CN116536264A - 一种结肠癌类器官无血清专用培养基 - Google Patents
一种结肠癌类器官无血清专用培养基 Download PDFInfo
- Publication number
- CN116536264A CN116536264A CN202210950210.1A CN202210950210A CN116536264A CN 116536264 A CN116536264 A CN 116536264A CN 202210950210 A CN202210950210 A CN 202210950210A CN 116536264 A CN116536264 A CN 116536264A
- Authority
- CN
- China
- Prior art keywords
- culture medium
- colon cancer
- culture
- medium
- serum
- 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
- 206010009944 Colon cancer Diseases 0.000 title claims abstract description 63
- 208000029742 colonic neoplasm Diseases 0.000 title claims abstract description 59
- 210000002220 organoid Anatomy 0.000 title claims abstract description 54
- 239000004017 serum-free culture medium Substances 0.000 title claims description 7
- 239000001963 growth medium Substances 0.000 claims abstract description 47
- 239000000654 additive Substances 0.000 claims abstract description 17
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims abstract description 12
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims abstract description 12
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims abstract description 11
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims abstract description 11
- 238000004113 cell culture Methods 0.000 claims abstract description 9
- 239000003102 growth factor Substances 0.000 claims abstract description 9
- 239000000243 solution Substances 0.000 claims abstract description 8
- 241000283690 Bos taurus Species 0.000 claims abstract description 6
- 102000029816 Collagenase Human genes 0.000 claims abstract description 6
- 108060005980 Collagenase Proteins 0.000 claims abstract description 6
- 102100028412 Fibroblast growth factor 10 Human genes 0.000 claims abstract description 6
- 102100035290 Fibroblast growth factor 13 Human genes 0.000 claims abstract description 6
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims abstract description 6
- 101000917237 Homo sapiens Fibroblast growth factor 10 Proteins 0.000 claims abstract description 6
- 102000004877 Insulin Human genes 0.000 claims abstract description 6
- 108090001061 Insulin Proteins 0.000 claims abstract description 6
- 229960002424 collagenase Drugs 0.000 claims abstract description 6
- 229960000890 hydrocortisone Drugs 0.000 claims abstract description 6
- 239000003112 inhibitor Substances 0.000 claims abstract description 6
- 229940125396 insulin Drugs 0.000 claims abstract description 6
- 230000001817 pituitary effect Effects 0.000 claims abstract description 6
- 150000003384 small molecules Chemical class 0.000 claims abstract description 6
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims abstract description 4
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 claims abstract description 4
- 239000007995 HEPES buffer Substances 0.000 claims abstract description 4
- 229960005322 streptomycin Drugs 0.000 claims abstract description 4
- 239000012583 B-27 Supplement Substances 0.000 claims abstract description 3
- 102000013814 Wnt Human genes 0.000 claims description 10
- 230000000996 additive effect Effects 0.000 claims description 10
- 239000003636 conditioned culture medium Substances 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 102000045246 noggin Human genes 0.000 claims description 9
- 108700007229 noggin Proteins 0.000 claims description 9
- 239000007640 basal medium Substances 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 6
- 210000001519 tissue Anatomy 0.000 claims description 6
- 238000011161 development Methods 0.000 claims description 5
- 102000034356 gene-regulatory proteins Human genes 0.000 claims description 5
- 108091006104 gene-regulatory proteins Proteins 0.000 claims description 5
- 230000001105 regulatory effect Effects 0.000 claims description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims 1
- 239000012679 serum free medium Substances 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 14
- 238000012136 culture method Methods 0.000 abstract description 9
- 238000000338 in vitro Methods 0.000 abstract description 5
- 230000001788 irregular Effects 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 18
- 238000011160 research Methods 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 238000007877 drug screening Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 238000009795 derivation Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000010874 in vitro model Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 102000010400 1-phosphatidylinositol-3-kinase activity proteins Human genes 0.000 description 1
- 210000003771 C cell Anatomy 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 102100021022 Gastrin Human genes 0.000 description 1
- 108010052343 Gastrins Proteins 0.000 description 1
- 101000851176 Homo sapiens Pro-epidermal growth factor Proteins 0.000 description 1
- 108010003272 Hyaluronate lyase Proteins 0.000 description 1
- 102000001974 Hyaluronidases Human genes 0.000 description 1
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 1
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 1
- 108091007960 PI3Ks Proteins 0.000 description 1
- 229960004308 acetylcysteine Drugs 0.000 description 1
- 239000012574 advanced DMEM Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- AOXOCDRNSPFDPE-UKEONUMOSA-N chembl413654 Chemical compound C([C@H](C(=O)NCC(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@H](CCSC)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](C)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]1N(CCC1)C(=O)CNC(=O)[C@@H](N)CCC(O)=O)C1=CC=C(O)C=C1 AOXOCDRNSPFDPE-UKEONUMOSA-N 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 description 1
- 229960002986 dinoprostone Drugs 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 229960002773 hyaluronidase Drugs 0.000 description 1
- 230000033607 mismatch repair Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- GVUGOAYIVIDWIO-UFWWTJHBSA-N nepidermin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C(C)C)C(C)C)C1=CC=C(O)C=C1 GVUGOAYIVIDWIO-UFWWTJHBSA-N 0.000 description 1
- 229960003966 nicotinamide Drugs 0.000 description 1
- 235000005152 nicotinamide Nutrition 0.000 description 1
- 239000011570 nicotinamide Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000005406 washing Methods 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
-
- 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
- C12N5/0679—Cells of the gastro-intestinal tract
-
- 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
-
- 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
- C12N2500/32—Amino acids
-
- 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
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
-
- 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
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- 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
- C12N2501/30—Hormones
- C12N2501/33—Insulin
-
- 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
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
-
- 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.)
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Oncology (AREA)
- Gastroenterology & Hepatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及肿瘤类器官培养领域。具体涉及一种结肠癌类器官无血清专用培养基和培养方法。所述培养基为一种无需添加重组蛋白的无血清专用培养基，由基础培养基和添加物组成，基础培养基为含有1％青‑链霉素、1％HEPES缓冲液、1％GlutaMax溶液的High GlucoseDMEM/F12培养基，添加物包括生长因子FGF2、FGF10，胰岛素，氢化可的松，小分子抑制剂Y‑27632、SB202190，牛垂体提取物(BPE)、B27supplement。将患者来源结肠癌样本用胶原酶消化，重悬于冷CultrexTM growth factor reduced BME type2中，加入培养板，37度固化后，每孔加入上述培养基，放入细胞培养箱，每2天更换培养基，一周即可获得所需结肠癌类器官。本发明所述培养基可支持结肠癌细胞体外生长，呈现典型球状或不规则团块肿瘤类器官形态，为后续进一步研究提供较好的研究样本。
Description
技术领域
本发明涉及一种肿瘤类器官培养技术领域，尤其涉及一种结肠癌类器官的无血清专用培养基和培养方法。
背景技术
结肠癌是消化道常见的恶性肿瘤，我国国家癌症中心2022年报告的发病人数超过40万，位列恶性肿瘤发病率的第四位，死亡人数将近20万，位列恶性肿瘤死亡率的第五位。随着分子生物学技术的进展，已经揭示了部分对结肠癌发病机制至关重要的信号通路，如WNT、PI3K、Ras-MAPK、P53等信号通路调节蛋白与结肠癌关系密切，同时也发现了DNA错配修复、突变和染色体易位与结肠癌的预后和治疗反应等密切相关。因此认为结肠癌是一种高度异质性疾病，不同亚型在遗传表型、病理特征和临床表现均不相同，虽然个体患者的基因组变化可以非常详细和低成本地评估疾病进展，但这些数据仍无法解释在临床治疗过程中患者的预后、药物反应或治疗结果的巨大差异。传统的结肠癌体外模型通常是使用结肠癌细胞系在二维平面培养条件下建立的，与体内肿瘤的三维生长环境不同，导致二维培养条件下的结肠癌细胞生物学特征与体内肿瘤真实情况有较大差异，因此需要构建基于患者来源的肿瘤类器官体外模型，用于分析个体患者基因型与表型之间的相关性和准确模拟体内肿瘤的生物学特征和对药物治疗的反应，结肠癌类器官模型对于个体化治疗、临床药敏检测、肿瘤研究、药物测试及高通量筛选有巨大的应用价值。
Van de Wetering et al.在2015年首先建立了一种结肠癌类器官的培养方法(Van de Wetering,M.,et al.(2015).Prospective derivation of a living organoidbiobank of colorectal cancer patients.Cell 161,933-945.)，来源于结肠癌患者的肿瘤类器官概括了结肠癌的体细胞拷贝数和突变谱，适合高通量药物筛选，允许个性化治疗，可用于高通量药物筛选，并建立了超过20个患者来源的结肠癌类器官样本库。专利CN202111156653(一种结肠癌类器官的培养基及其应用)亦公开了相似的结肠癌类器官培养基的组成和相似的培养方法。然而，这些设计存在以下问题和缺点：在结肠癌类器官培养基的主要成分中都需要单独制备Wnt条件培养基、R-Spondin条件培养基或Noggin条件培养基，或者需要添加Wnt信号调节蛋白R-Spondin或者发育调控蛋白Noggin等重组蛋白，单独生产条件培养基操作复杂，重组蛋白购买价格昂贵，且主要生产企业均位于国外，这导致结肠癌类器官专用培养基存在生产成本较高、主要添加成分需要进口、采购周期长且供应周期易受外部环境影响等问题。这些问题极大的阻碍了结肠癌类器官的大规模培养和更广范围的应用研究，因此，迫切希望开发更低成本、更易用的结肠癌类器官专用培养基的出现。
发明内容
为了解决上述问题，本发明提供了一种全新的结肠癌类器官无血清专用培养基的组成和培养方法，其目的在于，提供一种无需单独制备条件培养基且无需添加昂贵重组蛋白的结肠癌类器官无血清专用培养基。
为实现上述目的，本发明提供的方案是这样实现的：一方面，本发明所述结肠癌类器官无血清专用培养基由基础培养基和培养添加物两部分组成。基础培养基为含有1％青-链霉素、1％HEPES缓冲液、1％GlutaMax溶液的High Glucose DMEM/F12培养基。
进一步地，所述培养添加物包括生长因子FGF2、FGF10，胰岛素，氢化可的松，小分子抑制剂Y-27632、SB202190，牛垂体提取物(BPE)、B27 supplement。
进一步地，所述培养添加物的工作浓度为：FGF2：5-50ng/mL、FGF10：10-100ng/mL，胰岛素：0.5-50μg/mL，氢化可的松：50-500ng/mL，小分子抑制剂Y-27632：1-10μM、SB202190：0.1-1μM，牛垂体提取物：5-50ng/mL、B27 supplement：1％。
进一步地，所述结肠癌类器官无血清专用培养基在使用时配制，在已配制好的基础培养基中，按照工作浓度加入所有培养添加物，混合均匀后即可获得。
在本发明的另一个方面中，提供一种结肠癌类器官体外培养的方法，包括以下步骤：
(1)将结肠癌组织样本切成0.5mm×0.5mm大小，用无菌PBS处理清洗后，加入10mL含有0.5mg/mL胶原酶的基础培养基中消化，将消化后的细胞通过100μm孔径细胞滤网；
(2)细胞计数后，按照2×106个细胞/mL重悬于冷的CultrexTM growth factorreduced BME type2中，接种于细胞培养板，待含有细胞的BME固化；
(3)每孔加入上述结肠癌类器官无血清专用培养基400微升，放入5％CO2浓度、37℃细胞培养箱培养，每2-3天更换一次上述培养基，7天左右即可获得所需结肠癌类器官。
由于本发明采用的培养添加物中去除了价格昂贵的重组蛋白如Wnt、R-Spondin和Noggin成分，无需单独制备Wnt条件培养基、R-Spondin条件培养基和Noggin条件培养基，从而可以得到以下有益效果：
(1)本发明所述培养基对于结肠癌类器官的生长有较好的支持和促进作用，7天左右即可获得典型的结肠癌类器官，在体外较好的保留了患者来源肿瘤一致性和保留异质性，为进一步进行研究应用奠定了基础；
(2)本发明所述培养基的成本较其他方法有明显下降，主要原因在于不需要单独制备条件培养基，并去除了价格较为昂贵的Wnt信号调节蛋白R-Spondin或者发育调控蛋白Noggin等重组蛋白，使用其他成分替代，从而为以后进行大规模结肠癌类器官培养用于药物筛选、个体化治疗等研究应用提供了可能。
(3)本发明所述培养方法步骤清晰简便，操作人员对培养结果影响较小，提高了培养结果的一致性，为后续大规模培养提供了便利条件。
具体实施方式
实施例：
本发明的优选实施方式是，提供一种无需制备条件培养基、无需添加Wnt通路调控蛋白R-Spondin和发育调控蛋白Noggin等重组蛋白的结肠癌类器官无血清专用培养基及培养方法，所述培养基包括无血清基础培养基和培养添加物。所述基础培养基为含有1％青-链霉素、1％HEPES缓冲液、1％GlutaMax溶液的High Glucose DMEM/F12培养基，所述培养添加物由以下工作浓度的成分组成：FGF2：50ng/mL、FGF10：10ng/mL，胰岛素：50μg/mL，氢化可的松：500ng/mL，小分子抑制剂Y-27632：10μM、SB202190：1μM，牛垂体提取物：50ng/mL、B27supplement：1％。在使用时将所有培养添加物按照工作浓度加入所述基础培养基，即可获得所述结肠癌类器官无血清专用培养基。所述结肠癌类器官培养方法为，将患者来源结肠癌组织样本切成0.5mm×0.5mm左右大小，加入10mL无菌PBS清洗5次，将清洗过的肿瘤组织块加入10mL含有0.5mg/mL胶原酶的基础培养基中消化60分钟左右，将消化后的细胞通过100μm孔径的细胞滤网，细胞计数，按照2×106个细胞/mL的浓度重悬于冷的CultrexTMgrowth factor reduced BME type2中，接种于24孔细胞培养板，每孔加40微升，放入37度细胞培养箱20分钟，待含有细胞的BME固化后，每孔加入上述结肠癌类器官无血清专用培养基400微升，放入5％CO2浓度、37℃细胞培养箱培养，每2-3天更换一次上述结肠癌类器官无血清专用培养基，7天左右即可获得所需结肠癌类器官，可见所述结肠癌类器官无血清专用培养基可以有效支持结肠癌类器官的体外生长，且生长迅速，所形成的结肠癌类器官呈现典型的三维球状或三维不规则细胞团块，为后续进一步研究提供了较好的研究样本。
对比例：
本发明的对比例采用已公开发表文献(Van de Wetering,M.,et al.(2015).Prospective derivation of a living organoid biobank of colorectal cancerpatients.Cell 161,933-945.)中描述的方法，在基础培养基(Advanced DMEM/F12培养基+1％青-链霉素+10mM HEPES+1％Glutamax溶液)中加入50％Wnt条件培养基、20％R-Spondin条件培养基,、10％Noggin条件培养基，再加入1％B27,1.25mM N-Acetylcysteine,10mM尼克酰胺,50ng/ml human EGF,10nM Gastrin,500nM A83-01,3μM SB202190,10nM前列腺素E2和100mg/ml Primocin。具体培养方法为，将肿瘤切成碎片，与胶原酶II(1.5mg/ml)、透明质酸酶(20ug/ml)和Y-27632(10μM)在37℃孵育30分钟，通过100μM细胞过滤器，以除去大的碎片，随后以1,000转/分的速度离心3分钟，重悬于基础培养基中，再以1,000转/分的速度离心两次以去除碎片和胶原酶，将肿瘤细胞悬液重悬于CultrexTM growth factor reducedBME type2中，加在细胞培养板中，待BME固化后，放置于5％CO2、37℃细胞培养箱中。培养7-10天后即可获得结肠癌类器官。本发明采用对比例的方法也能够获得所需结肠癌类器官，所获得的结肠癌类器官呈现典型的肿瘤类器官三维结构，生长良好。
综上，本发明所述结肠癌类器官无血清专用培养基及培养方法能够有效支持结肠癌类器官的体外生长，且生长迅速，所形成的结肠癌类器官呈现典型的三维球状或三维不规则细胞团块，可以生长至直径较大水平依然保持肿瘤类器官形态。与已公开文献与专利所述方法比较，本专利所述培养基不需要添加单独制备的条件培养基，去除了价格昂贵的重组蛋白类添加成分，改用其他生长因子替代，从而极大地降低了专用培养基的成本，且培养效果与已公开的方法相比较为一致。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本领域的技术人员应该了解本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都属于本发明保护的范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (6)
1.一种结肠癌类器官无血清专用培养基，其特征在于：包括基础培养基和培养添加物，基础培养基为含有1％青-链霉素、1％HEPES缓冲液、1％GlutaMax溶液的High GlucoseDMEM/F12培养基，培养添加物包括生长因子FGF2、FGF10，胰岛素，氢化可的松，小分子抑制剂Y-27632、SB202190，牛垂体提取物(BPE)、B27supplement。
2.根据权利要求1所述的一种结肠癌类器官无血清专用培养基，其特征在于，所述培养添加物的工作浓度为：FGF2：5-50ng/mL、FGF10：10-100ng/mL，胰岛素：0.5-50μg/mL，氢化可的松：50-500ng/mL，小分子抑制剂Y-27632：1-10μM、SB202190：0.1-1μM，牛垂体提取物：5-50ng/mL、B27 supplement：1％。
3.根据权利要求1所述的一种结肠癌类器官无血清专用培养基，其特征在于，所述基础培养基为无血清培养基。
4.根据权利要求1或2所述的一种结肠癌类器官无血清专用培养基，其特征在于，所述培养基不需要添加Wnt信号调节重组蛋白R-Spondin或者发育调控蛋白Noggin等重组蛋白，亦不需要单独制备Wnt条件培养基、R-Spondin条件培养基或Noggin条件培养基。
5.根据权利要求1所述的一种结肠癌类器官无血清专用培养基，其特征在于，在已配制好的基础培养基中，按照工作浓度加入所有培养添加物，混合均匀后即可获得。
6.一种结肠癌类器官培养方法，其特征在于，将结肠癌组织样本切成0.5mm×0.5mm大小，用无菌PBS处理清洗后，加入10mL含有0.5mg/mL胶原酶的基础培养基中消化，将消化后的细胞通过100μm孔径细胞滤网，细胞计数后，按照2×106个细胞/mL重悬于冷的CultrexTMgrowth factor reduced BME type2中，接种于细胞培养板，待含有细胞的BME固化后，加入权利要求1-5任意一项所述的培养基，放入5％CO2浓度、37℃细胞培养箱培养，每2-3天更换一次上述培养基，7天左右即可获得所需结肠癌类器官。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210950210.1A CN116536264A (zh) | 2022-08-15 | 2022-08-15 | 一种结肠癌类器官无血清专用培养基 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210950210.1A CN116536264A (zh) | 2022-08-15 | 2022-08-15 | 一种结肠癌类器官无血清专用培养基 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116536264A true CN116536264A (zh) | 2023-08-04 |
Family
ID=87442311
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210950210.1A Pending CN116536264A (zh) | 2022-08-15 | 2022-08-15 | 一种结肠癌类器官无血清专用培养基 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116536264A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116836934A (zh) * | 2023-08-31 | 2023-10-03 | 北京大橡科技有限公司 | 骨肉瘤类器官培养液、培养试剂组合及培养方法 |
-
2022
- 2022-08-15 CN CN202210950210.1A patent/CN116536264A/zh active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116836934A (zh) * | 2023-08-31 | 2023-10-03 | 北京大橡科技有限公司 | 骨肉瘤类器官培养液、培养试剂组合及培养方法 |
| CN116836934B (zh) * | 2023-08-31 | 2023-11-24 | 北京大橡科技有限公司 | 骨肉瘤类器官培养液、培养试剂组合及培养方法 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112080472B (zh) | 一种培养专用于生物医药功能研究的人肺癌类器官3d模型的方法 | |
| CN108504625B (zh) | 一种小鼠成纤维细胞及其用途 | |
| CN114790446B (zh) | 一种宫颈腺癌类器官培养基及其构建方法 | |
| CN112522201A (zh) | 一种膀胱癌类器官的培养基及培养方法 | |
| CN113278586A (zh) | 一种用于培养甲状腺癌类器官的培养基和培养方法 | |
| CN112831471A (zh) | 一种甲状腺癌类器官的培养基、培养方法和检测方法 | |
| CN114181903A (zh) | 结直肠癌类器官培养基及无支架3d培养方法 | |
| CN115161283B (zh) | 一种用于肝门部胆管癌来源类器官定向分化和培养的组合物及应用 | |
| CN116536264A (zh) | 一种结肠癌类器官无血清专用培养基 | |
| CN114075539A (zh) | 构建原位原发膀胱癌动物模型的方法 | |
| Wang et al. | Establishment of a patient-derived organoid model and living biobank for nasopharyngeal carcinoma | |
| JP2005533512A (ja) | 細胞外マトリックスの製造方法および腫瘍細胞培養のためのその使用 | |
| Becker et al. | Characterization of primary breast carcinomas grown in three-dimensional cultures | |
| CN114958753B (zh) | 一种舌癌类器官的培养基、培养方法及鉴定方法 | |
| CN116590232A (zh) | 一种甲状腺癌类器官、培养基及培养方法 | |
| CN114774359B (zh) | 一种宫颈鳞癌类器官培养基及其构建方法 | |
| CN115094027A (zh) | 一种胰腺癌类器官无血清专用培养基 | |
| WO2023274043A1 (zh) | 一种人泪腺干细胞及其分化培养方法与应用 | |
| CN112760289B (zh) | 一种乳腺癌类器官专用培养基及3d培养方法 | |
| CN113583961A (zh) | 一种乳腺癌类器官无血清专用培养基 | |
| CN115418352A (zh) | 一种黑色素瘤类器官无血清专用培养基 | |
| CN114369573A (zh) | 构建原位原发鼻咽癌动物模型的方法 | |
| Jiang et al. | Organoids: opportunities and challenges of cancer therapy | |
| CN115386553A (zh) | 一种肺癌类器官无血清专用培养基 | |
| CN116590216A (zh) | 一种胰腺正常类器官培养基及胰腺癌类器官筛选培养基 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication |