CN117305243A - 一种类器官培养方法 - Google Patents
一种类器官培养方法 Download PDFInfo
- Publication number
- CN117305243A CN117305243A CN202210704242.3A CN202210704242A CN117305243A CN 117305243 A CN117305243 A CN 117305243A CN 202210704242 A CN202210704242 A CN 202210704242A CN 117305243 A CN117305243 A CN 117305243A
- Authority
- CN
- China
- Prior art keywords
- culture
- organoid
- cells
- washbuffer
- drug
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000002220 organoid Anatomy 0.000 title claims abstract description 59
- 238000000034 method Methods 0.000 title claims abstract description 24
- 210000004027 cell Anatomy 0.000 claims abstract description 79
- 239000003814 drug Substances 0.000 claims abstract description 59
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 50
- 229940079593 drug Drugs 0.000 claims abstract description 46
- 239000001963 growth medium Substances 0.000 claims abstract description 26
- 239000003112 inhibitor Substances 0.000 claims abstract description 13
- 238000012216 screening Methods 0.000 claims abstract description 11
- 238000012136 culture method Methods 0.000 claims abstract description 10
- 238000012545 processing Methods 0.000 claims abstract description 8
- 239000000872 buffer Substances 0.000 claims abstract description 7
- 210000003743 erythrocyte Anatomy 0.000 claims abstract description 6
- 239000003102 growth factor Substances 0.000 claims abstract description 6
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 5
- 239000000126 substance Substances 0.000 claims abstract description 5
- 108091000080 Phosphotransferase Proteins 0.000 claims abstract description 4
- 239000005557 antagonist Substances 0.000 claims abstract description 4
- 239000007640 basal medium Substances 0.000 claims abstract description 4
- 210000000988 bone and bone Anatomy 0.000 claims abstract description 4
- 230000000921 morphogenic effect Effects 0.000 claims abstract description 4
- 102000020233 phosphotransferase Human genes 0.000 claims abstract description 4
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 4
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 4
- 230000019491 signal transduction Effects 0.000 claims abstract description 4
- 239000000556 agonist Substances 0.000 claims abstract description 3
- 102000037983 regulatory factors Human genes 0.000 claims abstract description 3
- 108091008025 regulatory factors Proteins 0.000 claims abstract description 3
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 claims abstract 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims abstract 2
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 claims abstract 2
- 229960004308 acetylcysteine Drugs 0.000 claims abstract 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims abstract 2
- 229960002743 glutamine Drugs 0.000 claims abstract 2
- 210000001519 tissue Anatomy 0.000 claims description 44
- 108010082117 matrigel Proteins 0.000 claims description 30
- 206010006187 Breast cancer Diseases 0.000 claims description 22
- 208000026310 Breast neoplasm Diseases 0.000 claims description 22
- 238000005406 washing Methods 0.000 claims description 19
- 239000007788 liquid Substances 0.000 claims description 15
- 239000011534 wash buffer Substances 0.000 claims description 14
- 239000012981 Hank's balanced salt solution Substances 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 11
- 238000001514 detection method Methods 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 9
- 239000002609 medium Substances 0.000 claims description 8
- 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 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 230000029087 digestion Effects 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 5
- 102000029816 Collagenase Human genes 0.000 claims description 4
- 108060005980 Collagenase Proteins 0.000 claims description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 4
- 102100028071 Fibroblast growth factor 7 Human genes 0.000 claims description 4
- 229930182555 Penicillin Natural products 0.000 claims description 4
- 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 4
- 229960002424 collagenase Drugs 0.000 claims description 4
- 229940049954 penicillin Drugs 0.000 claims description 4
- 229960005322 streptomycin Drugs 0.000 claims description 4
- 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 3
- 239000007995 HEPES buffer Substances 0.000 claims description 3
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 3
- 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 3
- 229960003942 amphotericin b Drugs 0.000 claims description 3
- 239000002246 antineoplastic agent Substances 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 238000010899 nucleation Methods 0.000 claims description 3
- 102000009024 Epidermal Growth Factor Human genes 0.000 claims description 2
- 102100028412 Fibroblast growth factor 10 Human genes 0.000 claims description 2
- 108090001047 Fibroblast growth factor 10 Proteins 0.000 claims description 2
- 108090000385 Fibroblast growth factor 7 Proteins 0.000 claims description 2
- 101001060261 Homo sapiens Fibroblast growth factor 7 Proteins 0.000 claims description 2
- 102000048238 Neuregulin-1 Human genes 0.000 claims description 2
- 108090000556 Neuregulin-1 Proteins 0.000 claims description 2
- 101710098940 Pro-epidermal growth factor Proteins 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 102000045246 noggin Human genes 0.000 claims description 2
- 108700007229 noggin Proteins 0.000 claims description 2
- 101000825954 Homo sapiens R-spondin-1 Proteins 0.000 claims 1
- FABQUVYDAXWUQP-UHFFFAOYSA-N N4-(1,3-benzodioxol-5-ylmethyl)-6-(3-methoxyphenyl)pyrimidine-2,4-diamine Chemical compound COC1=CC=CC(C=2N=C(N)N=C(NCC=3C=C4OCOC4=CC=3)C=2)=C1 FABQUVYDAXWUQP-UHFFFAOYSA-N 0.000 claims 1
- 102100022762 R-spondin-1 Human genes 0.000 claims 1
- 230000001105 regulatory effect Effects 0.000 claims 1
- 239000000523 sample Substances 0.000 description 24
- 238000004140 cleaning Methods 0.000 description 19
- 238000007877 drug screening Methods 0.000 description 13
- 230000012010 growth Effects 0.000 description 11
- 230000035945 sensitivity Effects 0.000 description 11
- 230000000694 effects Effects 0.000 description 10
- 239000008188 pellet Substances 0.000 description 10
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 238000003860 storage Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 241000233866 Fungi Species 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 238000007664 blowing Methods 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 230000008595 infiltration Effects 0.000 description 4
- 238000001764 infiltration Methods 0.000 description 4
- 239000013049 sediment Substances 0.000 description 4
- MWWSFMDVAYGXBV-FGBSZODSSA-N (7s,9s)-7-[(2r,4s,5r,6s)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7h-tetracene-5,12-dione;hydron;chloride Chemical compound Cl.O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@@H](O)[C@H](C)O1 MWWSFMDVAYGXBV-FGBSZODSSA-N 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000012472 biological sample Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 3
- 229960004316 cisplatin Drugs 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 229960004679 doxorubicin Drugs 0.000 description 3
- 230000007071 enzymatic hydrolysis Effects 0.000 description 3
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 3
- 229960003265 epirubicin hydrochloride Drugs 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 206010059313 Anogenital warts Diseases 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 102000038379 digestive enzymes Human genes 0.000 description 2
- 108091007734 digestive enzymes Proteins 0.000 description 2
- 230000000857 drug effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 238000011532 immunohistochemical staining Methods 0.000 description 2
- 238000010874 in vitro model Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000017074 necrotic cell death Effects 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000011332 tumor tissue staining Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- AOJJSUZBOXZQNB-VTZDEGQISA-N 4'-epidoxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-VTZDEGQISA-N 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- HTIJFSOGRVMCQR-UHFFFAOYSA-N Epirubicin Natural products COc1cccc2C(=O)c3c(O)c4CC(O)(CC(OC5CC(N)C(=O)C(C)O5)c4c(O)c3C(=O)c12)C(=O)CO HTIJFSOGRVMCQR-UHFFFAOYSA-N 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 208000032420 Latent Infection Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 229930192392 Mitomycin Natural products 0.000 description 1
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 102100039313 Rho-associated protein kinase 1 Human genes 0.000 description 1
- 101710088411 Rho-associated protein kinase 1 Proteins 0.000 description 1
- 102100039314 Rho-associated protein kinase 2 Human genes 0.000 description 1
- 101710088493 Rho-associated protein kinase 2 Proteins 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 229940034982 antineoplastic agent Drugs 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 208000037771 disease arising from reactivation of latent virus Diseases 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229960001904 epirubicin Drugs 0.000 description 1
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 229960002949 fluorouracil Drugs 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- -1 nicotinamide glutamine acetylcysteine Chemical compound 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 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 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0631—Mammary cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
- G01N21/763—Bioluminescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/117—Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/119—Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/15—Transforming growth factor beta (TGF-β)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/40—Regulators of development
- C12N2501/405—Cell cycle regulated proteins, e.g. cyclins, cyclin-dependant kinases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/40—Regulators of development
- C12N2501/415—Wnt; Frizzeled
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
- C12N2501/72—Transferases (EC 2.)
- C12N2501/727—Kinases (EC 2.7.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N2015/1486—Counting the particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Oncology (AREA)
- Toxicology (AREA)
- Dermatology (AREA)
- Dispersion Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
一种类器官培养基以及培养方法以及使用类器官组织细胞进行药物模型筛选方法,培养基包括烟酰胺、谷氨酰胺、乙酰半胱氨酸、缓冲液、基础培养基、生长因子和/或调节因子、抗菌物质、Wnt激动剂、骨形态发生蛋白(BMPs)拮抗剂、Rho相关的线圈激酶(ROCK)抑制剂、TGFβ信号通路抑制剂、p38MAPK抑制剂;培养方法包括样本处理步骤、酶解步骤、红细胞破碎步骤、立体培养步骤;以及使用上述方法获得的类器官组织进行肿瘤药物筛选。
Description
技术领域
本发明属于药敏检测技术领域,具体地涉及一种基于类器官的药敏检测技术。
背景技术
对于乳腺癌的发生和发展机制,以及药物筛选大多利用体外和动物模型中的癌细胞株来研究的,但细胞系的平面培养特点不能真实反应肿瘤微环境,并且缺乏基因突变的复杂性,用于疾病研究和药效鉴定存在严重的局限性;并且,原代肿瘤细胞的平面培养成功率低,不能快速增殖,难以进行高通量的药敏检测;且平面培养不能有效的模拟药物在实体肿瘤组织中的作用效果。现有技术多利用肿瘤细胞系、原代肿瘤细胞和人源性组织异种移植模型来进行药物筛选。现有技术多利用体外培养癌细胞株来进行乳腺癌的药物筛选;例如用乳腺癌的原代细胞检测阿霉素、紫杉醇、氟尿嘧啶、丝裂霉素和长春新碱等的毒性,但是原代细胞培养成功率低,药敏检测结果与临床相关性低。
CN112760289B公开了一种乳腺癌肿瘤类器官培养基以及3D培养方法,然而该专利并未公开特定的清洗方式,无法保证细胞的活性;并且培养基成分有一定缺陷,例如缺少能够抑制细胞凋亡,促进干细胞的分裂和分化的ROCK-I和ROCK-II抑制剂,因此培养基优化存在一定不足。
CN110777116B公开了肿瘤组织预处理的清洗方法,但仅限于清洗和初步处理,并未公开针对特定肿瘤组织的立体培养方法。
发明内容
本发明涉及一种类器官培养方法,进一步的,涉及肿瘤类器官培养,更进一步的,涉及乳腺癌肿瘤类器官的培养方法。
样本的获得
一般而言,样本来自与医院合作,通过肿瘤手术样本或者穿刺样本中分离,以更加贴近患者的患病实际以及微环境,而并非选择体外细胞系。从医院收取肿瘤手术样本,使用样本储存液进行转移。
肿瘤组织并没有特别限定,但基于取样的难度,优选实体瘤,例如胃癌、肺癌、结肠癌、乳腺癌、食道癌等,但更有选可以是乳腺癌。对于样本储存,并不限于具体的储存液,其目的在于能够更好的保存细胞组织,例如可以选择DMEM/F12+1%三抗+1%HEPES作为更优。
样本的处理
获得样本之后,样本进行通过特定成分的WashBuffer进行清洗,以下5步处理:
采用第一清洗处理:WashBuffer4—WashBuffer2——WashBuffer3中顺序清洗
第一清洗处理后的样本优选进行剔除处理,以便去除脂肪、血液、坏死以及间质质量分数大于18~22%的组织
进一步的,所述第一清洗步骤包括(1)Wash Buffer 4,清洗2遍,每遍4-6分钟,优选5min(2)Wash Buffer 2清洗2-4分钟,优选3min。(3)Wash Buffer 3清洗3min。
将清洗后或者剔除处理后的组织进行第二清洗处理
第二清洗处理步骤,包括
WashBuffer1——WashBuffer2——WashBuffer3——WashBuffer4——WashBuffer5中顺序清洗
WashBuffer1中清洗5或6遍,每遍4~6分钟;
在WashBuffer2中清洗2~4分钟;
在WashBuffer3中清洗3分钟;
在WashBuffer4中、在4℃的条件下浸泡28~32分钟;
以及将经过WashBuffer4浸泡的组织在WashBuffer5中清洗5或6遍,每遍4~6分钟。
所述Wash Buffer成分优选以下组分:
以上样本处理过程中采用的5种Wash Buffer分别通过不同的原理去除微生物,降低样本培养污染的概率,同时通过剪刀剔除相关组织部位,一方面可以去除部分污染,使清洗过程更加彻底有效,另一方面剔除间质较多的部位可以缩短酶解时间,提高酶解效率,以获得活性更高的肿瘤细胞。
其中WashBuffer4清洗新鲜肿瘤组织,可以去除和抑制肿瘤样本表面携带的各种细菌和真菌,现有技术中使用的添加了100单位/mL的青霉素和100μg/mL链霉素的双抗的细胞清洗液,用在类器官的培养中导致真菌污染的概率较高,而本申请的方法采用的WashBuffer4,增加了两性霉素B来进行抗真菌的处理,同时使用10倍浓度的青霉素以及链霉素,对存在的细菌真菌污染进行冲击法处理,在保证组织细胞活性的前提下,最大限度地降低了类器官的污染概率,尤其适合培养类器官组织的前处理。
本申请的方法中,发明人创造性的采用了含有乙醇体积分数为75%的HBSS缓冲液2进行组织样本的清洗,有效杀灭了样本携带的细菌真菌,本领域技术人员常规所了解的是,75%乙醇会对细胞的产生伤害作用,因此75%乙醇不会用来处理原代组织,而本申请的发明人发现,采用清洗缓冲液2短时间(2~4分钟)处理肿瘤样本,不仅可以有效的杀灭样本表面的细菌和真菌,同时仍可以保留细胞活性,实现后续的培养。
含有乙酸体积分数为3%的HBSS的WashBuffer3现有技术中主要用于处理尖锐湿疣、尖锐湿疣亚临床表现或HPV潜伏感染的试验方法。而本申请的发明人发现了该清洗缓冲液的新用途,使用WashBuffer3处理原代肿瘤样本,可以一定程度地抑制真菌的生长,降低样本后续培养发生真菌污染的概率。
剪碎组织。将按照上述步骤清洗好的肿瘤组织放入培养皿中,加入5-6mL HBSS,眼科剪剔除脂肪,血液,坏死以及间质较多的部位,然后使用眼科剪和镊子将组织剪成小于1-2mm3的小块。
酶解
将上述全部溶液转移到容器中,加入消化酶配置成样本酶溶液,37℃条件下消化上述组织块,同时每隔一段时间在镜下观察酶解状态,及时收集并更换新的酶解液,防止过度消化,维持收集后的细胞活力,当观察到有较多的细胞解离出,且多为细胞团时即可停止酶解,之后使用滤网过滤得到滤液。
所述容器优选50mL离心管中;所述酶溶液优选约10mL样本酶解液;酶解过程可以是,将离心管放置在摇床中,37℃,300r/min酶解时间为60-120min较好;
间隔的观察时间可根据需要选择,一般10-30min为宜。作为优选,消化酶可以选择多种,只要能够实现分离效果即可,但优选是胶原酶,更优选胶原酶type I,其浓度优选1-3mg/ml,更优选1mg/ml;,述滤网孔径为100μm较好。
进一步的,还可以包括二次酶解的步骤,所述步骤为,将滤网上部的组织块重新转移至新的容器(例如离心管)中,加入一定量的样本酶解液(例如10mL),在一定条件下进行酶解,例如37℃,300r/min酶解60min-120min,酶解后再次过滤,例如使用100μm滤网过滤得到滤液。
作为优选,所述二次酶解采用和一次酶解相同或不同的酶;但为了充分酶解,并且避免第一次酶解时间过长,导致已经分离的细胞受到损伤,优选采用和第一次酶解相同的酶。
清洗
一次,或者两次、或者更多次酶解得到的滤液,通过离心重悬清洗后获得细胞沉淀。例如1000r/min,离心5min。离心后弃上清,使用1ⅹHBSS缓冲液重悬沉淀,清洗1次,离心后得到细胞沉淀。
破碎红细胞
将细胞沉淀用无菌水重悬,吹打混匀后,立即加入同体积的氯化钠水溶液,使其终浓度达到0.9%的生理盐水浓度,之后离心得到去除红细胞的细胞沉淀。
所述吹打步骤大约30s,所述氯化钠水溶液优选1.8%,
立体培养
将收集的细胞使用合适体积的基质胶混悬均匀,使用枪头点板,放置在37℃培养箱中,一定时间后,加入相应的培养基COM-1001 350uL。该培养基是在基础培养基的基础上添加了多种营养成分,多种生长因子以及相关小分子化合物等优化配方,可实现乳腺癌类器官的快速生长,以及传代,冻存,复苏等过程,建立相关生物样本库。
作为优选,基质胶和枪头4℃预冷,培养基37℃预热。
作为优选,基质胶加入后,在培养箱中孵育20-40min,优选30min。
作为优选,基质胶可以选择现有技术中已知基质胶,包括但不限于MatrigelMatrix。
培养基COM-1001成分如下:
烟酰胺谷氨酰胺乙酰半胱氨酸
缓冲液:HEPES、Tirs-HCl,优选HEPES
基础培养基:包括但不限于B27、DMEM、MEM、1640,优选B27
生长因子、调节因子:FGF-10,EGF,Neuregulin 1,FGF7/KGF
抗菌物质:Normocin
Wnt激动剂:R-spondin 1
骨形态发生蛋白(BMPs)拮抗剂:Noggin
Rho相关的线圈激酶(ROCK)抑制剂:Y-27632
TGFβ信号通路抑制剂:A8301
p38 MAPK抑制剂:SB202190
具体的,可以优选以下培养基组分:
种板将除清上清的细胞沉淀,加入一定量的基质胶重悬混匀后,接入孔板。
具体的,基质胶和枪头4℃预冷,培养基37℃预热。得到的细胞沉淀,应将上清尽量除尽。根据细胞沉淀的多少,加入40-200μL不等的基质胶重悬细胞沉淀(注意不要产生气泡)。重悬混匀后,将基质胶与细胞的混悬液种到二十四孔板(孔板需放置于细胞培养箱中过夜预热,便于基质胶的成型)的板孔中,每孔1滴,约50μL/well。
凝胶。将带有基质胶的孔板放入培养箱种凝固,添加特定的培养基。
具体的,将孔板平放于37℃细胞培养箱中,凝固约30min。取出,每孔添加350μL37℃预热的培养基(COM-1001),放入培养箱中培养。
优选的可以添加换液步骤,可跟踪细胞的生长情况,培养2-3天,根据需要更换新鲜的培养基。
优选的,一定时间后可进行传代步骤:并每间隔一段时间,对类器官的生长状态拍照记录。
药物模型筛选
一种药物模型筛选方法,取类器官3D培养的类器官组织和对照细胞,分别加入抗肿瘤药,消化后,对得到单细胞通过流式细胞检测阳性细胞的比例。
所述对照细胞可以是MCF-7细胞,加入的抗肿瘤药物可以是Doxorubicin。
肿瘤组织染色分别取患者来源的肿瘤组织及患者来源肿瘤组织建立的样本,经4%多聚甲醛固定后,制作石蜡切片,进行免疫组织化学染色,观察患者来源的肿瘤组织及其来源的PDO样本间遗传特性的相关性。
所述患者来源组织建立的样本为上述类器官培养得到的类器官组织样本。
乳腺癌肿瘤类器官的药物筛选及鉴定
使用基质胶与培养基的混合液重悬细胞,将细胞接种在孔板中,每孔种一基质胶球,孔板倒扣放入培养箱中,使胶凝固;添加预热的培养基,放入培养箱中培养,三天后进行加药处理;药物处理3天后,重新进行新鲜药液的更换;药物处理6天后,使用ATP检测试剂进行各个药物处理的化学发光强度值的检测。根据数据,计算相应药物对类器官的IC50值。所述基质胶与培养基的比例为1:1。
所述细胞接种密度优选0.4万cells/well;
所述药物可以为Cisplatin、Epirubicin、Hydrochloride之一。
有益效果
1本发明采用特定的培养基,在基础培养基的基础上添加了多种营养成分,多种生长因子以及相关小分子化合物等优化配方,可实现肿瘤类器官特别是乳腺癌类器官的快速生长,配合特定的清洗缓冲液的清洗步骤,有效提高细胞活性,配合传代,冻存,复苏等过程,便于建立相关生物样本库。
2本发明采用的培养技术成功建立了乳腺癌肿瘤类器官体外模型;为体内外实验的需要及建立生物样本库提供保障。
3.肿瘤类器官的三维培养模式能更准确地模拟患者体内的肿瘤微环境,如实施例2显示,药物进入类器官的效率与平面细胞系存在显著性差异,体现了其在用于疾病研究和药物筛选方面的显著优势。
4.患者来源的肿瘤组织建立的PDO样本保留了患者肿瘤组织表型和遗传特性(实施例3),在进行药物筛选时,不同的乳腺癌病人样本药物筛选存在明显的个体化差异,如实施例4中所示,患者P3、P4、P6对药物Epirubicin Hydrochloride更为敏感,而患者P5对药物Cisplatin更为敏感,能更真实反应患者对药物方案的敏感性,可以指导医生有目的性地用药,达到个性化辅助治疗的目的。
对于乳腺癌患者来说,可以通过肿瘤手术样本或者穿刺样本中分离出的细胞来培养类器官,通过体外培养乳腺癌肿瘤组织的类器官样本(PDO)的多种药物方案的筛选,来预测患者对相应药物方案的敏感性,可以指导医生有目的性地用药,达到个性化辅助治疗的目的。
附图说明
图1为实施例1中类器官培养P0代生长状态图
图2为实施例1中类器官P0代培养7天后进行传代,P1代生长状态图
图3为实施例2中药物筛选模型图
图4为实施例3中各代次类器官染色图
图5为实施例4药物敏感性测试图
图6类器官培养与药物筛选流程图
具体实施方式
实施例1肿瘤组织中细胞的分离和类器官的培养
1、转移:从医院收取乳腺癌的肿瘤手术样本,使用样本储存液(DMEM/F12+1%三抗+1%HEPES)进行转移。
2、清洗。将肿瘤样本转移至10mL的样本清洗液中,置于旋转混匀仪上,清洗3次,每次5min。清洗后更换新鲜的清洗液。((1)Wash Buffer 4,清洗2遍,每遍5min(2)WashBuffer 2清洗3min。(3)Wash Buffer 3清洗3min。(4)Wash Buffer1~5顺序清洗(WashBuffer1中清洗5或6遍,每遍4~6分钟;在WashBuffer2中清洗2~4分钟;在WashBuffer3中清洗3分钟;在WashBuffer4中、在4℃的条件下浸泡28~32分钟;以及将经过WashBuffer4浸泡的组织在WashBuffer5中清洗5或6遍,每遍4~6分钟)
3、剪碎组织。将清洗好的肿瘤组织放入培养皿中,加入5-6mL HBSS,使用眼科剪和镊子将组织剪成小于1-2mm3的小块。
4、酶解细胞。将全部液体转移至50mL离心管中,配成约10mL样本酶解液(1mg/mL胶原酶type I),将离心管放置在摇床中,37℃,300r/min酶解60min-120min。期间每隔30min取少量酶解液于显微镜下观察细胞酶解情况,并注意及时收集并更换新的酶解液,防止过度消化,维持收集后的细胞活力,当观察到有较多的细胞解离出,且多为细胞团时即可停止酶解,之后使用100μm滤网过滤得到滤液。
5、二次酶解。滤网上部的组织块重新转移至新的离心管中,加入10mL的样本酶解液,37℃,300r/min酶解60min-120min,酶解后使用100μm滤网过滤得到滤液。
6、清洗酶解液:两次酶解得到的滤液,1000r/min,离心5min。离心后弃上清,使用1ⅹHBSS缓冲液重悬沉淀,清洗1次,离心后得到细胞沉淀。
7、破碎红细胞。将细胞沉淀用无菌水重悬,吹打混匀约30s后,立即加入同体积的1.8%的氯化钠水溶液,使其终浓度达到0.9%的生理盐水浓度,之后离心得到去除红细胞的细胞沉淀。
8、种板。基质胶和枪头4℃预冷,培养基37℃预热。得到的细胞沉淀,应将上清尽量除尽。根据细胞沉淀的多少,加入40-200μL不等的基质胶重悬细胞沉淀(注意不要产生气泡)。重悬混匀后,将基质胶与细胞的混悬液种到二十四孔板(孔板需放置于细胞培养箱中过夜预热,便于基质胶的成型)的板孔中,每孔1滴,约50μL/well。
9、凝胶。将孔板平放于37℃细胞培养箱中,凝固约30min。取出,每孔添加350μL37℃预热的培养基(COM-1001),放入培养箱中培养。
10、换液。跟踪细胞的生长情况。培养2-3天,需要更换新鲜的培养基。
11、传代:培养7天后,单个类器官直径在300μm左右,进行传代操作。去除孔内培养基,加入细胞回收液400μm/well,4℃条件下放置3h,镜下观察类器官从基质胶中散落出来,将上述类器官转移至15mL离心管中,1ml移液器反复吹打20次,离心,使用10mL预冷的HBSS吹打清洗2次,离心去除上清,加入37℃预热的5mL TrypLE溶液,37℃处理5min,加入5mLHBSS缓冲液吹打清洗,离心收集类器官沉淀,计数,并按照500~1500cells/10μL基质胶的浓度加入相应基质胶进行重悬,按照培养操作,进行种板,放入37℃待基质胶凝固后,加入培养基,每3天更换培养基,观察类器官生长状态并拍照记录。
分别在培养1天,5天以及7天后观察类器官生长状态并拍照记录。
P0代生长状态,参见图1;培养7天后进行传代,P1代生长状态,参见图2。
实施例2药物模型筛选
取3D培养的乳腺癌类器官组织(PDO)和MCF-7细胞,分别分为CTRL、0.5h、1h、2h处理组四组(PDO约5×104个细胞球/组,MCF-7约1×106cells/组),在1.5mL EP管中,分别加入Doxorubicin(10μg/mL)处理0h、0.5h、1h、2h后,用1×PBS清洗2遍。然后按照PDO的消化步骤对PDO进行消化,对得到的PDO的单细胞和MCF-7通过流式细胞检测Doxorubicin阳性细胞的比例。
结果显示在0.5h的时候Doxorubicin对MCF-7的浸润效率已经达到100%,但是对PDO的浸润效率只有62.5%,至2h,对PDO的浸润效率也只有75.4%。因此与目前常用的平面细胞系药物筛选模型相对,用PDO模型进行药物筛选能更好的模拟药物在体内对肿瘤组织的浸润及作用情况,具有显著优势。具体结果见图3。
实施例3肿瘤组织染色
分别取患者来源的肿瘤组织及患者来源肿瘤组织建立的PDO样本,经4%多聚甲醛固定后,制作石蜡切片,进行免疫组织化学染色,观察患者来源的肿瘤组织及其来源的PDO样本间遗传特性的相关性。
患者来源的肿瘤组织建立的PDO样本保留了患者肿瘤组织表型和遗传特性。从肿瘤组织和肿瘤类器官HE染色结果可观察到,类器官维持了组织的中低分化特征,细胞及细胞核异质性大,核质比增大,呈现出典型的肿瘤特征,并且和肿瘤组织保持一致,同时,在ER和PR表达方面,肿瘤类器官和组织也保持较好的一致性。
结果参见图4。
实施例4乳腺癌肿瘤类器官的药物筛选及鉴定
根据观察,待类器官成球,且细胞球数量较多时,可以进行相应乳腺癌类器官的药物筛选。我们根据临床方案来决定体外使用类器官进行药物筛选的方案。
1、使用基质胶与培养基的混合液(基质胶:培养基=1:1)重悬细胞,将细胞接种在白色九十六孔板中,约0.4万cells/well,每孔种一个5μL的基质胶球。种好细胞后,孔板倒扣放入培养箱中,使胶凝固约30min。
2、待基质胶凝固后,每孔添加75μL 37℃预热的培养基,放入培养箱中培养。三天后进行加药处理。并设置对照组,同时保证每个药物至少6个浓度梯度,每个浓度梯度至少3个复孔,药物使用100%PPC作为最高浓度,两倍倍比稀释。药物添加的孔外围添加PBS,减少药液蒸发。
3、为保证药效,药物处理3天后,重新进行新鲜药液的更换。药物处理6天后,使用ATP检测试剂进行各个药物处理的化学发光强度值的检测。根据数据,计算相应药物对类器官的IC50值。
对不同患者来源的乳腺癌类器官组织(PDO)和MCF-7细胞进行Cisplatin、Epirubicin Hydrochloride药物的敏感性测试,计算对应药物对类器官的IC50值,以确定PDO或MCF-7对药物的敏感性。结果显示不同患者来源的乳腺癌类器官组织(PDO)对不同药物的敏感性存在显著性差异(患者P3、P4、P6对药物Epirubicin Hydrochloride更为敏感,而患者P5对药物Cisplatin更为敏感。),体现了不同患者的个体差异性,且与MCF-7对药物的敏感性存在显著差异,表明了以PDO来预测患者对相应药物方案的敏感性具有显著优势。
表1药物敏感性数据
趋势图参见图5。
根据以上实验结果,对于乳腺癌患者来说,我们可以通过肿瘤手术样本或者穿刺样本中分离出的细胞,通过酶解来进行3D培养类器官,通过对类器官的体外多种药物方案的筛选,来预测患者对相应药物方案的敏感性,可以指导医生有目的性地用药,达到个性化辅助治疗的目的,具体步骤参加图6。
Claims (10)
1.一种类器官培养基,包括烟酰胺、谷氨酰胺、乙酰半胱氨酸、缓冲液、基础培养基、生长因子和/或调节因子、抗菌物质、Wnt激动剂、骨形态发生蛋白(BMPs)拮抗剂、Rho相关的线圈激酶(ROCK)抑制剂、TGFβ信号通路抑制剂、p38 MAPK抑制剂。
2.权利要求1所述的培养基,其特征在于,所述缓冲液选自HEPES、Tirs-HCl之一;所述基础培养基选自B27、DMEM、MEM、1640等培养基之一;所述生长因子、调节因子选自FGF-10,EGF,Neuregulin 1,FGF7/KGF中至少一个。
3.权利要求1所述的培养基:其特征在于,所述抗菌物质可以为Normocin;Wnt激动剂可以为R-spondin 1;骨形态发生蛋白(BMPs)拮抗剂可以为Noggin;Rho相关的线圈激酶(ROCK)抑制剂可以为Y-27632;TGFβ信号通路抑制剂可以为A8301;p38 MAPK抑制剂可以为SB202190。
4.一种类器官培养方法,包括样本处理步骤、酶解步骤、红细胞破碎步骤、立体培养步骤,其中立体培养步骤包括将收集的细胞使用合适体积的基质胶混悬均匀,点板后,放置在培养箱中,一定时间后,加入权利要求1-3所述培养基。
5.权利要求4所述的培养方法,其中所述样本处理步骤使用Wash Buffer1-5的5种清洗液,所述Wash Buffer 1为包含浓度为100单位/mL的青霉素,浓度为100μg/mL的链霉素以及浓度为0.25μg/mL两性霉素B的HBSS缓冲溶液;Wash Buffer 2为包含体积分数为75%的无水乙醇的HBSS缓冲溶液;Wash Buffer 3为包含体积分数为3%的乙酸的HBSS缓冲溶液;Wash Buffer 4为包含浓度为1000单位/mL的青霉素、浓度为1000μg/mL的链霉素以及浓度为2.5μg/mL的两性霉素B的HBSS缓冲溶液;Wash Buffer 5为包含体积分数为1%的FBS的HBSS缓冲溶液。
6.权利要求5所述的培养方法,所述样本处理步骤包括第一清洗处理步骤和第二清洗处理步骤,所述第一清洗处理步骤包括在WashBuffer4—WashBuffer2——WashBuffer3中顺序清洗的步骤,所述第二清洗步骤包括在WashBuffer1——WashBuffer2——WashBuffer3——WashBuffer4——WashBuffer5中顺序清洗。
7.权利要求4所述的培养方法,所述酶解步骤包括采用胶原酶进行样本消化,酶解后过滤得到滤液,作为优选,滤网上遗留的组织块可继续进行酶解,并可重复该步骤若干次,优选重复一次。
8.根据权利要求1-3所述的培养基或权利要求4-7所述的培养方法,其特征在于,所述类器官为肿瘤类器官,优选乳腺癌。
9.一种药物模型筛选方法,取权利要求4-8的培养方法获得的类器官3D培养的类器官组织和对照细胞,分别加入抗肿瘤药,消化后,对得到单细胞通过流式细胞检测阳性细胞的比例。
10.一种药物模型筛选方法,使用基质胶与培养基的混合液重悬权利要求4-8的培养方法获得的类器官3D培养的类器官细胞,将细胞接种在孔板中,每孔种一基质胶球,孔板倒扣放入培养箱中,使胶凝固;添加预热的培养基,放入培养箱中培养,三天后进行加药处理;药物处理3天后,重新进行新鲜药液的更换;药物处理6天后,使用ATP检测试剂进行各个药物处理的化学发光强度值的检测。根据数据,计算相应药物对类器官的IC50值。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210704242.3A CN117305243A (zh) | 2022-06-21 | 2022-06-21 | 一种类器官培养方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210704242.3A CN117305243A (zh) | 2022-06-21 | 2022-06-21 | 一种类器官培养方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117305243A true CN117305243A (zh) | 2023-12-29 |
Family
ID=89283565
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210704242.3A Pending CN117305243A (zh) | 2022-06-21 | 2022-06-21 | 一种类器官培养方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117305243A (zh) |
-
2022
- 2022-06-21 CN CN202210704242.3A patent/CN117305243A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7461674B2 (ja) | 食道扁平上皮癌の上皮細胞用の培養培地、培養方法、及びその用途 | |
WO2023274338A1 (zh) | 用于快速培养肿瘤类器官的培养基、方法以及试剂盒 | |
CN112080472B (zh) | 一种培养专用于生物医药功能研究的人肺癌类器官3d模型的方法 | |
CN117402826A (zh) | 培养类器官的方法 | |
CN108504625B (zh) | 一种小鼠成纤维细胞及其用途 | |
CN114181903A (zh) | 结直肠癌类器官培养基及无支架3d培养方法 | |
CN117004572A (zh) | 病人来源的移植瘤类器官模型pdxo的构建方法与应用 | |
CN115011560A (zh) | 一种脑胶质瘤类器官、培养基及培养方法 | |
CN110499290B (zh) | 一株人尤文肉瘤细胞系 | |
CN114958753B (zh) | 一种舌癌类器官的培养基、培养方法及鉴定方法 | |
CN110117570B (zh) | 一种类风湿性关节炎滑膜成纤维细胞的原代培养方法 | |
CN117025505A (zh) | 胃粘膜上皮前体样细胞及其制备方法和应用 | |
CN117305243A (zh) | 一种类器官培养方法 | |
EP4368706A1 (en) | Culture medium and culture method for lung cancer epithelial cells, and application thereof | |
CN112608899B (zh) | 一种无血清培养基在培养癌组织起源球状体中的应用 | |
CN115558633A (zh) | 一种利用微基质胶球快速培养类器官的方法 | |
CN109370986A (zh) | 一种犬脂肪干细胞的提取方法及其制剂和应用 | |
CN111849904B (zh) | 神经母细胞瘤类器官的培养基、培养方法及移植体 | |
CN115161282A (zh) | 一种小鼠脑微血管内皮细胞与周细胞联合提取培养方法 | |
CN113957053A (zh) | 一种以人源肠癌类器官筛选抗肠癌干细胞中药活性成分的方法 | |
CN115820560B (zh) | 一种复发性呼吸道乳头状瘤病类器官的构建方法及其应用 | |
CN113201494B (zh) | 一种黏膜黑色素瘤细胞及其用途 | |
WO2024072157A1 (ko) | 오가노이드 제작 방법 | |
WO2015119107A1 (ja) | がん細胞の取得方法 | |
CN114369576A (zh) | 一种基于微流控芯片培养的肝癌患者源性肿瘤组织球的建立方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication |