing whether obvious erythrocyte precipitation exists in the cell precipitation, if so, treating the cells with 3-5 mL of RBC lysate for 3-5 min, then stopping the lysis with 5-10 mL of PBS, 2000rpm for 5min, centrifuging, and discarding the supernatant; continuing the next step without cracking red;
(3) Preparing single cell suspension: adding a proper amount of DPBS buffer solution to resuspend cell sediment (after the end of the erythrocyte, preparing single cell suspension by a 70 mu m filter screen, then 2000rpm for 5min, discarding the supernatant, and washing the DPBS buffer solution for 2 times;
(4) Cell count: the cells were resuspended by adding an appropriate amount of DPBS buffer and counted for use using a cytometer.
1.3 Primary cell culture
(1) Cell culture dish pretreatment
Diluting 50-100 mu L matrigel into DPBS (differential pressure BS) pre-cooled by 5 mL (0-4 ℃), transferring the 5 mL mixed solution into a 10 cm cell culture dish to uniformly cover the whole dish bottom, and standing in a cell culture incubator at 37 ℃ for later use, wherein the temperature of the DPBS is higher than 4-8 h;
(2) Primary cell culture
Proper amount (about 1x 10) 6 Dish) the primary cells prepared in 1.2 are inoculated into a 10 cm cell culture dish which is pretreated and coated by matrigel, then 8-10 mL primary cell culture medium is added for primary cell culture and marked as F0 generation, and the primary cell culture is carried out under the conditions of 37 ℃,5% CO2 and 95% relative saturation humidity;
(3) Primary cell replacement fluid
The first inoculation culture (F0 generation) can be changed for the first time 5-7 days after inoculation, and the culture is continued and the growth state is observed after the liquid change;
(4) Primary cell passaging and cryopreservation
And (5) when the cell confluence reaches about 90%, the primary cells can be passaged. The primary cells are collected by trypsinization in a 15 mL centrifuge tube at 1000 rpm for 5min, the supernatant is discarded, and then the primary cells are cultured according to a ratio of 1:2 (marked as F1,2,3 … … n, n is less than or equal to 10, and the primary cells are generally ensured to be in biological consistency with F0 generation samples, and F5 generation or less cell samples are used for routine research) or are subjected to gradient cooling and freezing storage at-80 ℃ and then are transferred to a liquid nitrogen tank for long-term storage.
1.4 Organoid culture (direct ascites cell precipitation culture and ascites primary cell culture are used as contrast)
(1) Organoid inoculation: taking appropriate amount of hydrothorax and ascites cell sediment (F0) or primary cultured cells (F1-Fn) according to cell counting result, thawing overnight at 4deg.C with matrigel, and re-suspending cell sediment according to 5-8X10 4 cell/point/well inoculating gel drop mixed with cells into 24-well cell culture plate (the matrigel needs to be carefully operated on ice during use), and avoiding air bubbles during inoculation;
(2) Organoid culture: reversing 24-well plate inoculated with cell-containing gel ball in cell incubator, adding 1 mL/well organoid culture solution (see above 1.1 (4)) after matrix gel is sufficiently coagulated (1-2 h), and heating at 37deg.C and 5% CO 2 Relative saturation humidityCulturing under the condition of 95%;
(3) Organoid exchange: the culture condition should be followed in time during the sample culture period, the liquid is changed for the first time after 5-7 days of culture, and the liquid is changed every 3-5 days for observing the growth state of organoids during the culture process;
(4) Organoid passaging and cryopreservation: discarding the culture medium, adding 200 mu L of type II collagenase with the working concentration of 1-2mg/mL into each hole, blowing by using a pipetting gun, placing in an incubator for digestion for about 30 minutes under the condition of 5% CO2 at 37 ℃, digesting large organoids into small organoids suitable for enlarged culture growth, transferring the small organoids into a 1.5 mL EP tube by using a pipetting gun after digestion, centrifuging at 2000rpm for 5 minutes to obtain organoids suitable for culture, washing 3 times by using the organoid culture medium, freezing again matrigel/cell spheres (specific operation reference step 1.4 (2)) or using an organoid frozen stock solution-80 ℃ gradient, and then transferring the organoids into a liquid nitrogen tank for long-term storage.
(5) Organoid characterization: the pathological results of the agar paraffin embedding and the slice HE staining (shown in figure 2) show that the cultured organoid samples all keep the cancer tissue morphology of tissue sources, prove that the organoid is successfully cultured, and the cultured organoids can be used for screening clinical medicines to guide personalized treatment of patients or used as a better in vitro research model for other preclinical researches.
Compared with the direct use of the precipitated cell culture, the organoid model constructed by the primary cells cultured from the ascites of the patient has better growth state; and a higher uniformity of organoids using primary cell culture and a greater number of organoids were observed with successful culture (fig. 1 c).
EXAMPLE 2 drug screening
2.1 construction of sample sources and clinical data for organoids:
sample 1: malignant pleural effusion of patients with gastric cancer and pleural metastasis;
sample 2: malignant ascites of patients with gastric cancer and peritoneal metastasis;
sample 3: malignant ascites of patients with rectal cancer accompanied by peritoneal metastasis;
sample 4: malignant ascites of patients with colon cancer and peritoneal metastasis.
(patients were all from Beijing tumor Hospital, and sample collection was informed consent and approved by the Hospital ethical committee)
Samples 1-4 are all patients with advanced (stage IV) clinical diagnosis of tumors, and according to the previous medical history data of the patients, the patient 1 and the patient 2 are treated by oxaliplatin and have a certain control effect on the tumors; patients 3 and 4 also received chemotherapy of the peritoneal perfusion of albumin paclitaxel with some effect on ascites control.
The above samples 1 to 4 were subjected to model construction culture using the culture system described in example 1, and then to drug screening test, respectively.
2.2 Drug preparation
2.3 Screening step
(1) Inoculating the organoid sample derived from the hydrothorax and ascites after the expansion culture into a 96-well plate, and controlling the organoid in each well to be between 20 and 30, wherein the culture medium in each well is 100 mu L;
(2) Concentration gradients of 1000×, 10000× were set for each group of 3 biological replicates, and 100 μl of drug was added for treatment. Simultaneously setting a blank control group without adding medicine, wherein the volume of a culture system of each hole is 200 uL;
(3) Performing conventional culture, performing organoid proliferation activity detection on the 4 th to 6 th days after drug treatment, and evaluating drug sensitivity according to organoid activity, wherein the organoid proliferation activity detection comprises the following specific steps:
(1) detection reagent:
cell viability assay kit (Biyun C0068, cellTiter-Lumi ™ Plus luminescence assay kit) of CellTiter-Lumi ™ Plus luminescence assay was selected for detecting proliferation of cells in a sample.
(2) Cell viability assay
a. The cell culture plates were removed and equilibrated at room temperature for 10-15 minutes.
b. 50-100. Mu.L of CellTiter-Lumi ™ Plus luminometric assay reagents were added to each well of a 96-well plate.
c. Shaking for 2 min at room temperature to promote cell lysis.
d. Incubation at room temperature (about 25 ℃) for 10-15 minutes stabilizes the luminescent signal.
e. Then transferred to a 96-well plate suitable for chemiluminescent detection while cell-free culture wells are set as negative controls.
f. And (3) performing chemiluminescence detection by using a multifunctional enzyme-labeled instrument with a chemiluminescence detection function.
g. The relative viability of the cells was directly calculated from the chemiluminescent readings.
2.4 Screening results:
the drug sensitivity evaluation results are shown in FIG. 3. The results show that case 1: malignant pleural effusion source samples of patients with gastric cancer and pleural metastasis are mainly sensitive to 5-fluorouracil; case 2: malignant ascites source samples of patients with gastric cancer and peritoneal metastasis are mainly sensitive to cisplatin; case 3: the malignant ascites source sample of the patient with rectal cancer and peritoneal metastasis is mainly sensitive to irinotecan; case 4: malignant ascites samples of patients with colon cancer and peritoneal metastasis are mainly sensitive to paclitaxel.
Comparing with the past medical history data of patients, the organoid model constructed by the culture system provided by the invention can be used for guiding the operation feasibility of the individuation treatment by clinical medicine screening.
While the invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Various modifications or changes may be made to the exemplary embodiments of the present disclosure without departing from the scope or spirit of the invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.
Claims (8)
1. The construction method of the primary cell organoid model is characterized by comprising the following steps:
(1) Centrifuging malignant hydrothorax and ascites samples from digestive tract tumor patients, collecting cell sediment, and preparing single-cell suspension;
(2) Primary culturing the single cell suspension to obtain primary cultured cells of malignant hydrothorax and ascites sources of the digestive tract tumor;
(3) And mixing the primary cultured cells with matrigel for organoid culture to obtain a primary cell organoid model.
2. The method of claim 1, further comprising the step of lysing the red blood cells after collecting the cell pellet.
3. The construction method according to claim 1, the medium of the primary culture comprising: IMDM basal medium; 10% fetal bovine serum; 8-12 mu M Y-27632;0.05-0.15w/w% penicillin-streptomycin; 0.05-0.15w/w% beta-mercaptoethanol and 0.5-1.5w/w% MEM optional amino acid solution.
4. The method of claim 1, wherein the organoid culture medium comprises: DMEM/F12 basal medium; 3% -5% of fetal bovine serum; 50-100 ng/mL EGF;50-200 ng/mL FGF-10;50-200 ng/mL R-Spondin-1;50-150 ng/mL Noggin;50-100 ng/mL Wnt-3 a, 3-7w/w% KSR serum replacement; 8-12 mu M Y-27632;0.05-0.15w/w% penicillin-streptomycin; 0.05-0.15w/w% beta-mercaptoethanol; 5-1.5w/w% MEM optional amino acid solution.
5. An organoid model constructed by the construction method of any of claims 1-4.
6. Use of the organoid model of claim 5 in drug screening.
7. The use according to claim 6, wherein the medicament is a chemotherapeutic agent.
8. The use according to claim 7, wherein the chemotherapeutic agent comprises oxaliplatin, cisplatin, fluorouracil, capecitabine, paclitaxel, docetaxel, and irinotecan.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311515256.1A CN117247905A (en) | 2023-11-15 | 2023-11-15 | Construction method and application of hydrothorax and ascites-derived digestive tract tumor cell organoid model |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311515256.1A CN117247905A (en) | 2023-11-15 | 2023-11-15 | Construction method and application of hydrothorax and ascites-derived digestive tract tumor cell organoid model |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117247905A true CN117247905A (en) | 2023-12-19 |
Family
ID=89129809
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311515256.1A Pending CN117247905A (en) | 2023-11-15 | 2023-11-15 | Construction method and application of hydrothorax and ascites-derived digestive tract tumor cell organoid model |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117247905A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110373388A (en) * | 2019-04-24 | 2019-10-25 | 博奥生物集团有限公司 | A kind of culture medium and organoid cultural method for organoid culture |
| CN111172111A (en) * | 2019-12-26 | 2020-05-19 | 浙江科途医学科技有限公司 | Method for preparing suspension tumor cell organoid by using malignant pleural effusion and ascites |
| CN111575237A (en) * | 2020-05-22 | 2020-08-25 | 江苏信安佳医疗科技有限公司 | Special culture medium and culture method for breast cancer stentless organoid |
| CN114426949A (en) * | 2022-02-25 | 2022-05-03 | 重庆嘉士腾生物科技有限公司 | Culture medium for establishing pancreas or pancreatic cancer organoid, method and application |
| WO2022115433A1 (en) * | 2020-11-25 | 2022-06-02 | Xilis, Inc. | Micro-organospheres for use in personalized medicine and drug development |
-
2023
- 2023-11-15 CN CN202311515256.1A patent/CN117247905A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110373388A (en) * | 2019-04-24 | 2019-10-25 | 博奥生物集团有限公司 | A kind of culture medium and organoid cultural method for organoid culture |
| CN111172111A (en) * | 2019-12-26 | 2020-05-19 | 浙江科途医学科技有限公司 | Method for preparing suspension tumor cell organoid by using malignant pleural effusion and ascites |
| CN111575237A (en) * | 2020-05-22 | 2020-08-25 | 江苏信安佳医疗科技有限公司 | Special culture medium and culture method for breast cancer stentless organoid |
| WO2022115433A1 (en) * | 2020-11-25 | 2022-06-02 | Xilis, Inc. | Micro-organospheres for use in personalized medicine and drug development |
| CN114426949A (en) * | 2022-02-25 | 2022-05-03 | 重庆嘉士腾生物科技有限公司 | Culture medium for establishing pancreas or pancreatic cancer organoid, method and application |
Non-Patent Citations (3)
| Title |
|---|
| ANDREA MAZZOCCHI等: "Pleural Effusion Aspirate for Use in 3D Lung Cancer Modeling and Chemotherapy Screening", ACS BIOMATER SCI ENG, vol. 5, no. 4, pages 1937 - 1943 * |
| 刘丹等: "UGT1A在伊立替康治疗转移性结直肠癌中的疗效与不良反应预测价值", 临床肿瘤学杂志, vol. 21, no. 7, pages 654 - 660 * |
| 曾云丽等: "1231例结直肠癌类器官培养成功率的影响因素分析", 中华胃肠外科杂志, vol. 26, no. 8, pages 780 - 786 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2021208130A1 (en) | Culture medium for esophageal squamous cell carcinoma epithelial cells, culture method, and application thereof | |
| CN110592022B (en) | Special culture medium for lung tumor organoid and 3D culture method without stent | |
| WO2021088119A1 (en) | Primary breast epithelial cell culture medium, culture method, and use thereof | |
| CN107151645A (en) | A kind of method and culture medium that in vitro individuation drug test is provided for lung cancer | |
| WO2021184408A1 (en) | Culture medium for primary cells of gastric cancer, and cultivation method therefor | |
| CN109837242A (en) | A kind of cultural method of the foreign bodies for scientific experiment | |
| CN114317444B (en) | Intestinal cancer organoid culture solution, culture reagent composition and culture method | |
| CN112831471A (en) | Culture medium, culture method and detection method for thyroid cancer organoid | |
| CN113278586A (en) | Culture medium and culture method for culturing thyroid cancer organoid | |
| CN112852714A (en) | Method for constructing in-situ primary lung cancer animal model | |
| Zhang et al. | A pre‑clinical model combining cryopreservation technique with precision‑cut slice culture method to assess the in vitro drug response of hepatocellular carcinoma | |
| CN102329775B (en) | A kind of human pancreatic cancer cell to gemcitabine resistance and application thereof | |
| CN114075539A (en) | Method for constructing in-situ primary bladder cancer animal model | |
| CN114908039A (en) | Culture medium for stomach cancer organoid and culture method without bracket thereof | |
| CN109355261B (en) | Urinary bladder cancer cell culture medium and urinary bladder cancer cell in-vitro culture method | |
| CN113943755A (en) | Method for constructing in-situ primary esophageal cancer animal model | |
| CN117247905A (en) | Construction method and application of hydrothorax and ascites-derived digestive tract tumor cell organoid model | |
| US20130196349A1 (en) | In Vitro Tumor Metastasis Model | |
| CN115992095A (en) | Method for constructing digestive tract tumor patient-derived organoids based on endoscopic biopsy tumor tissue samples | |
| CN114874989B (en) | Method for capturing circulating tumor cells | |
| EP4368706A1 (en) | Culture medium and culture method for lung cancer epithelial cells, and application thereof | |
| CN106929475B (en) | Method for counting liver cancer circulating tumor cells in vitro based on 3D temperature-sensitive type biological gel hanging drop culture method | |
| CN111440769A (en) | Human hepatocyte hepatoma cell strain and application thereof | |
| CN114891735B (en) | Mammary gland epithelial organoid model construction method | |
| CN117467616B (en) | Special culture medium for preparing oral cancer micro-tumor model, related method product and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |