WO2023060774A1 - Culture medium for cervical cancer organoids, and culture method and application thereof - Google Patents
Culture medium for cervical cancer organoids, and culture method and application thereof Download PDFInfo
- Publication number
- WO2023060774A1 WO2023060774A1 PCT/CN2021/142274 CN2021142274W WO2023060774A1 WO 2023060774 A1 WO2023060774 A1 WO 2023060774A1 CN 2021142274 W CN2021142274 W CN 2021142274W WO 2023060774 A1 WO2023060774 A1 WO 2023060774A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cervical cancer
- alkyl
- culture
- medium
- organoids
- Prior art date
Links
- 210000002220 organoid Anatomy 0.000 title claims abstract description 116
- 206010008342 Cervix carcinoma Diseases 0.000 title claims abstract description 100
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 title claims abstract description 100
- 201000010881 cervical cancer Diseases 0.000 title claims abstract description 100
- 239000001963 growth medium Substances 0.000 title claims abstract description 35
- 238000012136 culture method Methods 0.000 title claims abstract description 9
- 101001066435 Homo sapiens Hepatocyte growth factor-like protein Proteins 0.000 claims abstract description 24
- 101000880431 Homo sapiens Serine/threonine-protein kinase 4 Proteins 0.000 claims abstract description 24
- 239000000654 additive Substances 0.000 claims abstract description 23
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims abstract description 17
- 229940043355 kinase inhibitor Drugs 0.000 claims abstract description 17
- 239000003757 phosphotransferase inhibitor Substances 0.000 claims abstract description 17
- 102000003972 Fibroblast growth factor 7 Human genes 0.000 claims abstract description 12
- 108090000385 Fibroblast growth factor 7 Proteins 0.000 claims abstract description 12
- 102000004864 Fibroblast growth factor 10 Human genes 0.000 claims abstract description 11
- 108090001047 Fibroblast growth factor 10 Proteins 0.000 claims abstract description 11
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 claims abstract description 11
- 230000000996 additive effect Effects 0.000 claims abstract description 11
- 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 10
- 229960003966 nicotinamide Drugs 0.000 claims abstract description 10
- 235000005152 nicotinamide Nutrition 0.000 claims abstract description 10
- 239000011570 nicotinamide Substances 0.000 claims abstract description 10
- 102000009024 Epidermal Growth Factor Human genes 0.000 claims abstract description 9
- 101800003838 Epidermal growth factor Proteins 0.000 claims abstract description 9
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(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]([C@@H](C)O)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](CCCNC(N)=N)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)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)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=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 claims abstract description 8
- 238000004113 cell culture Methods 0.000 claims abstract description 7
- 229940116977 epidermal growth factor Drugs 0.000 claims abstract description 7
- 238000011282 treatment Methods 0.000 claims abstract description 7
- 102100032813 Hepatocyte growth factor-like protein Human genes 0.000 claims abstract 6
- 239000002609 medium Substances 0.000 claims description 70
- 210000001519 tissue Anatomy 0.000 claims description 38
- 210000004027 cell Anatomy 0.000 claims description 34
- 229940079593 drug Drugs 0.000 claims description 21
- 239000003814 drug Substances 0.000 claims description 21
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 17
- 150000001875 compounds Chemical class 0.000 claims description 14
- 206010028980 Neoplasm Diseases 0.000 claims description 13
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 12
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 10
- 102000003745 Hepatocyte Growth Factor Human genes 0.000 claims description 10
- 125000000171 (C1-C6) haloalkyl group Chemical group 0.000 claims description 8
- 125000005913 (C3-C6) cycloalkyl group Chemical group 0.000 claims description 8
- 229910052739 hydrogen Inorganic materials 0.000 claims description 7
- 239000001257 hydrogen Substances 0.000 claims description 7
- 229930182555 Penicillin Natural products 0.000 claims description 6
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 6
- 125000001316 cycloalkyl alkyl group Chemical group 0.000 claims description 6
- 229940049954 penicillin Drugs 0.000 claims description 6
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 6
- 229960005322 streptomycin Drugs 0.000 claims description 6
- 229910052736 halogen Inorganic materials 0.000 claims description 5
- 150000002367 halogens Chemical class 0.000 claims description 5
- 150000002431 hydrogen Chemical class 0.000 claims description 5
- 239000003112 inhibitor Substances 0.000 claims description 5
- 102000045246 noggin Human genes 0.000 claims description 5
- 108700007229 noggin Proteins 0.000 claims description 5
- 239000012453 solvate Substances 0.000 claims description 5
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 claims description 4
- -1 C1-C6 alkylhydroxyl Chemical group 0.000 claims description 4
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 4
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 claims description 4
- 229960003942 amphotericin b Drugs 0.000 claims description 4
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 125000001544 thienyl group Chemical group 0.000 claims description 4
- 230000035899 viability Effects 0.000 claims description 4
- 125000004191 (C1-C6) alkoxy group Chemical group 0.000 claims description 3
- 239000003242 anti bacterial agent Substances 0.000 claims description 3
- 229940088710 antibiotic agent Drugs 0.000 claims description 3
- 239000012592 cell culture supplement Substances 0.000 claims description 3
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 claims description 3
- 125000006273 (C1-C3) alkyl group Chemical group 0.000 claims description 2
- 102000018233 Fibroblast Growth Factor Human genes 0.000 claims description 2
- 108050007372 Fibroblast Growth Factor Proteins 0.000 claims description 2
- 102000041829 R-spondin family Human genes 0.000 claims description 2
- 108091078718 R-spondin family Proteins 0.000 claims description 2
- 102000013814 Wnt Human genes 0.000 claims description 2
- 108050003627 Wnt Proteins 0.000 claims description 2
- 239000000556 agonist Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 claims description 2
- 229940126864 fibroblast growth factor Drugs 0.000 claims description 2
- 125000001072 heteroaryl group Chemical group 0.000 claims description 2
- 125000001624 naphthyl group Chemical group 0.000 claims description 2
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims 3
- 101000844802 Lacticaseibacillus rhamnosus Teichoic acid D-alanyltransferase Proteins 0.000 claims 1
- 230000010261 cell growth Effects 0.000 claims 1
- 210000002615 epidermis Anatomy 0.000 claims 1
- 125000001153 fluoro group Chemical group F* 0.000 claims 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims 1
- 230000001575 pathological effect Effects 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 2
- 102100021866 Hepatocyte growth factor Human genes 0.000 abstract 1
- 102100037629 Serine/threonine-protein kinase 4 Human genes 0.000 description 18
- 230000029087 digestion Effects 0.000 description 16
- 230000000694 effects Effects 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- 239000007640 basal medium Substances 0.000 description 12
- 239000000203 mixture Substances 0.000 description 12
- 238000002360 preparation method Methods 0.000 description 12
- 230000035755 proliferation Effects 0.000 description 10
- 229940125904 compound 1 Drugs 0.000 description 7
- 238000012258 culturing Methods 0.000 description 7
- 102000029816 Collagenase Human genes 0.000 description 6
- 108060005980 Collagenase Proteins 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 229960002424 collagenase Drugs 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 108010082117 matrigel Proteins 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- OOEJZUUVEWCSEG-UHFFFAOYSA-N 4-[[7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-7H-pteridin-2-yl]amino]benzenesulfonamide Chemical compound NS(=O)(=O)C1=CC=C(NC2=NC=C3N(C(=O)C(N(C)C3=N2)C2=C(F)C=CC=C2F)C)C=C1 OOEJZUUVEWCSEG-UHFFFAOYSA-N 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 229930012538 Paclitaxel Natural products 0.000 description 4
- VSRXQHXAPYXROS-UHFFFAOYSA-N azanide;cyclobutane-1,1-dicarboxylic acid;platinum(2+) Chemical compound [NH2-].[NH2-].[Pt+2].OC(=O)C1(C(O)=O)CCC1 VSRXQHXAPYXROS-UHFFFAOYSA-N 0.000 description 4
- 229960001467 bortezomib Drugs 0.000 description 4
- GXJABQQUPOEUTA-RDJZCZTQSA-N bortezomib Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)B(O)O)NC(=O)C=1N=CC=NC=1)C1=CC=CC=C1 GXJABQQUPOEUTA-RDJZCZTQSA-N 0.000 description 4
- 229960004562 carboplatin Drugs 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 4
- 229960004316 cisplatin Drugs 0.000 description 4
- 239000012043 crude product Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 230000002055 immunohistochemical effect Effects 0.000 description 4
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 4
- 239000012074 organic phase Substances 0.000 description 4
- 229960001592 paclitaxel Drugs 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 239000006228 supernatant Substances 0.000 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 description 4
- APZCECSEMLNKFK-UHFFFAOYSA-N 2-chloro-7-(2,6-difluorophenyl)-7,8-dihydro-5H-pteridin-6-one Chemical compound ClC1=NC=2NC(C(NC=2C=N1)=O)C1=C(C=CC=C1F)F APZCECSEMLNKFK-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- 102000016911 Deoxyribonucleases Human genes 0.000 description 3
- 108010053770 Deoxyribonucleases Proteins 0.000 description 3
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 3
- 108010003272 Hyaluronate lyase Proteins 0.000 description 3
- 102000001974 Hyaluronidases Human genes 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 210000000601 blood cell Anatomy 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000007877 drug screening Methods 0.000 description 3
- 229910052731 fluorine Inorganic materials 0.000 description 3
- 239000011737 fluorine Substances 0.000 description 3
- 229960002773 hyaluronidase Drugs 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 2
- JEEZAZSTUWUWQK-UHFFFAOYSA-N 2-chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7H-pteridin-6-one Chemical compound ClC1=NC=2N(C(C(N(C=2C=N1)C)=O)C1=C(C=CC=C1F)F)C JEEZAZSTUWUWQK-UHFFFAOYSA-N 0.000 description 2
- OHCQJHSOBUTRHG-KGGHGJDLSA-N FORSKOLIN Chemical compound O=C([C@@]12O)C[C@](C)(C=C)O[C@]1(C)[C@@H](OC(=O)C)[C@@H](O)[C@@H]1[C@]2(C)[C@@H](O)CCC1(C)C OHCQJHSOBUTRHG-KGGHGJDLSA-N 0.000 description 2
- 101000880439 Homo sapiens Serine/threonine-protein kinase 3 Proteins 0.000 description 2
- 102100022762 R-spondin-1 Human genes 0.000 description 2
- 102100037628 Serine/threonine-protein kinase 3 Human genes 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- BTANRVKWQNVYAZ-UHFFFAOYSA-N butan-2-ol Chemical compound CCC(C)O BTANRVKWQNVYAZ-UHFFFAOYSA-N 0.000 description 2
- 238000012054 celltiter-glo Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- IGMWMQOJLFFZRK-UHFFFAOYSA-N methyl 2-[(2-chloro-5-nitropyrimidin-4-yl)amino]-2-(2,6-difluorophenyl)acetate Chemical compound ClC1=NC=C(C(=N1)NC(C(=O)OC)C1=C(C=CC=C1F)F)[N+](=O)[O-] IGMWMQOJLFFZRK-UHFFFAOYSA-N 0.000 description 2
- GXMZVLXPZSATTL-UHFFFAOYSA-N methyl 2-amino-2-(2,6-difluorophenyl)acetate Chemical compound COC(=O)C(N)C1=C(F)C=CC=C1F GXMZVLXPZSATTL-UHFFFAOYSA-N 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- AICOOMRHRUFYCM-ZRRPKQBOSA-N oxazine, 1 Chemical compound C([C@@H]1[C@H](C(C[C@]2(C)[C@@H]([C@H](C)N(C)C)[C@H](O)C[C@]21C)=O)CC1=CC2)C[C@H]1[C@@]1(C)[C@H]2N=C(C(C)C)OC1 AICOOMRHRUFYCM-ZRRPKQBOSA-N 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 239000013630 prepared media Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- FYSNRJHAOHDILO-UHFFFAOYSA-N thionyl chloride Chemical compound ClS(Cl)=O FYSNRJHAOHDILO-UHFFFAOYSA-N 0.000 description 2
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- INUSQTPGSHFGHM-UHFFFAOYSA-N 2,4-dichloro-5-nitropyrimidine Chemical compound [O-][N+](=O)C1=CN=C(Cl)N=C1Cl INUSQTPGSHFGHM-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- UOXJNGFFPMOZDM-UHFFFAOYSA-N 2-[di(propan-2-yl)amino]ethylsulfanyl-methylphosphinic acid Chemical compound CC(C)N(C(C)C)CCSP(C)(O)=O UOXJNGFFPMOZDM-UHFFFAOYSA-N 0.000 description 1
- UQFQFMHLBQOCLY-UHFFFAOYSA-N 2-azaniumyl-2-(2,6-difluorophenyl)acetate Chemical compound OC(=O)C(N)C1=C(F)C=CC=C1F UQFQFMHLBQOCLY-UHFFFAOYSA-N 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- KUTQQJJKLIZJFV-UHFFFAOYSA-N CC(N=C(N=C1NC(C(O)=O)C(C(F)=CC=C2)=C2F)Cl)=C1[N+]([O-])=O Chemical compound CC(N=C(N=C1NC(C(O)=O)C(C(F)=CC=C2)=C2F)Cl)=C1[N+]([O-])=O KUTQQJJKLIZJFV-UHFFFAOYSA-N 0.000 description 1
- AQGNHMOJWBZFQQ-UHFFFAOYSA-N CT 99021 Chemical compound CC1=CNC(C=2C(=NC(NCCNC=3N=CC(=CC=3)C#N)=NC=2)C=2C(=CC(Cl)=CC=2)Cl)=N1 AQGNHMOJWBZFQQ-UHFFFAOYSA-N 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 241000766026 Coregonus nasus Species 0.000 description 1
- SUZLHDUTVMZSEV-UHFFFAOYSA-N Deoxycoleonol Natural products C12C(=O)CC(C)(C=C)OC2(C)C(OC(=O)C)C(O)C2C1(C)C(O)CCC2(C)C SUZLHDUTVMZSEV-UHFFFAOYSA-N 0.000 description 1
- 102100028071 Fibroblast growth factor 7 Human genes 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 101001060261 Homo sapiens Fibroblast growth factor 7 Proteins 0.000 description 1
- 101000825954 Homo sapiens R-spondin-1 Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 description 1
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 description 1
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 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
- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 101710110302 R-spondin-1 Proteins 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- DHXVGJBLRPWPCS-UHFFFAOYSA-N Tetrahydropyran Chemical compound C1CCOCC1 DHXVGJBLRPWPCS-UHFFFAOYSA-N 0.000 description 1
- OKJPEAGHQZHRQV-UHFFFAOYSA-N Triiodomethane Natural products IC(I)I OKJPEAGHQZHRQV-UHFFFAOYSA-N 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 102000044880 Wnt3A Human genes 0.000 description 1
- 108700013515 Wnt3A Proteins 0.000 description 1
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 239000012574 advanced DMEM Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000004037 angiogenesis inhibitor Substances 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 210000003679 cervix uteri Anatomy 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- OHCQJHSOBUTRHG-UHFFFAOYSA-N colforsin Natural products OC12C(=O)CC(C)(C=C)OC1(C)C(OC(=O)C)C(O)C1C2(C)C(O)CCC1(C)C OHCQJHSOBUTRHG-UHFFFAOYSA-N 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229960005309 estradiol Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000004996 female reproductive system Anatomy 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000011990 functional testing Methods 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 230000000683 nonmetastatic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- UUEVFMOUBSLVJW-UHFFFAOYSA-N oxo-[[1-[2-[2-[2-[4-(oxoazaniumylmethylidene)pyridin-1-yl]ethoxy]ethoxy]ethyl]pyridin-4-ylidene]methyl]azanium;dibromide Chemical compound [Br-].[Br-].C1=CC(=C[NH+]=O)C=CN1CCOCCOCCN1C=CC(=C[NH+]=O)C=C1 UUEVFMOUBSLVJW-UHFFFAOYSA-N 0.000 description 1
- 229960002566 papillomavirus vaccine Drugs 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000011338 personalized therapy Methods 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 125000003386 piperidinyl group Chemical group 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 239000003761 preservation solution Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 239000012312 sodium hydride Substances 0.000 description 1
- 229910000104 sodium hydride Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 229940124530 sulfonamide Drugs 0.000 description 1
- 150000003456 sulfonamides Chemical class 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 101150068520 wnt3a gene Proteins 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
Definitions
- the invention belongs to the field of biotechnology, and in particular relates to a culture medium for cervical cancer organoids and a method for cultivating cervical cancer organoids using the medium.
- Cervical cancer is a malignant tumor that originates in the cervix and is the most common malignant tumor in the female reproductive system. Cervical cancer ranks fourth among female malignancies worldwide. The highest incidence of cervical cancer is between 30 and 55 years old, and the incidence rate in my country is only behind breast cancer. Due to the popularity of cervical cancer screening and the promotion of HPV vaccine, cervical cancer has largely become a preventable disease, but its 5-year survival rate is still only about 60%. For early non-metastatic cervical cancer, surgery, radiotherapy and chemotherapy are the main treatment methods, but for metastatic or recurrent cervical cancer, traditional treatment methods have not achieved satisfactory results. With the use of targeted therapy and immunotherapy drugs such as anti-angiogenic drugs and immune checkpoint inhibitors, the survival time of these patients has been significantly prolonged, but the final cure effect has not yet been achieved. Cells serve as research models to discover new drug targets and guide personalized therapy.
- Organoids which belong to three-dimensional (3D) cell cultures, are mainly derived from human embryonic stem cells, induced pluripotent stem cells and adult stem cells with differentiation ability. Endogenous tissue stem cells exist in different tissues and organs, and play an important role in maintaining the functional morphology of various organs. Under certain induction conditions in vitro, these stem cells can self-organize to form a miniature structure with a diameter of only a few millimeters.
- Tumor organoids are obtained from primary tumors in patients, and some miniature 3D tumor cell models are cultivated in the laboratory. Tumor organoids highly simulate the characteristics of the source tumor tissue, retain the tumor heterogeneity among individuals, and can be used for functional testing, such as drug screening and individualized precision therapy.
- cervical cancer organoid culture methods mostly use expensive protein factors such as R-spondin-1, WNT3A, and Noggin, resulting in high cost of organoid culture; and this technology is complicated to operate and technically difficult, leading to its large-scale commercialization Application is limited. Therefore, there is a need to develop a low-cost, simple and high success rate organoid culture method and medium.
- the present invention provides a culture medium and a culture method for rapid expansion of cervical cancer organoids in vitro.
- One aspect of the present invention is to provide a culture medium for cervical cancer organoids, said culture medium comprising an MST1/2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, hepatocyte growth factor, SB202190, Y27632 , A83-01, Epidermal Growth Factor, Fibroblast Growth Factor 10, Keratinocyte Growth Factor, GlutaMAX, and Niacinamide.
- the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof,
- R 1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted by 1-2 independent R (such as phenyl and naphthyl, etc.), aryl C1-C6 alkyl (such as benzyl, etc.) and heteroaryl (such as thienyl, etc.);
- R 2 and R 3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;
- R 4 and R 5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxyl, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl (the heterocyclyl is selected from, for example, piperidinyl, tetrahydropyran base, etc.);
- R is selected from halogen (preferably fluorine and chlorine, more preferably fluorine), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoro methyl).
- halogen preferably fluorine and chlorine, more preferably fluorine
- C1-C6 alkyl preferably methyl
- C1-C6 alkoxy preferably methoxy
- C1-C6 haloalkyl preferably trifluoro methyl
- the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt thereof, or a solvate,
- R is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently R6 , thienyl optionally substituted by 1-2 independently R6 , and optionally substituted by 1 -2 independently R6 substituted benzyl, R1 is more preferably optionally 1-2 independently R6 substituted phenyl;
- R 5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R 5 is more preferably hydrogen;
- R 6 is each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, and R 6 is more preferably fluorine, methyl or trifluoromethyl.
- the MST1/2 inhibitor is at least one selected from the following compounds or pharmaceutically acceptable salts or solvates thereof.
- the MST1/2 kinase inhibitor of the present invention is Compound 1.
- the content of each component in the culture medium of the present invention satisfies any one or more or all of the following:
- the concentration of the MST1/2 kinase inhibitor is preferably 2.5-10 ⁇ M
- the concentration of hepatocyte growth factor is preferably 5-40 ng/mL
- the concentration of SB202190 is preferably 200-1000nM
- the concentration of Y27632 is preferably 2.5-10 ⁇ M
- the concentration of A83-01 is preferably 200-1000nM
- the concentration of epidermal growth factor is preferably 1-40 ng/mL;
- the concentration of fibroblast growth factor 10 is preferably 10-100 ng/mL
- the concentration of keratinocyte growth factor is preferably 2-40 ng/mL;
- the volume ratio of GlutaMAX to the medium is preferably 1:50 to 1:200;
- the concentration of nicotinamide is preferably 1 to 10 mM.
- the medium also contains an initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one selected from streptomycin/penicillin, amphotericin B and Primocin one or more antibiotics.
- the streptomycin concentration ranges from 25 to 400 ⁇ g/mL, and the penicillin concentration ranges from 25 to 400 U/mL; when the antibiotic is selected from amphotericin B, The concentration range is 0.25-4 ⁇ g/mL, and when the antibiotic is selected from Primocin, the concentration range is 25-400 ⁇ g/mL.
- the invention also provides a method for culturing cervical cancer organoids.
- cervical cancer organoids are cultured using the cervical cancer organoid culture medium of the present invention.
- the method for culturing cervical cancer organoids of the present invention includes the following steps.
- the process includes the following steps:
- tissue samples Separate cervical cancer tissue samples, add basal culture medium and tissue digestion solution at a ratio of 1:1 (the amount of tissue digestion solution is about 10 mL of tissue digestion solution per 1 g of tumor tissue) and place in a constant temperature shaker for digestion , the digestion temperature is 4-37°C, the shaker speed is 200rpm-300rpm, and the digestion time is 3-6 hours;
- the basal medium formula includes an initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one or more antibiotics selected from streptomycin/penicillin, amphotericin B and Primocin.
- the formula of tissue digestion solution includes 1640 medium, collagenase II (1-2 mg/mL), collagenase IV (1-2 mg/mL), DNase (50-100 U/mL), hyaluronidase (0.5-1 mg /mL), calcium chloride (1 ⁇ 5mM), bovine serum albumin BSA (5 ⁇ 10mg/mL).
- the cervical cancer primary cells obtained in the above step 1 Resuspend the cervical cancer primary cells obtained in the above step 1 with the cervical cancer organoid culture medium of the present invention and count them, dilute the cell density to 5-10 ⁇ 105 cells/mL, take out the diluted cell suspension and add Mix in an equal volume of Matrigel matrigel, then inoculate the mixture on a multi-well plate, put the inoculated multi-well plate in the incubator for 30-60 minutes, wait until the Matrigel is completely solidified, and then add cervical cancer organoid medium for expansion culture.
- a method for assessing or screening a drug for the treatment of cervical cancer characterized in that it comprises the following steps:
- the culture cost is controllable, and the medium does not need to add expensive Wnt agonists, R-spondin family proteins and Noggin proteins;
- the number of cervical cancer organoids obtained by the technique is large, which is suitable for high-throughput screening of candidate compounds and providing patients with high-throughput drug sensitivity functional tests in vitro.
- 1A-1K are graphs showing the effects of different concentrations of factors added to the cervical cancer organoid medium of the present invention on the proliferation of cervical cancer organoids.
- Figures 2A-2D are photographs of cervical cancer organoids cultured using the cervical cancer organoid medium of the present invention observed under a microscope, wherein Figure 2A shows photos of organoids obtained from sample OC1 cultured for 5 days; Figure 2B shows photos obtained from sample OC1 Photos of organoids obtained from OC1 after 14 days of culture; Figure 2C and 2D show photos of different fields of view of organoids obtained from sample OC2 after 7 days of culture.
- Fig. 3 is the result of pathological and immunohistochemical identification of the original tissue sample OC4 and the cervical cancer organoid obtained by using the cervical cancer organoid culture medium culture sample OC4 of the present invention.
- Figures 4A and 4B are the comparison results of using the cervical cancer organoid culture medium of the present invention and the culture medium of the prior art to culture cervical cancer organoids, wherein Figure 4A shows the photos after culturing for 10 days with the COM medium of the present invention; Fig. 4B shows photographs after 10 days of culture with literature medium ROM.
- Fig. 5 is a graph showing the results of drug concentration sensitivity testing of cervical cancer organoids cultured using the cervical cancer organoid medium of the present invention.
- an MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1/2 signal transduction.
- MST1/2 kinase inhibitors for example, bind to MST1/2 kinase and reduce its activity. Due to the similarity in the structures of MST1 and MST2, MST1/2 kinase inhibitors may also be, for example, compounds that bind to MST1 or MST2 and reduce their activity.
- 2-Amino-2-(2,6-difluorophenyl)acetic acid methyl ester (A2): In a round bottom flask was added 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) Methanol (30 mL) was then added, followed by the dropwise addition of thionyl chloride (1.2 mL) under ice-cooling. The reaction system was reacted overnight at 85°C. After the reaction, the system was evaporated to dryness under reduced pressure to obtain a white solid, which was directly used in the next step.
- MST1/2 inhibitor compounds of the present invention were synthesized according to a method similar to compound 1, and their structures and mass spectrometry data are shown in the table below.
- the initial medium can be selected from DMEM/F12, DMEM, F12 or RPMI-1640 commonly used in the art.
- the formulation of the basal medium is: DMEM/F12 medium (purchased from Corning Company)+100 ⁇ g/mL Primocin (purchased from InvivoGen Company, 0.2% (v/v), commercially available product concentration 50mg/ml ).
- Intraoperative samples were obtained from patients by professional medical staff of professional medical institutions, and all patients signed informed consent. Intraoperative samples of 5-10 mm 3 were stored and transported in commercial tissue preservation solution (manufacturer: Miltenyi Biotec).
- Tissue digestion solution formula 1640 medium (Corning, 10-040-CVR), collagenase II (2mg/mL), collagenase IV (2mg/mL), DNase (50U/mL), hyaluronidase (0.75 mg/mL), calcium chloride (3.3mM), bovine serum albumin BSA (10mg/mL).
- Collagenase II, collagenase IV, DNase, and hyaluronidase mentioned above were all purchased from Sigma; calcium chloride and BSA were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.
- the medium added with this additive has the effect of promoting the proliferation of at least two cases of cervical cancer organoids isolated from cervical cancer tissue;
- “-” means that the medium with this additive added
- the culture medium has an inhibitory effect on at least one case of cervical cancer organoids isolated from cervical cancer tissues;
- “ ⁇ ” indicates that the medium added with this additive has an effect on at least one of the cervical cancer organoids Proliferation was not significantly affected in both cases.
- FGF10 prepared on the basis of formula 7 is added to the 96-well plate inoculated with organoids, and the final concentrations of FGF10 are 10 ng/mL, 20 ng/mL, and 100 ng respectively. /mL; and use the medium of formula 7 to set up control wells (BC).
- a ratio greater than 1 indicates that the prepared medium containing different concentrations of factors or small molecular compounds has a better effect on promoting proliferation than the culture medium of the control well; a ratio less than 1 indicates that the prepared medium containing different concentrations of factors or small molecular compounds promotes proliferation The effect was weaker than that of the culture medium in the control well.
- the volume concentration of B27 is preferably 1:25-1:100; the content of hepatocyte growth factor HGF is preferably 5-40 ng/mL; the content of SB202190 is preferably 200-1000 nM; the content of Y27632 is preferably The content of A83-01 is preferably 200-1000nM; the content of epidermal growth factor EGF is preferably 1-40ng/mL; the content of fibroblast growth factor 10 is preferably 10-100ng/mL; The content of growth factors is preferably 2-40ng/mL; the volume concentration of GlutaMAX is preferably 1:50-1:200; the content of MST1/2 kinase inhibitor compound 1 is preferably 2.5-10 ⁇ M; the content of nicotinamide is preferably 1- 10mM.
- the cervical cancer primary cells (OC1, OC2 and OC4) obtained according to the method described in (2) of Example 1 were resuspended and counted with the cervical cancer organoid medium COM of the present invention, and the cell density was diluted to 5-10 ⁇ 10 cells/mL, take 400 ⁇ L of the diluted cell suspension and add it to an equal volume of Matrigel (Corning) to mix gently, and then inoculate the mixture in a 24-well plate at 40 ⁇ L/well. Put the inoculated culture plate into the incubator for 30 minutes, wait until the Matrigel is completely solidified, then add the cervical cancer organoid medium COM that has been returned to room temperature in advance, 500 ⁇ L per well, and expand the culture by replacing the medium every five days.
- Figures 2A-2D are the cervical cancers obtained after culture of samples OC1 (day 5), OC1 (day 14), OC2 (day 7), and OC2 (day 7, another field of view) under a 4x objective lens Photos of organs. As shown, organoids grow in size during culture; different types of organoids can be formed from the same sample, which can mimic tumor heterogeneity in vitro.
- Pathological and immunohistochemical identifications were performed on the cultured cervical cancer organoids, and the corresponding original tissue samples were also identified pathologically and immunohistochemically to compare the consistency of organoids and histopathological indicators.
- Figure 3 is the results of pathological and immunohistochemical identification of the original tissue sample OC4 and the cervical cancer organoids obtained after its in vitro culture, which are pictures taken under a 20x objective lens.
- the results show that the structural morphology of the organoid is a cancerous tissue morphology; according to the immunohistochemical indicators, it is judged that the cells obtained after culturing the organoids in this case are cervical cancer cells.
- This result shows that the cervical cancer organoid cultured using the medium COM of the present invention is consistent with the diagnostic result of the cervical cancer tissue before culture.
- Figures 4A and 4B are photographs of organoids cultured in COM medium and ROM medium for 10 days under a 4x objective lens.
- COM medium can significantly promote the formation and expansion of cervical cancer organoids.
- Example 5 Using the culture medium of the present invention to amplify the cervical cancer organoids for drug screening
- Cervical cancer primary cells were isolated from the cervical cancer intraoperative sample (CCa5) according to the method of (2) of Example 1, and the organoids were cultured in COM medium, and drug screening was performed when the diameter of the cervical cancer organoids exceeded 50 ⁇ m.
- cisplatin was prepared into 6 different concentrations of additives, the highest concentration was 9.5 ⁇ M, and then diluted with a 2-fold dilution ratio to obtain 4.75 ⁇ M, 2.38 ⁇ M, 1.19 ⁇ M, 0.59 ⁇ M, 0.3 ⁇ M ⁇ M of different concentrations of additives.
- Carboplatin was prepared into 6 different concentrations of storage solution, the highest concentration was 137.92 ⁇ M, and then diluted by 2 times dilution ratio to obtain 68.96 ⁇ M, 34.48 ⁇ M, 17.24 ⁇ M, 8.62 ⁇ M, 4.31 ⁇ M ⁇ M of different concentrations of additives.
- Paclitaxel was prepared into 6 different concentrations of additive solutions, the highest concentration was 11.51 ⁇ M, and then diluted with a 2-fold dilution ratio to obtain different concentrations of 5.76 ⁇ M, 2.88 ⁇ M, 1.44 ⁇ M, 0.72 ⁇ M, and 0.36 ⁇ M. concentration of additives.
- bortezomib was prepared into 6 different concentrations of additives, the highest concentration was 2 ⁇ M, and then diluted by 2 times dilution ratio to obtain 1 ⁇ M, 0.5 ⁇ M, 0.25 ⁇ M, 0.125 ⁇ M, 0.0625 ⁇ M ⁇ M of different concentrations of additives.
- step (1) Take out the prepared drug additive solution and place it at room temperature. Take out the organoids obtained by culturing according to step (1) from the incubator, remove the medium in the culture wells, and slowly pour the additives containing different concentrations of drugs into the 96-well transparent culture plate along the well wall at 100 ⁇ L per well. After the drug addition, the surface of the 96-well plate was sterilized and moved to the incubator to continue culturing, and the viability of the organoids was measured 5 days later.
- the drug inhibition rate (%) 100%-(the chemiluminescence value drug treatment group of the culture well on the fifth day/the chemiluminescence value drug treatment group of the culture well on the zero day)/(the chemiluminescence value DMSO of the culture well on the fifth day/the The chemiluminescent value of the zero-day culture wells (DMSO )*100% was calculated to obtain the inhibition rates of different drugs, and the results are shown in FIG. 5 .
- Fig. 5 is the inhibition rate curve of different concentrations of test drugs inhibiting the growth of cervical cancer organoids.
- bortezomib has a strong inhibitory effect on organoid growth at 6 concentrations
- paclitaxel has the same inhibitory efficiency at 6 concentrations
- the inhibitory effects of different concentrations of cisplatin and carboplatin are significant. Certain differences were dose-dependent, suggesting that organoids from the same patient have different effectiveness and sensitivity to different drugs. According to the results, the effectiveness and effective dosage of the drug can be judged when the cervical cancer patients are clinically used.
- the invention provides a culture medium and a culture method for cervical cancer organoids, and the cultured organoids can be applied to the efficacy evaluation and screening of drugs.
- the present invention is suitable for industrial applications.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
A culture medium for cervical cancer organoids, comprising an MST1/2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, hepatocyte growth factor, SB202190, Y27632, A83-01, epidermal growth factor, fibroblast growth factor 10, keratinocyte growth factor, GlutaMAX, and niacinamide. Also provided is a culture method for cervical cancer organoids. By using the culture medium for cervical cancer organoids, effective and quick expansion of cervical cancer organoids can be achieved, the expanded organoids maintain the pathological characteristics of patients, the culture success rate and expansion rate of cervical cancer organoids are improved, and a research basis can be provided for personalized treatment of the patients.
Description
本发明属于生物技术领域,具体涉及一种用于宫颈癌类器官的培养基、使用该培养基培养宫颈癌类器官的方法。The invention belongs to the field of biotechnology, and in particular relates to a culture medium for cervical cancer organoids and a method for cultivating cervical cancer organoids using the medium.
宫颈癌是原发于子宫颈部位的恶性肿瘤,为女性生殖系统最常见的恶性肿瘤。目前宫颈癌在全世界女性恶性肿瘤中排名第四。宫颈癌高发于30~55岁,在我国发病率仅位于乳腺癌之后。由于宫颈癌筛查的普及和HPV疫苗的推广,宫颈癌很大程度上已成为一种可预防的疾病,然而目前其5年生存率仍只有60%左右。对于早期无转移的宫颈癌,手术及放化疗是主要的治疗手段,但是对于转移性或复发性宫颈癌,传统的治疗方法并未取得满意的疗效。随着抗血管生成药物、免疫检查点抑制剂等靶向治疗和免疫治疗药物的使用,这部分患者的生存时间得到了显著延长,但仍未达到最终治愈的效果,亟需借助于肿瘤原代细胞作为研究模型来探究新的药物靶点和指导个性化治疗。Cervical cancer is a malignant tumor that originates in the cervix and is the most common malignant tumor in the female reproductive system. Cervical cancer ranks fourth among female malignancies worldwide. The highest incidence of cervical cancer is between 30 and 55 years old, and the incidence rate in my country is only behind breast cancer. Due to the popularity of cervical cancer screening and the promotion of HPV vaccine, cervical cancer has largely become a preventable disease, but its 5-year survival rate is still only about 60%. For early non-metastatic cervical cancer, surgery, radiotherapy and chemotherapy are the main treatment methods, but for metastatic or recurrent cervical cancer, traditional treatment methods have not achieved satisfactory results. With the use of targeted therapy and immunotherapy drugs such as anti-angiogenic drugs and immune checkpoint inhibitors, the survival time of these patients has been significantly prolonged, but the final cure effect has not yet been achieved. Cells serve as research models to discover new drug targets and guide personalized therapy.
传统临床药物敏感性检测大多采用二维细胞培养。然而,二维培养的细胞仅在有限程度上模拟组织生理条件,缺乏体内真实的组织结构,易导致低分化水平和细胞生理功能的丢失,进而导致获得的实验结果很难预测临床实际结果。类器官,属于三维(3D)细胞培养物,主要来源于人体具有分化能力的胚胎干细胞、诱导多潜能干细胞和成体干细胞。不同组织器官都存在内源组织干细胞,在维持各器官的功能形态发挥着重要作用。这些干细胞在体外一定的诱导条件下,可以自组织形成一个直径仅为几毫米的迷你结构。肿瘤类器官是用取自患者体内原发性肿瘤,在实验室中培养出一些微型的3D肿瘤细胞模型。肿瘤类器官高度模拟了来源肿瘤组织的特征,保留了个体之间的肿瘤异质性,可用于功能性的测试,如进行药物筛选和个体化精准治疗。Traditional clinical drug sensitivity testing mostly uses two-dimensional cell culture. However, two-dimensional cultured cells can only simulate the physiological conditions of tissues to a limited extent, and lack the real tissue structure in vivo, which will easily lead to low differentiation level and loss of cell physiological functions, which makes it difficult to predict the actual clinical results from the obtained experimental results. Organoids, which belong to three-dimensional (3D) cell cultures, are mainly derived from human embryonic stem cells, induced pluripotent stem cells and adult stem cells with differentiation ability. Endogenous tissue stem cells exist in different tissues and organs, and play an important role in maintaining the functional morphology of various organs. Under certain induction conditions in vitro, these stem cells can self-organize to form a miniature structure with a diameter of only a few millimeters. Tumor organoids are obtained from primary tumors in patients, and some miniature 3D tumor cell models are cultivated in the laboratory. Tumor organoids highly simulate the characteristics of the source tumor tissue, retain the tumor heterogeneity among individuals, and can be used for functional testing, such as drug screening and individualized precision therapy.
当前,宫颈癌类器官培养方法多采用R-spondin-1、WNT3A和Noggin等昂贵的蛋白因子,导致类器官培养成本较高;且这项技术操作复杂和技术难度大,导致其大规模商业化应用受到限制。因此,需 要开发一种低成本、简单且成功率高的类器官培养方法和培养基。Currently, cervical cancer organoid culture methods mostly use expensive protein factors such as R-spondin-1, WNT3A, and Noggin, resulting in high cost of organoid culture; and this technology is complicated to operate and technically difficult, leading to its large-scale commercialization Application is limited. Therefore, there is a need to develop a low-cost, simple and high success rate organoid culture method and medium.
发明内容Contents of the invention
为了解决上述技术问题,本发明提供了一种用于在体外快速扩增宫颈癌类器官的培养基及培养方法。In order to solve the above technical problems, the present invention provides a culture medium and a culture method for rapid expansion of cervical cancer organoids in vitro.
本发明的一个方面在于提供一种宫颈癌类器官的培养基,所述培养基包含MST1/2激酶抑制剂、选自N2和B27的至少一种细胞培养添加剂、肝细胞生长因子、SB202190、Y27632、A83-01、表皮细胞生长因子、成纤维细胞生长因子10、角化细胞生长因子、GlutaMAX和烟酰胺。其中,所述MST1/2激酶抑制剂包括式(I)的化合物或其药学可接受的盐、或溶剂化物,One aspect of the present invention is to provide a culture medium for cervical cancer organoids, said culture medium comprising an MST1/2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, hepatocyte growth factor, SB202190, Y27632 , A83-01, Epidermal Growth Factor, Fibroblast Growth Factor 10, Keratinocyte Growth Factor, GlutaMAX, and Niacinamide. Wherein, the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof,
其中,in,
R
1选自C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C2-C6螺环烷基、以及任选地被1-2个独立地R
6取代的芳基(例如苯基和萘基等)、芳基C1-C6烷基(例如苯甲基等)和杂芳基(例如噻吩基等);
R 1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted by 1-2 independent R (such as phenyl and naphthyl, etc.), aryl C1-C6 alkyl (such as benzyl, etc.) and heteroaryl (such as thienyl, etc.);
R
2和R
3各自独立地选自C1-C6烷基,优选C1-C3烷基,更优选甲基;
R 2 and R 3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;
R
4和R
5各自独立地选自氢、C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C1-C6烷基羟基、C1-C6卤代烷基、C1-C6烷基氨基C1-C6烷基、C1-C6烷氧基C1-C6烷基、和C3-C6杂环基C1-C6烷基(所述杂环基选自例如哌啶基、四氢吡喃基等);
R 4 and R 5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxyl, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl (the heterocyclyl is selected from, for example, piperidinyl, tetrahydropyran base, etc.);
R
6选自卤素(优选氟和氯,更优选氟)、C1-C6烷基(优选甲基)、C1-C6烷氧基(优选甲氧基)、和C1-C6卤代烷基(优选三氟甲基)。
R is selected from halogen (preferably fluorine and chlorine, more preferably fluorine), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoro methyl).
优选的实施方式中,MST1/2激酶抑制剂包括式(Ia)的化合物或其 药学可接受的盐、或溶剂化物,In a preferred embodiment, the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt thereof, or a solvate,
其中,in,
R
1选自C1-C6烷基、任选地被1-2个独立地R
6取代的苯基、任选地被1-2个独立地R
6取代的噻吩基、和任选地被1-2个独立地R
6取代的苯甲基,R
1更优选为任选地被1-2个独立地R
6取代的苯基;
R is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently R6 , thienyl optionally substituted by 1-2 independently R6 , and optionally substituted by 1 -2 independently R6 substituted benzyl, R1 is more preferably optionally 1-2 independently R6 substituted phenyl;
R
5选自氢、C1-C6烷基、和C3-C6环烷基,R
5更优选为氢;
R 5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R 5 is more preferably hydrogen;
R
6各自独立地选自卤素、C1-C6烷基、和C1-C6卤代烷基,R
6更优选为氟、甲基或三氟甲基。
R 6 is each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, and R 6 is more preferably fluorine, methyl or trifluoromethyl.
优选地,所述MST1/2抑制剂是选自以下化合物或其药学可接受的盐、或溶剂化物中的至少一种。Preferably, the MST1/2 inhibitor is at least one selected from the following compounds or pharmaceutically acceptable salts or solvates thereof.
最优选地,本发明的MST1/2激酶抑制剂为化合物1。Most preferably, the MST1/2 kinase inhibitor of the present invention is Compound 1.
在本发明的实施方式中,本发明的培养基中各成分的含量满足以下任意一项或多项或全部满足:In an embodiment of the present invention, the content of each component in the culture medium of the present invention satisfies any one or more or all of the following:
(1)MST1/2激酶抑制剂的浓度优选为2.5~10μM;(1) The concentration of the MST1/2 kinase inhibitor is preferably 2.5-10 μM;
(2)B27或N2细胞培养添加剂相对于培养基的体积比为1:25~1:100;(2) The volume ratio of B27 or N2 cell culture supplement to the medium is 1:25~1:100;
(3)肝细胞生长因子的浓度优选为5~40ng/mL;(3) The concentration of hepatocyte growth factor is preferably 5-40 ng/mL;
(4)SB202190的浓度优选为200~1000nM;(4) The concentration of SB202190 is preferably 200-1000nM;
(5)Y27632的浓度优选为2.5~10μM;(5) The concentration of Y27632 is preferably 2.5-10 μM;
(6)A83-01的浓度优选为200~1000nM;(6) The concentration of A83-01 is preferably 200-1000nM;
(7)表皮细胞生长因子的浓度优选为1~40ng/mL;(7) The concentration of epidermal growth factor is preferably 1-40 ng/mL;
(8)成纤维细胞生长因子10的浓度优选为10~100ng/mL;(8) The concentration of fibroblast growth factor 10 is preferably 10-100 ng/mL;
(9)角化细胞生长因子的浓度优选为2~40ng/mL;(9) The concentration of keratinocyte growth factor is preferably 2-40 ng/mL;
(10)GlutaMAX相对于培养基的体积比优选为1:50~1:200;(10) The volume ratio of GlutaMAX to the medium is preferably 1:50 to 1:200;
(11)烟酰胺的浓度优选为1~10mM。(11) The concentration of nicotinamide is preferably 1 to 10 mM.
在本发明的实施方式中,所述培养基还含有选自DMEM/F12、DMEM、F12或RPMI-1640的初始培养基;和选自链霉素/青霉素、两性霉素B和Primocin中的一种或多种的抗生素。In an embodiment of the present invention, the medium also contains an initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one selected from streptomycin/penicillin, amphotericin B and Primocin one or more antibiotics.
在优选的实施方式中,当抗生素选自链霉素/青霉素时,链霉素浓度范围为25~400μg/mL,青霉素浓度范围为25~400U/mL,当抗生素选自两性霉素B时,浓度范围为0.25~4μg/mL,当抗生素选自Primocin时,浓度范围为25~400μg/mL。In a preferred embodiment, when the antibiotic is selected from streptomycin/penicillin, the streptomycin concentration ranges from 25 to 400 μg/mL, and the penicillin concentration ranges from 25 to 400 U/mL; when the antibiotic is selected from amphotericin B, The concentration range is 0.25-4 μg/mL, and when the antibiotic is selected from Primocin, the concentration range is 25-400 μg/mL.
本发明还提供一种宫颈癌类器官的培养方法。在本发明的宫颈癌类器官的培养方法中,使用本发明的宫颈癌类器官培养基对宫颈癌类器官进行培养。The invention also provides a method for culturing cervical cancer organoids. In the method for culturing cervical cancer organoids of the present invention, cervical cancer organoids are cultured using the cervical cancer organoid culture medium of the present invention.
本发明的宫颈癌类器官培养方法包括以下步骤。The method for culturing cervical cancer organoids of the present invention includes the following steps.
1.从宫颈癌实体瘤组织分离样本,获得宫颈癌原代细胞。该处理过程包括以下步骤:1. Separating samples from cervical cancer solid tumor tissues to obtain primary cervical cancer cells. The process includes the following steps:
(1)分离宫颈癌组织样本,加入1:1比例的基础培养基和组织消化液(组织消化液的加入量是每1g肿瘤组织使用约10mL组织消化液)中置于恒温摇床中进行消化,消化温度为4~37℃,摇床转速为200rpm~300rpm,消化时间为3~6小时;(1) Separate cervical cancer tissue samples, add basal culture medium and tissue digestion solution at a ratio of 1:1 (the amount of tissue digestion solution is about 10 mL of tissue digestion solution per 1 g of tumor tissue) and place in a constant temperature shaker for digestion , the digestion temperature is 4-37°C, the shaker speed is 200rpm-300rpm, and the digestion time is 3-6 hours;
(2)消化完成后,离心后弃去上清液,离心转速为1200~1600rpm,离心时间为2~6分钟。(2) After the digestion is completed, discard the supernatant after centrifugation, the centrifugation speed is 1200-1600 rpm, and the centrifugation time is 2-6 minutes.
其中,基础培养基配方包括选自DMEM/F12、DMEM、F12或RPMI-1640的初始培养基;和选自链霉素/青霉素、两性霉素B和Primocin中的一种或多种的抗生素。组织消化液配方包括1640培养基、胶原酶Ⅱ(1~2mg/mL)、胶原酶Ⅳ(1~2mg/mL)、DNA酶(50~100 U/mL)、透明质酸酶(0.5~1mg/mL)、氯化钙(1~5mM)、牛血清白蛋白BSA(5~10mg/mL)。Wherein, the basal medium formula includes an initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one or more antibiotics selected from streptomycin/penicillin, amphotericin B and Primocin. The formula of tissue digestion solution includes 1640 medium, collagenase Ⅱ (1-2 mg/mL), collagenase Ⅳ (1-2 mg/mL), DNase (50-100 U/mL), hyaluronidase (0.5-1 mg /mL), calcium chloride (1~5mM), bovine serum albumin BSA (5~10mg/mL).
2.配制本发明的宫颈癌类器官培养基,并对上述步骤获得的宫颈癌原代细胞进行培养。2. Prepare the cervical cancer organoid culture medium of the present invention, and culture the primary cervical cancer cells obtained in the above steps.
将上述步骤1中获得的宫颈癌原代细胞用本发明的宫颈癌类器官培养基重悬并计数,将细胞密度稀释为5~10×10
5个/mL,取出稀释后的细胞悬液加入等体积的Matrigel基质胶中混匀,然后将混合物接种于多孔板,将接种好的多孔板放入培养箱30-60分钟,待Matrigel完全凝固,然后加入宫颈癌类器官培养基进行扩大培养。
Resuspend the cervical cancer primary cells obtained in the above step 1 with the cervical cancer organoid culture medium of the present invention and count them, dilute the cell density to 5-10× 105 cells/mL, take out the diluted cell suspension and add Mix in an equal volume of Matrigel matrigel, then inoculate the mixture on a multi-well plate, put the inoculated multi-well plate in the incubator for 30-60 minutes, wait until the Matrigel is completely solidified, and then add cervical cancer organoid medium for expansion culture.
在本发明的其他方面,还提供一种用于评估或筛选治疗宫颈癌的药物的方法,其特征在于,包括以下步骤:In other aspects of the present invention, there is also provided a method for assessing or screening a drug for the treatment of cervical cancer, characterized in that it comprises the following steps:
(1)使用本发明的宫颈癌类器官的培养方法培养宫颈癌类器官;(1) Cultivate cervical cancer organoids using the culture method of cervical cancer organoids of the present invention;
(2)选定需要检测的药物并按照所需浓度梯度进行稀释;(2) Select the drug to be detected and dilute it according to the required concentration gradient;
(3)对(1)中培养得到的宫颈癌类器官添加稀释后的所述药物;和(3) adding the diluted drug to the cervical cancer organoids cultured in (1); and
(4)进行类器官大小或类器官活力检测。(4) Perform organoid size or organoid viability detection.
本发明的有益效果包括:The beneficial effects of the present invention include:
(1)提高宫颈癌组织来源类器官培养的成功率,成功率达到85%以上;(1) Improve the success rate of cervical cancer tissue-derived organoid culture, with a success rate of over 85%;
(2)保证体外原代培养的宫颈癌类器官能够保持病人的病理特性;(2) Ensure that the cervical cancer organoids cultured in vitro can maintain the pathological characteristics of the patient;
(3)扩增效率高,能快速培养出宫颈癌类器官,扩增出的宫颈癌类器官还可以连续传代;(3) The amplification efficiency is high, and cervical cancer organoids can be rapidly cultivated, and the amplified cervical cancer organoids can also be continuously passaged;
(4)培养成本可控,培养基无需加入价格昂贵的Wnt激动剂、R-spondin家族蛋白和Noggin蛋白;(4) The culture cost is controllable, and the medium does not need to add expensive Wnt agonists, R-spondin family proteins and Noggin proteins;
(5)所述技术养获得的宫颈癌类器官数量多,适合高通量筛选候选化合物和为病人提供高通量药物体外敏感性功能测试。(5) The number of cervical cancer organoids obtained by the technique is large, which is suitable for high-throughput screening of candidate compounds and providing patients with high-throughput drug sensitivity functional tests in vitro.
图1A-1K为显示本发明的宫颈癌类器官培养基所添加因子的不同浓度对宫颈癌类器官增殖的影响的图。1A-1K are graphs showing the effects of different concentrations of factors added to the cervical cancer organoid medium of the present invention on the proliferation of cervical cancer organoids.
图2A-2D为利用显微镜观察使用本发明的宫颈癌类器官培养基培养得到的宫颈癌类器官的照片,其中图2A显示由样本OC1获得的类器官培养5天的照片;图2B显示由样本OC1获得的类器官培养14天后的照片;图2C和2D显示由样本OC2获得的类器官培养7天后的不同视野的照片。Figures 2A-2D are photographs of cervical cancer organoids cultured using the cervical cancer organoid medium of the present invention observed under a microscope, wherein Figure 2A shows photos of organoids obtained from sample OC1 cultured for 5 days; Figure 2B shows photos obtained from sample OC1 Photos of organoids obtained from OC1 after 14 days of culture; Figure 2C and 2D show photos of different fields of view of organoids obtained from sample OC2 after 7 days of culture.
图3为对原始组织样本OC4及使用本发明的宫颈癌类器官培养基培养样本OC4得到的宫颈癌类器官进行病理和免疫组化鉴定的结果。Fig. 3 is the result of pathological and immunohistochemical identification of the original tissue sample OC4 and the cervical cancer organoid obtained by using the cervical cancer organoid culture medium culture sample OC4 of the present invention.
图4A和4B为使用本发明的宫颈癌类器官培养基与现有技术培养基对宫颈癌类器官进行培养的比较结果,其中图4A显示用本发明的COM培养基培养10天后的照片;图4B显示用文献培养基ROM培养10天后的照片。Figures 4A and 4B are the comparison results of using the cervical cancer organoid culture medium of the present invention and the culture medium of the prior art to culture cervical cancer organoids, wherein Figure 4A shows the photos after culturing for 10 days with the COM medium of the present invention; Fig. 4B shows photographs after 10 days of culture with literature medium ROM.
图5为显示对使用本发明的宫颈癌类器官培养基培养得到宫颈癌类器官进行药物浓度敏感性测试的结果的图。Fig. 5 is a graph showing the results of drug concentration sensitivity testing of cervical cancer organoids cultured using the cervical cancer organoid medium of the present invention.
为更好地理解本发明,下面结合实施例及附图对本发明作进一步描述。以下实施例仅是对本发明进行说明而非对其加以限定。In order to better understand the present invention, the present invention will be further described below in conjunction with the embodiments and accompanying drawings. The following examples are only to illustrate the present invention but not to limit it.
[MST1/2激酶抑制剂的制备实施例][Preparation Example of MST1/2 Kinase Inhibitor]
本说明书中,MST1/2激酶抑制剂是指直接或间接地对MST1/2信号传导进行负调节的任意的抑制剂。一般来说,MST1/2激酶抑制剂例如与MST1/2激酶结合并降低其活性。由于MST1和MST2的结构具有相似性,MST1/2激酶抑制剂也可以是例如与MST1或MST2结合并降低其活性的化合物。In the present specification, an MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1/2 signal transduction. In general, MST1/2 kinase inhibitors, for example, bind to MST1/2 kinase and reduce its activity. Due to the similarity in the structures of MST1 and MST2, MST1/2 kinase inhibitors may also be, for example, compounds that bind to MST1 or MST2 and reduce their activity.
1.MST1/2激酶抑制剂化合物1的制备1. Preparation of MST1/2 Kinase Inhibitor Compound 1
4-((7-(2,6-二氟苯基)-5,8-二甲基-6-氧代-5,6,7,8-四氢蝶啶-2-基)氨基)苯4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzene
磺酰胺1Sulfonamide 1
2-氨基-2-(2,6-二氟苯基)乙酸甲酯(A2):在圆底烧瓶中加入2-氨基-2-(2,6-二氟苯基)乙酸(2.0克)后加入甲醇(30毫升),随后冰浴下滴加二氯亚砜(1.2毫升)。反应体系在85℃反应过夜。反应结束后,体系在减压下蒸干溶剂,所得白色固体,直接用于下一步。2-Amino-2-(2,6-difluorophenyl)acetic acid methyl ester (A2): In a round bottom flask was added 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) Methanol (30 mL) was then added, followed by the dropwise addition of thionyl chloride (1.2 mL) under ice-cooling. The reaction system was reacted overnight at 85°C. After the reaction, the system was evaporated to dryness under reduced pressure to obtain a white solid, which was directly used in the next step.
2-((2-氯-5-硝基嘧啶-4-基)氨基)-2-(2,6-二氟苯基)乙酸甲酯(A3):在圆底烧瓶中加入2-氨基-2-(2,6-二氟苯基)乙酸甲酯(2克)后加入丙酮(30毫升)和碳酸钾(2.2克),然后用冰盐浴使体系冷却到-10℃,接着缓慢加入2,4-二氯-5-硝基嘧啶(3.1克)的丙酮溶液。反应体系在室温搅拌过夜。反应结束后,过滤,滤液在减压下除去溶剂,残留物经加压硅胶柱层析提纯后得化合物A3。LC/MS:M+H 359.0。Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (A3): Add 2-amino- After adding acetone (30 ml) and potassium carbonate (2.2 g) to 2-(2,6-difluorophenyl) methyl acetate (2 g), the system was cooled to -10 ° C with an ice-salt bath, and then slowly added 2,4-Dichloro-5-nitropyrimidine (3.1 g) in acetone. The reaction was stirred overnight at room temperature. After the reaction was completed, it was filtered, and the solvent was removed from the filtrate under reduced pressure, and the residue was purified by pressurized silica gel column chromatography to obtain compound A3. LC/MS: M+H 359.0.
2-氯-7-(2,6-二氟苯基)-7,8-二氢蝶啶-6(5H)-酮(A4):在圆底烧瓶中加入2-((2-氯-5-硝基嘧啶-4-基)氨基)-2-(2,6-二氟苯基)乙酸甲酯(2.5克)后加入醋酸(50毫升)和铁粉(3.9克)。反应体系在60℃搅拌两小时。反应结束后,体系在减压下蒸干溶剂,所得物用饱和碳酸氢钠中和至碱性。乙酸乙酯萃取,有机相分别用水、饱和食盐水洗涤后用无水硫酸钠干燥。有机相经过滤,减压蒸干后得粗品。粗品经乙醚洗涤后得化合物A4。LC/MS:M+H 297.0。2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (A4): add 2-((2-chloro- Methyl 5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (2.5 g) was added followed by acetic acid (50 ml) and iron powder (3.9 g). The reaction system was stirred at 60°C for two hours. After the reaction, the system was evaporated to dryness under reduced pressure, and the resultant was neutralized to alkaline with saturated sodium bicarbonate. Extracted with ethyl acetate, the organic phase was washed with water and saturated brine, and dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to obtain a crude product. The crude product was washed with ether to obtain compound A4. LC/MS: M+H 297.0.
2-氯-7-(2,6-二氟苯基)-5,8-二甲基-7,8-二氢蝶啶-6(5H)-酮(A5):在圆底烧瓶中加入2-氯-7-(2,6-二氟苯基)-7,8-二氢蝶啶-6(5H)-酮(2克)和N,N-二甲基乙酰胺(10毫升),冷却至-35℃,加入碘甲烷(0.9毫升),随后加入氢化钠(615毫克),反应体系继续搅拌两小时。反应结束后,加水淬灭,乙酸乙酯萃取,有机相分别用水、饱和食盐水洗涤后用无水硫酸钠干燥。有机相经过滤,减压蒸干后得粗品。粗品经乙醚洗涤后得化合物A5。LC/MS:M+H 325.0。2-Chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (A5): add 2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (2 g) and N,N-dimethylacetamide (10 mL) , cooled to -35°C, iodomethane (0.9 ml) was added, followed by sodium hydride (615 mg), and the reaction system was stirred for two hours. After the reaction was completed, it was quenched by adding water, extracted with ethyl acetate, and the organic phase was washed with water and saturated brine respectively, and dried with anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to obtain a crude product. The crude product was washed with ether to obtain compound A5. LC/MS: M+H 325.0.
4-((7-(2,6-二氟苯基)-5,8-二甲基-6-氧代-5,6,7,8-四氢蝶啶-2-基)氨 基)苯磺酰胺(1):在圆底烧瓶中加入2-氯-7-(2,6-二氟苯基)-5,8-二甲基-7,8-二氢蝶啶-6(5H)-酮(100毫克)、磺胺(53毫克)、对甲苯磺酸(53毫克)和仲丁醇(5毫升)。反应体系在120℃搅拌过夜。反应结束后,过滤,甲醇和乙醚洗涤得化合物1。LC/MS:M+H 461.1。4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzene Sulfonamide (1): Add 2-chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridine-6(5H) to a round bottom flask - Ketone (100 mg), sulfonamide (53 mg), p-toluenesulfonic acid (53 mg) and sec-butanol (5 ml). The reaction system was stirred overnight at 120°C. After the reaction, filter and wash with methanol and ether to obtain compound 1. LC/MS: M+H 461.1.
2.本发明的其他MST1/2抑制剂化合物的制备2. Preparation of other MST1/2 inhibitor compounds of the present invention
本发明的其他MST1/2抑制剂化合物按照与化合物1类似的方法合成,其结构及质谱数据如下表所示。Other MST1/2 inhibitor compounds of the present invention were synthesized according to a method similar to compound 1, and their structures and mass spectrometry data are shown in the table below.
实施例1宫颈癌类器官培养基中各添加因子对宫颈癌类器官增殖的影响Example 1 Effects of Added Factors in Cervical Cancer Organoid Culture Medium on Proliferation of Cervical Cancer Organoids
(1)宫颈癌类器官培养基的配制(1) Preparation of cervical cancer organoid culture medium
首先配制含有初始培养基的基础培养基。初始培养基可选自本领域常用的DMEM/F12、DMEM、F12或RPMI-1640。在本实施例中,基础培养基的配方为:DMEM/F12培养基(购自Corning公司)+100μg/mL Primocin(购自InvivoGen公司,0.2%(v/v),市售产品浓度50mg/ml)。First prepare the basal medium containing the initial medium. The initial medium can be selected from DMEM/F12, DMEM, F12 or RPMI-1640 commonly used in the art. In this embodiment, the formulation of the basal medium is: DMEM/F12 medium (purchased from Corning Company)+100 μg/mL Primocin (purchased from InvivoGen Company, 0.2% (v/v), commercially available product concentration 50mg/ml ).
在基础培养基内分别加入不同种类的添加剂(参见表1)配制成含有不同添加成分的宫颈癌类器官培养基。Different types of additives (see Table 1) were added to the basal medium to prepare cervical cancer organoid culture medium containing different additive components.
(2)宫颈癌原代细胞的分离和处理(2) Isolation and processing of primary cervical cancer cells
1样品选择1 sample selection
宫颈癌实体瘤组织样品(术中)由专业医疗机构的专业医务人员从患者获取,患者均签署了知情同意书。术中样本5-10mm
3,采用商品化组织保存液(生产厂家:Miltenyi Biotec)存储运输。
Cervical cancer solid tumor tissue samples (intraoperative) were obtained from patients by professional medical staff of professional medical institutions, and all patients signed informed consent. Intraoperative samples of 5-10 mm 3 were stored and transported in commercial tissue preservation solution (manufacturer: Miltenyi Biotec).
2材料准备2 material preparation
15mL无菌离心管、移液枪、10mL移液管、无菌枪头等表面消毒后放入超净工作台中紫外照射30分钟。提前30分钟从4℃冰箱取出基础培养基,提前30分钟从-20℃冰箱取出组织消化液。After surface disinfection of 15mL sterile centrifuge tubes, pipette guns, 10mL pipettes, sterile pipette tips, etc., put them in the ultra-clean workbench and irradiate them with ultraviolet light for 30 minutes. Take out the basal medium from the 4°C refrigerator 30 minutes in advance, and take out the tissue digestion solution from the -20°C refrigerator 30 minutes in advance.
组织消化液配方:1640培养基(Corning,10-040-CVR)、胶原酶Ⅱ(2mg/mL)、胶原酶Ⅳ(2mg/mL)、DNA酶(50U/mL)、透明质酸酶(0.75mg/mL)、氯化钙(3.3mM)、牛血清白蛋白BSA(10mg/mL)。Tissue digestion solution formula: 1640 medium (Corning, 10-040-CVR), collagenase Ⅱ (2mg/mL), collagenase Ⅳ (2mg/mL), DNase (50U/mL), hyaluronidase (0.75 mg/mL), calcium chloride (3.3mM), bovine serum albumin BSA (10mg/mL).
以上提及的胶原酶Ⅱ、胶原酶Ⅳ、DNA酶、透明质酸酶均购自Sigma公司;氯化钙和BSA购自生工生物工程(上海)股份有限公司。Collagenase II, collagenase IV, DNase, and hyaluronidase mentioned above were all purchased from Sigma; calcium chloride and BSA were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.
3样品分离3 Sample Separation
3.1超净台中取组织样品于培养皿中,去除带血液的组织,用基础培养基冲洗2次,将组织转移至另一培养皿中用无菌手术刀进行机械分离,将组织块分割为1*2*1mm
3大小;
3.1 Take the tissue samples in the culture dish in the ultra-clean bench, remove the tissue with blood, wash it twice with the basal medium, transfer the tissue to another culture dish for mechanical separation with a sterile scalpel, and divide the tissue block into 1 *2*1mm 3 size;
3.2将切割后的术中组织吸至15mL离心管中,加入5mL基础培养基,混匀,于1500rpm离心3分钟;3.2 Aspirate the cut intraoperative tissue into a 15mL centrifuge tube, add 5mL basal medium, mix well, and centrifuge at 1500rpm for 3 minutes;
3.3弃上清,加入1:1比例的基础培养基和组织消化液(注:组织消化液的加入量是1g肿瘤组织使用约10mL组织消化液),标记样品名称及编号,用封口膜密封,在37℃下于300rpm摇床(知楚仪器ZQLY-180N)中进行消化,期间每30分钟观察消化是否完成,判断依据为无肉眼可见的颗粒物;3.3 Discard the supernatant, add basal medium and tissue digestion solution in a ratio of 1:1 (note: the amount of tissue digestion solution added is about 10 mL of tissue digestion solution for 1 g of tumor tissue), mark the sample name and number, and seal it with a parafilm. Digestion was carried out at 37°C on a 300rpm shaker (ZQLY-180N), during which the digestion was observed every 30 minutes to see if there were no particles visible to the naked eye;
3.4消化完成后,经70μm滤网过滤掉未消化的组织团块,滤网上的组织团块用基础培养基冲洗入离心管中以减少细胞损失,室温1500rpm离心3分钟;3.4 After the digestion is completed, filter the undigested tissue mass through a 70 μm filter, wash the tissue mass on the filter with the basal medium into the centrifuge tube to reduce cell loss, and centrifuge at room temperature for 3 minutes at 1500 rpm;
3.5弃上清,观察是否有血细胞,若有血细胞,加8mL血细胞裂解液(购自Sigma公司),混匀,4℃裂解20分钟,期间颠倒混匀一次,室温1500rpm离心4分钟;3.5 Discard the supernatant and observe whether there are blood cells. If there are blood cells, add 8 mL of blood cell lysate (purchased from Sigma Company), mix well, and lyse at 4°C for 20 minutes.
3.6弃上清,加入2mL基础培养基重悬细胞,备用。3.6 Discard the supernatant, add 2mL basal medium to resuspend the cells, and set aside.
4细胞计数及处理4 Cell Counting and Processing
4.1镜下观察:移取少量重悬细胞平铺于培养皿中,显微镜(CNOPTEC,BDS400)下观察癌细胞密度和形态;4.1 Observation under a microscope: transfer a small amount of resuspended cells to a culture dish, and observe the density and shape of cancer cells under a microscope (CNOPTEC, BDS400);
4.2活细胞计数:取重悬的细胞悬液12μL,12μL台盼蓝染液(生产厂家:生工生物工程(上海)股份有限公司)充分混合后,取20μL加入细胞计数板(生产厂家:Countstar,规格:50片/盒),细胞计数仪(Countstar,IC1000)下计算出活的大细胞(细胞粒径>10μm)百分率=活细胞数/总细胞数*100%。4.2 Viable cell counting: Take 12 μL of the resuspended cell suspension and 12 μL of trypan blue staining solution (manufacturer: Sangon Bioengineering (Shanghai) Co., Ltd.) and mix thoroughly, then take 20 μL and add it to a cell counting plate (manufacturer: Countstar , specification: 50 pieces/box), the percentage of living large cells (cell size > 10 μm) calculated by a cell counter (Countstar, IC1000) = number of viable cells/number of total cells * 100%.
(3)宫颈癌类器官的培养(3) Cultivation of cervical cancer organoids
将上述步骤中获得的宫颈癌原代细胞用预冷的DMEM/F12重悬并计数,将细胞密度稀释为5~10×10
5个/mL,取出400μL稀释后的细胞悬液加入等体积的Matrigel基质胶(Corning)中轻轻混匀,然后将混合物按照8μL/孔接种于96孔板。将接种好的培养板放入培养箱30分钟,待Matrigel完全凝固,然后分别加入事先恢复到室温的表1所示培养基,按照每五天更换一次培养基进行扩大培养。10天后对所培养的类器官进行拍照,并测量统计类器官的直径大小,比较各因子对宫颈癌类器官增殖的促进作用。其中,作为实验对照,使用未添加任何添加剂的基础培养基,将实验结果示于表1。
Resuspend and count the primary cervical cancer cells obtained in the above steps with pre-cooled DMEM/F12, dilute the cell density to 5-10× 105 cells/mL, take out 400 μL of the diluted cell suspension and add an equal volume of Matrigel Matrigel (Corning) was mixed gently, and then the mixture was inoculated in a 96-well plate at 8 μL/well. Put the inoculated culture plate into the incubator for 30 minutes, wait until the Matrigel is completely solidified, then add the culture medium shown in Table 1 that has been returned to room temperature beforehand, and expand the culture by replacing the culture medium every five days. After 10 days, the cultured organoids were photographed, and the diameters of the organoids were measured and counted, and the promotion effects of various factors on the proliferation of cervical cancer organoids were compared. Among them, as an experimental control, a basal medium without any additives was used, and the experimental results are shown in Table 1.
表1培养基中的添加成分及促类器官增殖效果Table 1 Added ingredients in the medium and the effect of promoting organoid proliferation
序号serial number | 培养基添加剂种类Types of medium additives | 供应商supplier | 终浓度Final concentration |
促增殖程度分级Grading of |
|
11 | N2N2 | GibcoGibco | 1:501:50 | ++ | |
22 | R-spondin1R-spondin1 | 北京义翘Beijing Yiqiao | 100ng/mL100ng/mL | ○○ | |
33 |
成纤维细胞生长因子10 |
北京义翘Beijing Yiqiao | 100ng/mL100ng/mL | ++ | |
44 | B27B27 | GibcoGibco | 1:501:50 | ++ | |
55 | A8301A8301 | MCEMCE | 500nM500nM | ++ | |
66 | SB202190SB202190 | MCEMCE | 500nM500nM | ++ | |
77 | 成纤维细胞生长因子/FGFFibroblast growth factor/FGF | 北京义翘Beijing Yiqiao | 10ng/mL10ng/mL | ○○ | |
88 | 肝细胞生长因子HGFhepatocyte growth factor HGF | 北京义翘Beijing Yiqiao | 10ng/mL10ng/mL | ++ | |
99 | NogginNoggin | 北京义翘Beijing Yiqiao | 100ng/mL100ng/mL | ○○ | |
1010 |
胎牛血清/FBSFetal bovine serum/ | ExcellExcell | 5%5% | ++ | |
1111 | 角化细胞生长因子/KGFKeratinocyte Growth Factor/KGF | 北京义翘Beijing Yiqiao | 10ng/mL10ng/mL | ++ | |
1212 | GlutaMAXGlutaMAX | GibcoGibco | 1:1001:100 | ++ | |
1313 | 烟酰胺Nicotinamide | MCEMCE | 2.5mM2.5mM | ++ | |
1414 | 表皮细胞生长因子EGFepidermal growth factor EGF | 北京义翘Beijing Yiqiao | 10ng/mL10ng/mL | ++ | |
1515 | Y27632Y27632 | MCEMCE | 10μM10μM | ++ | |
1616 | ITS细胞培养添加剂ITS Cell Culture Supplement | GibcoGibco | 1:1001:100 | ○○ |
1717 |
化合物1 |
制备例Preparation example | 5μM5μM | ++ |
1818 | CHIR99021CHIR99021 | MCEMCE | 2.5μM2.5μM | -- |
其中,“+”表示与基础培养基相比,加入该添加剂的培养基对从宫颈癌组织分离出的宫颈癌类器官中的至少两例有促进增殖的作用;“-”表示添加该添加剂的培养基对从宫颈癌组织分离出的宫颈癌类器官中的至少一例显示有抑制增殖的作用;“○”表示添加该添加剂的培养基对从宫颈癌组织分离出的宫颈癌类器官中的至少两例的增殖没有明显的影响。Among them, "+" means that compared with the basal medium, the medium added with this additive has the effect of promoting the proliferation of at least two cases of cervical cancer organoids isolated from cervical cancer tissue; "-" means that the medium with this additive added The culture medium has an inhibitory effect on at least one case of cervical cancer organoids isolated from cervical cancer tissues; "○" indicates that the medium added with this additive has an effect on at least one of the cervical cancer organoids Proliferation was not significantly affected in both cases.
根据以上结果,拟选择化合物1、Y27632、SB202190、角化细胞生长因子(KGF)、肝细胞生长因子(HGF)、A83-01、B27、GlutaMAX、成纤维细胞生长因子10(FGF10)、烟酰胺、表皮细胞生长因子(EGF)等因子进行进一步培养实验。According to the above results, it is proposed to select compound 1, Y27632, SB202190, keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), A83-01, B27, GlutaMAX, fibroblast growth factor 10 (FGF10), nicotinamide , epidermal growth factor (EGF) and other factors for further culture experiments.
实施例2培养基添加因子的不同浓度对宫颈癌类器官的增殖作用Example 2 Effects of Different Concentrations of Factors Added to Medium on the Proliferation of Cervical Cancer Organoids
按照实施例1之(2)的方法从术中组织样本(编号为OC1、OC2)获得宫颈癌原代细胞,并使用下表2中的培养基配方进行类器官培养。According to the method of (2) of Example 1, primary cervical cancer cells were obtained from intraoperative tissue samples (numbered OC1, OC2), and organoids were cultured using the medium formula in Table 2 below.
表2培养基配方(浓度为终浓度)Table 2 medium formula (concentration is the final concentration)
在使用配方1的培养基时,在接种有类器官的96孔板中在配方1的基础上分别添加配制好的B27每孔200μL,B27的终浓度分别为1:25、1:50、1:100;并使用配方1的培养基设置对照孔(BC)。该系列的培养基中其他添加因子的终浓度与COM培养基相同。以下配方1-11的实验也以同样的方式进行,不再赘述。When the medium of formula 1 was used, 200 μL of B27 prepared on the basis of formula 1 was added to the 96-well plate inoculated with organoids, and the final concentrations of B27 were 1:25, 1:50, 1 : 100; and use the medium of formula 1 to set up control wells (BC). The final concentration of other added factors in this series of media is the same as that of COM media. The experiments of the following recipes 1-11 were also carried out in the same manner, so no further description was given.
在使用配方2的培养基时,在接种有类器官的96孔板中在配方2的基础上分别添加配制好的HGF每孔200μL,HGF的终浓度分别为40ng/mL、10ng/mL、5ng/mL;并使用配方2的培养基设置对照孔(BC)。When using the medium of formula 2, add 200 μL of HGF prepared on the basis of formula 2 to the 96-well plate inoculated with organoids, and the final concentrations of HGF are 40 ng/mL, 10 ng/mL, and 5 ng respectively. /mL; and use the medium of formula 2 to set up control wells (BC).
在使用配方3的培养基时,在接种有类器官的96孔板中在配方3的基础上分别添加配制好的SB202190细胞培养添加剂每孔200μL,SB202190细胞培养添加剂终浓度分别为200nM、500nM、1000nM;并使用配方3的培养基设置对照孔(BC)。When using the medium of Formula 3, 200 μL of SB202190 cell culture additive prepared on the basis of Formula 3 was added to the 96-well plate inoculated with organoids, and the final concentration of SB202190 cell culture additive was 200 nM, 500 nM, 1000 nM; and set up control wells (BC) using the medium of recipe 3.
在使用配方4的培养基时,在接种有类器官的96孔板中在配方4的基础上分别添加配制好的Y27632每孔200μL,Y27632的终浓度分别为2.5μM、5μM、10μM;并使用配方4的培养基设置对照孔(BC)。When using the medium of formula 4, add 200 μL of Y27632 prepared on the basis of formula 4 to the 96-well plate inoculated with organoids, and the final concentrations of Y27632 are 2.5 μM, 5 μM, and 10 μM respectively; and use The media of formulation 4 set up control wells (BC).
在使用配方5的培养基时,在接种有类器官的96孔板中在配方5的基础上分别添加配制好的A83-01每孔200μL,A83-01的终浓度分别为200nM、500nM、1000nM;并使用配方5的培养基设置对照孔(BC)。When using the medium of Formula 5, add 200 μL of A83-01 prepared on the basis of Formula 5 to the 96-well plate inoculated with organoids, and the final concentrations of A83-01 are 200 nM, 500 nM, and 1000 nM respectively. ; and set up control wells (BC) using the medium of formula 5.
在使用配方6的培养基时,在接种有类器官的96孔板中在配方6的基础上分别添加配制好的EGF每孔200μL,EGF终浓度分别为1ng/mL、5ng/mL、40ng/mL;并使用配方6的培养基设置对照孔(BC)。When using the medium of Formula 6, add 200 μL of EGF prepared on the basis of Formula 6 to the 96-well plate inoculated with organoids, and the final concentrations of EGF are 1 ng/mL, 5 ng/mL, 40 ng/mL, respectively. mL; and set up control wells (BC) using the medium of recipe 6.
在使用配方7的培养基时,在接种有类器官的96孔板中在配方7的基础上分别添加配制好的FGF10每孔200μL,FGF10的终浓度分别为10ng/mL、20ng/mL、100ng/mL;并使用配方7的培养基设置对照孔(BC)。When the medium of formula 7 is used, 200 μL of FGF10 prepared on the basis of formula 7 is added to the 96-well plate inoculated with organoids, and the final concentrations of FGF10 are 10 ng/mL, 20 ng/mL, and 100 ng respectively. /mL; and use the medium of formula 7 to set up control wells (BC).
在使用配方8的培养基时,在接种有类器官的96孔板中在配方8的基础上分别添加配制好的KGF每孔200μL,KGF的终浓度分别为2ng/mL、10ng/mL、40ng/mL;并使用配方8的培养基设置对照孔(BC)。When using the medium of Formula 8, add 200 μL of KGF prepared on the basis of Formula 8 to the 96-well plate inoculated with organoids, and the final concentrations of KGF are 2 ng/mL, 10 ng/mL, and 40 ng respectively. /mL; and set up control wells (BC) using the medium of formula 8.
在使用配方9的培养基时,在接种有类器官的96孔板中在配方9的基础上分别添加配制好的GlutaMAX每孔200μL,GlutaMAX的终 浓度分别为1:200、1:100、1:50;并使用配方9的培养基设置对照孔(BC)。When using the medium of Formula 9, add 200 μL per well of GlutaMAX prepared on the basis of Formula 9 to the 96-well plate inoculated with organoids, and the final concentrations of GlutaMAX are 1:200, 1:100, 1 : 50; and use the medium of formula 9 to set up control wells (BC).
在使用配方10的培养基时,在接种有类器官的96孔板中在配方10的基础上分别添加配制好的化合物1每孔200μL,化合物1的终浓度分别为2.5μM、5μM、10μM;并使用配方10的培养基设置对照孔(BC)。When the medium of Formula 10 was used, 200 μL of Compound 1 prepared on the basis of Formula 10 was added to the 96-well plate seeded with organoids, and the final concentrations of Compound 1 were 2.5 μM, 5 μM, and 10 μM; And set up control wells (BC) using the medium of formulation 10.
在使用配方11的培养基时,在接种有类器官的96孔板中在配方11的基础上分别添加配制好的烟酰胺每孔200μL,烟酰胺的终浓度分别为1mM、2.5mM、10mM;并使用配方11的培养基设置对照孔(BC)。When using the medium of Formula 11, add 200 μL of prepared nicotinamide to each well of the 96-well plate inoculated with organoids on the basis of Formula 11, and the final concentrations of nicotinamide are 1 mM, 2.5 mM, and 10 mM respectively; And set up control wells (BC) using the medium of formulation 11.
12天后对所培养的类器官进行拍照,并测量统计类器官的直径大小,比较各因子浓度对宫颈癌类器官增殖的促进作用。将2例样本收集的数据汇总示于图1A~1K。图1A~1K中,比值为使用各培养基培养12天得到的类器官直径与对应的BC对照孔培养12天得到的类器官直径的比。比值大于1说明配制的含不同浓度的因子或小分子化合物的培养基促增殖效果优于对照孔培养基;比值小于1,则说明配制的含不同浓度的因子或小分子化合物的培养基促增殖效果较对照孔培养基促增殖效果弱。After 12 days, the cultured organoids were photographed, and the diameters of the organoids were measured and counted, and the promotion effect of each factor concentration on the proliferation of cervical cancer organoids was compared. The data collected from the two samples are summarized in Figures 1A-1K. In Figures 1A-1K, the ratios are the diameters of the organoids cultured for 12 days using each medium to the diameter of the organoids cultured for 12 days in the corresponding BC control wells. A ratio greater than 1 indicates that the prepared medium containing different concentrations of factors or small molecular compounds has a better effect on promoting proliferation than the culture medium of the control well; a ratio less than 1 indicates that the prepared medium containing different concentrations of factors or small molecular compounds promotes proliferation The effect was weaker than that of the culture medium in the control well.
根据图1A~1K的结果,B27的体积浓度优选为1:25~1:100;肝细胞生长因子HGF的含量优选为5~40ng/mL;SB202190的含量优选为200~1000nM;Y27632的含量优选为2.5~10μM;A83-01的含量优选为200~1000nM;表皮细胞生长因子EGF的含量优选为1~40ng/mL;成纤维细胞生长因子10的含量优选为10~100ng/mL;角化细胞生长因子的含量优选为2~40ng/mL;GlutaMAX的体积浓度优选为1:50~1:200;MST1/2激酶抑制剂化合物1的含量优选为2.5~10μM;烟酰胺的含量优选为1~10mM。According to the results in Figures 1A-1K, the volume concentration of B27 is preferably 1:25-1:100; the content of hepatocyte growth factor HGF is preferably 5-40 ng/mL; the content of SB202190 is preferably 200-1000 nM; the content of Y27632 is preferably The content of A83-01 is preferably 200-1000nM; the content of epidermal growth factor EGF is preferably 1-40ng/mL; the content of fibroblast growth factor 10 is preferably 10-100ng/mL; The content of growth factors is preferably 2-40ng/mL; the volume concentration of GlutaMAX is preferably 1:50-1:200; the content of MST1/2 kinase inhibitor compound 1 is preferably 2.5-10μM; the content of nicotinamide is preferably 1- 10mM.
实施例3宫颈癌类器官培养及鉴定Example 3 Cervical Cancer Organoid Culture and Identification
将按照实施例1之(2)所述方法获得的宫颈癌原代细胞(OC1、OC2和OC4)用本发明的宫颈癌类器官培养基COM重悬并计数,将细胞密度稀释为5~10×10
5个/mL,取出400μL稀释后的细胞悬液加入等体积的Matrigel基质胶(Corning)中轻轻混匀,然后将混合物按 照40μL/孔接种于24孔板。将接种好的培养板放入培养箱30分钟,待Matrigel完全凝固,然后加入事先恢复到室温的宫颈癌类器官培养基COM,每孔500μL,按照每五天更换一次培养基进行扩大培养。
The cervical cancer primary cells (OC1, OC2 and OC4) obtained according to the method described in (2) of Example 1 were resuspended and counted with the cervical cancer organoid medium COM of the present invention, and the cell density was diluted to 5-10 × 10 cells/mL, take 400 μL of the diluted cell suspension and add it to an equal volume of Matrigel (Corning) to mix gently, and then inoculate the mixture in a 24-well plate at 40 μL/well. Put the inoculated culture plate into the incubator for 30 minutes, wait until the Matrigel is completely solidified, then add the cervical cancer organoid medium COM that has been returned to room temperature in advance, 500 μL per well, and expand the culture by replacing the medium every five days.
在第5-14天,使用显微镜(Invitrogen公司EVOS M500)观察培养得到的宫颈癌类器官。图2A-2D的是4倍物镜下拍摄样本OC1(第5天)、OC1(第14天)、OC2(第7天)、OC2(第7天,另一视野)培养后得到的宫颈癌类器官的照片。如图所示,类器官在培养过程中体积不断增大;同一个样本可以形成不同类型的类器官,可以在体外模拟肿瘤的异质性。On days 5-14, the cultured cervical cancer organoids were observed using a microscope (EVOS M500 from Invitrogen). Figures 2A-2D are the cervical cancers obtained after culture of samples OC1 (day 5), OC1 (day 14), OC2 (day 7), and OC2 (day 7, another field of view) under a 4x objective lens Photos of organs. As shown, organoids grow in size during culture; different types of organoids can be formed from the same sample, which can mimic tumor heterogeneity in vitro.
对培养得到的宫颈癌类器官进行病理和免疫组化鉴定,同时将对应的原始组织样本也进行病理和免疫组化鉴定,比较类器官和组织病理指标的一致性。Pathological and immunohistochemical identifications were performed on the cultured cervical cancer organoids, and the corresponding original tissue samples were also identified pathologically and immunohistochemically to compare the consistency of organoids and histopathological indicators.
图3为对原始组织样本OC4及对其体外培养后得到的宫颈癌类器官进行病理和免疫组化鉴定的结果,分别为20倍物镜下拍照的图片。如图所示,结果显示类器官的结构形态为癌组织形态;根据免疫组化指标判断该例样本类器官培养后得到的细胞为宫颈癌细胞。这一结果表明使用本发明的培养基COM培养的宫颈癌类器官与培养前的宫颈癌组织的诊断结果一致。Figure 3 is the results of pathological and immunohistochemical identification of the original tissue sample OC4 and the cervical cancer organoids obtained after its in vitro culture, which are pictures taken under a 20x objective lens. As shown in the figure, the results show that the structural morphology of the organoid is a cancerous tissue morphology; according to the immunohistochemical indicators, it is judged that the cells obtained after culturing the organoids in this case are cervical cancer cells. This result shows that the cervical cancer organoid cultured using the medium COM of the present invention is consistent with the diagnostic result of the cervical cancer tissue before culture.
实施例4与现有技术培养基培养效果的比较Embodiment 4 and the comparison of culture medium culture effect of prior art
(1)文献培养基的配制(1) Preparation of literature medium
配制文献(Lohmussaar等,2021,Cell Stem Cell 28,1380–1396)中使用的培养基,其配方为Advanced DMEM/F12培养基(购自Invitrogen公司)+1:100青霉素/链霉素(购自Corning公司)+50μg/mL Primocin(购自Invivogen公司)+1:100GlutaMAX(购自Corning公司)+10mM HEPES(购自赛默飞公司)+1:50B27(购自Gibco公司)+1.25mmol/L N-乙酰半胱氨酸(购自MCE公司)+5mmol/L烟酰胺(购自MCE公司)+500ng/mL R-Spondin 1(购自sino biological公司)+25ng/mL角化细胞生长因子(购自sino biological公司)+50ng/mL表皮细胞生长因子(购自sino biological公司)+25ng/ml FGF7(购自sino biological公司)+100ng/ml FGF10(购自sino biological公司)+100ng/ml Noggin (购自sino biologica公司)+1μmol/L SB202190(购自MCE公司)+500nmol/L A8301(购自MCE公司)+10μmol/L Y27632(购自MCE公司)+10μmol/L forskolin(购自MCE公司)+100nMβ-雌二醇(购自MCE公司)+0.3mM CHIR(购自MCE公司)。以下简称为ROM培养基。Prepare the medium used in literature (Lohmussaar et al., 2021, Cell Stem Cell 28, 1380–1396), the formula of which is Advanced DMEM/F12 medium (purchased from Invitrogen) + 1:100 penicillin/streptomycin (purchased from Corning)+50μg/mL Primocin (purchased from Invivogen)+1:100GlutaMAX (purchased from Corning)+10mM HEPES (purchased from Thermo Fisher)+1:50B27 (purchased from Gibco)+1.25mmol/L N-acetylcysteine (purchased from MCE company)+5mmol/L nicotinamide (purchased from MCE company)+500ng/mL R-Spondin 1 (purchased from sino biological company)+25ng/mL keratinocyte growth factor ( Purchased from Sino biological company)+50ng/mL epidermal growth factor (purchased from sino biological company)+25ng/ml FGF7 (purchased from sino biological company)+100ng/ml FGF10 (purchased from sino biological company)+100ng/ml Noggin (purchased from Sino Biologica Company)+1 μmol/L SB202190 (purchased from MCE Company)+500nmol/L A8301 (purchased from MCE Company)+10 μmol/L Y27632 (purchased from MCE Company)+10 μmol/L forskolin (purchased from MCE Company) )+100nM β-estradiol (purchased from MCE Company)+0.3mM CHIR (purchased from MCE Company). Hereinafter referred to as ROM medium for short.
(2)宫颈癌类器官培养(2) Cervical cancer organoid culture
按照实施例1之(2)的方法从术中组织样本OC6获得宫颈癌原代细胞,并分别用COM培养基和ROM培养基按照实施例3的方法进行类器官培养。According to the method of (2) of Example 1, primary cervical cancer cells were obtained from the intraoperative tissue sample OC6, and the organoids were cultured according to the method of Example 3 using COM medium and ROM medium respectively.
在培养第10天,使用显微镜(Invitrogen公司EVOS M500)观察培养得到的宫颈癌类器官。图4A和4B是4倍物镜下拍摄分别由COM培养基和ROM培养基培养10天得到的类器官的照片。On the 10th day of culture, the cultured cervical cancer organoids were observed using a microscope (EVOS M500 from Invitrogen). Figures 4A and 4B are photographs of organoids cultured in COM medium and ROM medium for 10 days under a 4x objective lens.
根据图4A和4B的结果可知,与ROM培养基相比,COM培养基能显著促进宫颈癌类器官的形成和扩增培养。According to the results in Figures 4A and 4B, compared with ROM medium, COM medium can significantly promote the formation and expansion of cervical cancer organoids.
实施例5使用本发明的培养基扩增得到的宫颈癌类器官用于药物筛选Example 5 Using the culture medium of the present invention to amplify the cervical cancer organoids for drug screening
(1)宫颈癌类器官培养(1) Cervical cancer organoid culture
从宫颈癌术中样本(CCa5)按照实施例1之(2)的方法分离得到宫颈癌原代细胞,并使用COM培养基进行类器官培养,待宫颈癌类器官直径超过50μm时进行药物筛选。Cervical cancer primary cells were isolated from the cervical cancer intraoperative sample (CCa5) according to the method of (2) of Example 1, and the organoids were cultured in COM medium, and drug screening was performed when the diameter of the cervical cancer organoids exceeded 50 μm.
(2)筛选药物配制(2) Screen drug preparation
按照下表配制6个浓度梯度的4种药物(顺铂、卡铂、紫杉醇和硼替佐米;均购自MCE公司),保存待用。According to the table below, 4 drugs (cisplatin, carboplatin, paclitaxel and bortezomib; all purchased from MCE Company) with 6 concentration gradients were prepared and stored for later use.
不同浓度顺铂添加液的配制:将顺铂配制成6个不同浓度的添加液,最高浓度为9.5μM,然后2倍稀释比例进行稀释,得到4.75μM、2.38μM、1.19μM、0.59μM、0.3μM不同浓度的添加液。Preparation of different concentrations of cisplatin additives: cisplatin was prepared into 6 different concentrations of additives, the highest concentration was 9.5 μM, and then diluted with a 2-fold dilution ratio to obtain 4.75 μM, 2.38 μM, 1.19 μM, 0.59 μM, 0.3 μM μM of different concentrations of additives.
不同浓度卡铂添加液的配制:将卡铂配制成6个不同浓度的储存液,最高浓度为137.92μM,然后2倍稀释比例进行稀释,得到68.96μM、34.48μM、17.24μM、8.62μM、4.31μM不同浓度的添加液。Preparation of different concentrations of carboplatin addition solution: Carboplatin was prepared into 6 different concentrations of storage solution, the highest concentration was 137.92 μM, and then diluted by 2 times dilution ratio to obtain 68.96 μM, 34.48 μM, 17.24 μM, 8.62 μM, 4.31 μM μM of different concentrations of additives.
不同浓度紫杉醇添加液的配制:将紫杉醇配制成6个不同浓度的添加液,最高浓度为11.51μM,然后2倍稀释比例进行稀释,得到5.76 μM、2.88μM、1.44μM、0.72μM、0.36μM不同浓度的添加液。Preparation of paclitaxel additive solutions with different concentrations: Paclitaxel was prepared into 6 different concentrations of additive solutions, the highest concentration was 11.51 μM, and then diluted with a 2-fold dilution ratio to obtain different concentrations of 5.76 μM, 2.88 μM, 1.44 μM, 0.72 μM, and 0.36 μM. concentration of additives.
不同浓度硼替佐米添加液的配制:将硼替佐米配制成6个不同浓度的添加液,最高浓度为2μM,然后2倍稀释比例进行稀释,得到1μM、0.5μM、0.25μM、0.125μM、0.0625μM不同浓度的添加液。Preparation of different concentrations of bortezomib additives: Bortezomib was prepared into 6 different concentrations of additives, the highest concentration was 2 μM, and then diluted by 2 times dilution ratio to obtain 1 μM, 0.5 μM, 0.25 μM, 0.125 μM, 0.0625 μM μM of different concentrations of additives.
(3)加药(3) Dosing
取出配制好的药物添加液,置于室温。从孵箱取出按照步骤(1)培养获得的类器官,去除培养孔中的培养基,将含有不同浓度药物的添加剂按照每孔100μL沿着孔壁慢慢将入到96孔透明培养板中。加药结束后,96孔板表面消毒后移至培养箱中继续培养,5天后测定类器官活力。Take out the prepared drug additive solution and place it at room temperature. Take out the organoids obtained by culturing according to step (1) from the incubator, remove the medium in the culture wells, and slowly pour the additives containing different concentrations of drugs into the 96-well transparent culture plate along the well wall at 100 μL per well. After the drug addition, the surface of the 96-well plate was sterilized and moved to the incubator to continue culturing, and the viability of the organoids was measured 5 days later.
(4)类器官活力测试(4) Organoid Viability Test
4℃冰箱取出CellTiter-Glo发光试剂(购自Promega公司),取10毫升试剂于加样槽中,培养箱中取出待检测96孔板,每孔加入50μL CellTiter-Glo发光试剂,静置30分钟后观察96孔板中细胞状态,若细胞大部分已经裂解,则轻轻震荡混匀,吸取100μL至另外一块白色96孔板中,使用多功能酶标仪(Perkin Elmer公司Envision)检测。Take out the CellTiter-Glo luminescent reagent (purchased from Promega) from the refrigerator at 4°C, put 10 ml of the reagent in the sampling tank, take out the 96-well plate to be tested in the incubator, add 50 μL of the CellTiter-Glo luminescent reagent to each well, and let stand for 30 minutes Then observe the state of the cells in the 96-well plate. If most of the cells have been lysed, gently shake and mix, pipette 100 μL into another white 96-well plate, and use a multi-functional microplate reader (Envision of Perkin Elmer Company) to detect.
(5)数据处理(5) Data processing
按照公式药物抑制率(%)=100%-(第五天培养孔化学发光数值
药物处理组/第零天培养孔化学发光数值
药物处理组)/(第五天培养孔化学发光数值
DMSO/第零天培养孔化学发光数值
DMSO)*100%,计算得到不同药物的抑制率,将结果示于图5。图5为不同浓度的测试药物抑制宫颈癌类器官生长的抑制率曲线。四个抗肿瘤药物中,硼替佐米在6个浓度下都具有较强的抑制类器官生长的效果,紫杉醇在6个浓度下抑制效率保持一致,不同浓度的顺铂和卡铂的抑制效果有一定的差异,呈剂量依赖性,这表明同一病人的类器官对不同药物的有效性和敏感性不同。根据结果可以判断宫颈癌病人在临床使用该种药物时的有效性及有效用量。
According to the formula, the drug inhibition rate (%)=100%-(the chemiluminescence value drug treatment group of the culture well on the fifth day/the chemiluminescence value drug treatment group of the culture well on the zero day)/(the chemiluminescence value DMSO of the culture well on the fifth day/the The chemiluminescent value of the zero-day culture wells (DMSO )*100% was calculated to obtain the inhibition rates of different drugs, and the results are shown in FIG. 5 . Fig. 5 is the inhibition rate curve of different concentrations of test drugs inhibiting the growth of cervical cancer organoids. Among the four antineoplastic drugs, bortezomib has a strong inhibitory effect on organoid growth at 6 concentrations, paclitaxel has the same inhibitory efficiency at 6 concentrations, and the inhibitory effects of different concentrations of cisplatin and carboplatin are significant. Certain differences were dose-dependent, suggesting that organoids from the same patient have different effectiveness and sensitivity to different drugs. According to the results, the effectiveness and effective dosage of the drug can be judged when the cervical cancer patients are clinically used.
工业应用性Industrial Applicability
本发明提供一种用于宫颈癌类器官培养的培养基及培养方法,可将培养得到的类器官应用于药物的疗效评估和筛选。因而,本发明适 于工业应用。The invention provides a culture medium and a culture method for cervical cancer organoids, and the cultured organoids can be applied to the efficacy evaluation and screening of drugs. Thus, the present invention is suitable for industrial applications.
尽管本文对本发明作了详细说明,但本发明不限于此,本技术领域的技术人员可以根据本发明的原理进行修改,因此,凡按照本发明的原理进行的各种修改都应当理解为落入本发明的保护范围。Although the present invention has been described in detail herein, the present invention is not limited thereto, and those skilled in the art can make modifications according to the principle of the present invention. Therefore, all modifications made according to the principle of the present invention should be understood as falling within protection scope of the present invention.
Claims (10)
- 一种用于宫颈癌类器官的培养基,其特征在于包括MST1/2激酶抑制剂、选自N2和B27的至少一种细胞培养添加剂、肝细胞生长因子、SB202190、Y27632、A83-01、表皮细胞生长因子、成纤维细胞生长因子10、角化细胞生长因子、GlutaMAX和烟酰胺,A culture medium for cervical cancer organoids, characterized by comprising MST1/2 kinase inhibitors, at least one cell culture additive selected from N2 and B27, hepatocyte growth factor, SB202190, Y27632, A83-01, epidermis Cell Growth Factor, Fibroblast Growth Factor 10, Keratinocyte Growth Factor, GlutaMAX, and Niacinamide,其中,所述MST1/2激酶抑制剂包括式(I)的化合物或其药学可接受的盐、或溶剂化物,Wherein, the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof,其中,in,R 1选自C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C2-C6螺环烷基、以及任选地被1-2个独立地R 6取代的芳基、芳基C1-C6烷基和杂芳基; R 1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted by 1-2 independent R Base, aryl C1-C6 alkyl and heteroaryl;R 2和R 3各自独立地选自C1-C6烷基; R 2 and R 3 are each independently selected from C1-C6 alkyl;R 4和R 5各自独立地选自氢、C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C1-C6烷基羟基、C1-C6卤代烷基、C1-C6烷基氨基C1-C6烷基、C1-C6烷氧基C1-C6烷基、和C3-C6杂环基C1-C6烷基; R 4 and R 5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxyl, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl;R 6选自卤素、C1-C6烷基、C1-C6烷氧基、和C1-C6卤代烷基。 R 6 is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.
- 如权利要求1所述的培养基,其中culture medium as claimed in claim 1, whereinR 1选自C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C2-C6螺环烷基、以及任选地被1-2个独立地R 6取代的苯基、萘基、苯甲基和噻吩基; R is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and benzene optionally substituted by 1-2 independently R phenyl, naphthyl, benzyl and thienyl;R 2和R 3各自独立地选自C1-C3烷基; R 2 and R 3 are each independently selected from C1-C3 alkyl;R 4和R 5各自独立地选自氢、C1-C6烷基、C3-C6环烷基、C4-C8 环烷基烷基、C1-C6烷基羟基、C1-C6卤代烷基、C1-C6烷基氨基C1-C6烷基、C1-C6烷氧基C1-C6烷基、哌啶基C1-C6烷基、和四氢吡喃基C1-C6烷基; R 4 and R 5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxyl, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, piperidinyl C1-C6 alkyl, and tetrahydropyranyl C1-C6 alkyl;R 6选自卤素、C1-C6烷基、C1-C6烷氧基、和C1-C6卤代烷基。 R 6 is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.
- 如权利要求1所述的培养基,其中所述MST1/2激酶抑制剂包括式(Ia)的化合物或其药学可接受的盐、或溶剂化物,The culture medium according to claim 1, wherein the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt thereof, or a solvate,其中,in,R 1选自C1-C6烷基、任选地被1-2个独立地R 6取代的苯基、任选地被1-2个独立地R 6取代的噻吩基、和任选地被1-2个独立地R 6取代的苯甲基; R is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently R6 , thienyl optionally substituted by 1-2 independently R6 , and optionally substituted by 1 -2 independently R6 substituted benzyl groups;R 5选自氢、C1-C6烷基、和C3-C6环烷基; R is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl;R 6各自独立地选自卤素、C1-C6烷基、和C1-C6卤代烷基。 Each R 6 is independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl.
- 如权利要求3所述的培养基,其中culture medium as claimed in claim 3, whereinR 1为任选地被1-2个独立地R 6取代的苯基; R 1 is phenyl optionally substituted by 1-2 independently R 6 ;R 5为氢; R is hydrogen;R 6优选为氟、甲基或三氟甲基。 R6 is preferably fluoro, methyl or trifluoromethyl.
- 如权利要求1~5中任一项所述的培养基,其特征在于所述培养基中各成分的含量满足以下任意一项或多项或全部满足:The culture medium according to any one of claims 1 to 5, characterized in that the content of each component in the culture medium satisfies any one or more or all of the following:所述MST1/2激酶抑制剂的浓度为2.5~10μM;The concentration of the MST1/2 kinase inhibitor is 2.5-10 μM;所述B27或N2细胞培养添加剂相对于培养基的体积比为1:25~1:100;The volume ratio of the B27 or N2 cell culture supplement to the medium is 1:25 to 1:100;所述肝细胞生长因子的浓度为5~40ng/mL;The concentration of the hepatocyte growth factor is 5-40 ng/mL;所述SB202190的浓度为200~1000nM;The concentration of the SB202190 is 200-1000nM;所述Y27632的浓度为2.5~10μM;The concentration of Y27632 is 2.5-10 μM;所述A83-01的浓度为200~1000nM;The concentration of the A83-01 is 200-1000nM;所述表皮细胞生长因子的浓度为1~40ng/mL;The concentration of the epidermal growth factor is 1-40 ng/mL;所述成纤维细胞生长因子10的浓度为10~100ng/mL;The concentration of the fibroblast growth factor 10 is 10-100 ng/mL;所述角化细胞生长因子的浓度为2~40ng/mL;The concentration of the keratinocyte growth factor is 2-40 ng/mL;所述GlutaMAX相对于培养基的体积比为1:50~1:200;The volume ratio of the GlutaMAX to the medium is 1:50 to 1:200;所述烟酰胺的浓度为1~10mM。The concentration of the nicotinamide is 1-10mM.
- 如权利要求1~6中任一项所述的培养基,其特征在于还包括:The culture medium according to any one of claims 1 to 6, further comprising:选自DMEM/F12、DMEM、F12或RPMI-1640的初始培养基;和An initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and选自链霉素/青霉素、两性霉素B和Primocin中的一种或多种的抗生素。One or more antibiotics selected from streptomycin/penicillin, amphotericin B and Primocin.
- 如权利要求1~7中任一项所述的培养基,其特征在于所述培养基不含Wnt激动剂、R-spondin家族蛋白、Noggin蛋白、和BMP抑制剂。The medium according to any one of claims 1-7, characterized in that the medium does not contain Wnt agonists, R-spondin family proteins, Noggin proteins, and BMP inhibitors.
- 一种宫颈癌类器官的培养方法,其特征在于包括以下步骤:A culture method for cervical cancer organoids, characterized in that it comprises the following steps:(1)从宫颈癌实体瘤组织分离样本,获得宫颈癌原代细胞;(1) Separating samples from cervical cancer solid tumor tissues to obtain primary cervical cancer cells;(2)配制根据权利要求1~8中任一项所述的用于宫颈癌类器官的培养基,并对步骤(1)获得的宫颈癌原代细胞进行类器官培养。(2) preparing the culture medium for cervical cancer organoids according to any one of claims 1-8, and performing organoid culture on the primary cervical cancer cells obtained in step (1).
- 一种用于评估或筛选治疗宫颈癌的药物的方法,其特征在于,包括以下步骤:A method for assessing or screening a drug for the treatment of cervical cancer, comprising the following steps:(1)使用如权利要求9所述的宫颈癌类器官的培养方法培养宫颈癌类器官;(1) using the culture method of cervical cancer organoids as claimed in claim 9 to cultivate cervical cancer organoids;(2)选定需要检测的药物并按照所需浓度梯度进行稀释;(2) Select the drug to be detected and dilute it according to the required concentration gradient;(3)对(1)中培养得到的宫颈癌类器官添加稀释后的所述药物;和(3) adding the diluted drug to the cervical cancer organoids cultured in (1); and(4)进行类器官大小或类器官活力检测。(4) Perform organoid size or organoid viability detection.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111201783 | 2021-10-15 | ||
CN202111201783.6 | 2021-10-15 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2023060774A1 true WO2023060774A1 (en) | 2023-04-20 |
Family
ID=85956854
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2021/142274 WO2023060774A1 (en) | 2021-10-15 | 2021-12-29 | Culture medium for cervical cancer organoids, and culture method and application thereof |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN115975939A (en) |
WO (1) | WO2023060774A1 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116716236A (en) * | 2023-06-29 | 2023-09-08 | 重庆大学附属肿瘤医院 | Construction method and application of cervical cancer organoids |
CN116555165A (en) * | 2023-06-29 | 2023-08-08 | 重庆大学附属肿瘤医院 | Cervical cancer organoid culture medium and application thereof |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110734894A (en) * | 2019-10-11 | 2020-01-31 | 陈璞 | Universal cancer organoid in vitro culture medium |
CN111039944A (en) * | 2018-10-12 | 2020-04-21 | 中国科学院合肥物质科学研究院 | MST1 kinase inhibitors and uses thereof |
CN113122500A (en) * | 2021-03-18 | 2021-07-16 | 上海诺典生物科技有限公司 | Culture and application of metastatic intestinal cancer organoid |
CN113308437A (en) * | 2021-05-28 | 2021-08-27 | 丹望医疗科技(上海)有限公司 | Culture medium, method and kit for rapidly culturing bone metastasis cancer organoids |
CN113528444A (en) * | 2020-04-15 | 2021-10-22 | 合肥中科普瑞昇生物医药科技有限公司 | Culture medium for esophageal squamous carcinoma epithelial cells, culture method and application of culture medium |
-
2021
- 2021-12-29 WO PCT/CN2021/142274 patent/WO2023060774A1/en unknown
- 2021-12-29 CN CN202111635449.1A patent/CN115975939A/en active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111039944A (en) * | 2018-10-12 | 2020-04-21 | 中国科学院合肥物质科学研究院 | MST1 kinase inhibitors and uses thereof |
CN110734894A (en) * | 2019-10-11 | 2020-01-31 | 陈璞 | Universal cancer organoid in vitro culture medium |
CN113528444A (en) * | 2020-04-15 | 2021-10-22 | 合肥中科普瑞昇生物医药科技有限公司 | Culture medium for esophageal squamous carcinoma epithelial cells, culture method and application of culture medium |
CN113122500A (en) * | 2021-03-18 | 2021-07-16 | 上海诺典生物科技有限公司 | Culture and application of metastatic intestinal cancer organoid |
CN113308437A (en) * | 2021-05-28 | 2021-08-27 | 丹望医疗科技(上海)有限公司 | Culture medium, method and kit for rapidly culturing bone metastasis cancer organoids |
Non-Patent Citations (1)
Title |
---|
WANG HAINA, WANG RUI, HUANG DAN, LI SIHAN, GAO BEIBEI, KANG ZHIJIE, TANG BO, XIE JIAJUN, YAN FANZHI, LIANG RUI, LI HUA, YAN JINSON: "Homoharringtonine Exerts Anti-tumor Effects in Hepatocellular Carcinoma Through Activation of the Hippo Pathway", FRONTIERS IN PHARMACOLOGY, vol. 12, XP093056583, DOI: 10.3389/fphar.2021.592071 * |
Also Published As
Publication number | Publication date |
---|---|
CN115975939A (en) | 2023-04-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP4137565A1 (en) | Culture medium for esophageal squamous cell carcinoma epithelial cells, culture method, and application thereof | |
WO2023060774A1 (en) | Culture medium for cervical cancer organoids, and culture method and application thereof | |
WO2023035299A1 (en) | Culture medium for hepatoma organoid culture, hepatoma organoid culture method, and application of hepatoma organoid | |
WO2023004888A1 (en) | Culture medium for culturing oral cancer organoids, and culture method therefor and use thereof | |
WO2023060676A1 (en) | Culture medium and culture method for ovarian cancer organoid, and application thereof | |
WO2023060764A1 (en) | Culture medium for primary cell of gastric carcinoma, and culture method therefor | |
EP4368706A1 (en) | Culture medium and culture method for lung cancer epithelial cells, and application thereof | |
WO2022241845A1 (en) | Culture medium of primary hepatocellular carcinoma cells and culture method thereof | |
WO2023060684A1 (en) | Culture medium for lung cancer organoids, culture method and application thereof | |
WO2023060695A1 (en) | Culture medium and culture method for suspended culture of liver cancer organoids | |
WO2023060709A1 (en) | Esophageal cancer organoid culture medium and culture method and use thereof | |
WO2022227110A1 (en) | Culture medium for primary cells of oral cancer, and culturing method | |
WO2023060710A1 (en) | Culture medium and culture method for gastric cancer organoids | |
WO2023060711A1 (en) | Culture medium and culture method for organoid derived from pleural fluids from lung cancer, and use of organoid | |
WO2023060643A1 (en) | Culture medium and culture method for intestinal cancer organoid | |
WO2023060820A1 (en) | Culture medium and culture method for gastric carcinoma primary cells | |
WO2023060642A1 (en) | Culture medium for intestinal cancer primary cells, in-vitro culture method, and use | |
WO2023060696A1 (en) | Culture medium for primary ovarian cancer cells, culture method and application thereof | |
WO2023060681A1 (en) | Culture medium and culture method for primary cervical cancer cells | |
CN113969262B (en) | Culture medium for lung cancer epithelial cells, culture method and application thereof | |
WO2023060677A1 (en) | Culture medium, culture method and use of primary ovarian cancer cells | |
EP4159845A1 (en) | Culture medium for laryngeal cancer epithelial cells, culture method, and application thereof | |
JP2024524537A (en) | Culture medium and culture method for lung cancer epithelial cells, and uses thereof | |
CN115806936A (en) | Culture medium for lung cancer epithelial cells, culture method and application thereof | |
CN117736992A (en) | Culture medium, culture method and application of primary cells of neuroblastoma |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 21960495 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |