CN117286108B - Special culture medium for breast cancer organoids and culture method - Google Patents
Special culture medium for breast cancer organoids and culture method Download PDFInfo
- Publication number
- CN117286108B CN117286108B CN202311578139.XA CN202311578139A CN117286108B CN 117286108 B CN117286108 B CN 117286108B CN 202311578139 A CN202311578139 A CN 202311578139A CN 117286108 B CN117286108 B CN 117286108B
- Authority
- CN
- China
- Prior art keywords
- organoid
- tissue
- breast cancer
- cells
- matrigel
- 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.)
- Active
Links
- 210000002220 organoid Anatomy 0.000 title claims abstract description 132
- 206010006187 Breast cancer Diseases 0.000 title claims abstract description 70
- 208000026310 Breast neoplasm Diseases 0.000 title claims abstract description 70
- 239000001963 growth medium Substances 0.000 title claims abstract description 36
- 238000012136 culture method Methods 0.000 title abstract description 8
- 102000007237 Forkhead Box Protein M1 Human genes 0.000 claims abstract description 11
- 108010008599 Forkhead Box Protein M1 Proteins 0.000 claims abstract description 11
- 102100035423 POU domain, class 5, transcription factor 1 Human genes 0.000 claims abstract description 11
- 101710126211 POU domain, class 5, transcription factor 1 Proteins 0.000 claims abstract description 11
- 102000004266 Collagen Type IV Human genes 0.000 claims abstract description 9
- 108010042086 Collagen Type IV Proteins 0.000 claims abstract description 9
- 230000010261 cell growth Effects 0.000 claims abstract description 7
- 239000003102 growth factor Substances 0.000 claims abstract description 7
- -1 small molecule compounds Chemical class 0.000 claims abstract description 5
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000007995 HEPES buffer Substances 0.000 claims abstract description 4
- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 4
- 229940088710 antibiotic agent Drugs 0.000 claims abstract description 4
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 claims abstract description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 3
- 210000002966 serum Anatomy 0.000 claims abstract description 3
- 210000001519 tissue Anatomy 0.000 claims description 69
- 206010028980 Neoplasm Diseases 0.000 claims description 19
- 238000012258 culturing Methods 0.000 claims description 19
- 108010082117 matrigel Proteins 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 15
- 239000006228 supernatant Substances 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 13
- 239000002609 medium Substances 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 10
- 201000011510 cancer Diseases 0.000 claims description 9
- 239000013049 sediment Substances 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 238000004140 cleaning Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 7
- 229960003942 amphotericin b Drugs 0.000 claims description 6
- 238000007664 blowing Methods 0.000 claims description 6
- 238000007710 freezing Methods 0.000 claims description 6
- 230000008014 freezing Effects 0.000 claims description 6
- 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 6
- 230000029087 digestion Effects 0.000 claims description 5
- 230000001079 digestive effect Effects 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 239000011148 porous material Substances 0.000 claims description 4
- 239000003761 preservation solution Substances 0.000 claims description 4
- 238000010257 thawing Methods 0.000 claims description 4
- 108090000385 Fibroblast growth factor 7 Proteins 0.000 claims description 3
- 102100028071 Fibroblast growth factor 7 Human genes 0.000 claims description 3
- 101000825960 Homo sapiens R-spondin-3 Proteins 0.000 claims 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 description 3
- 229930182555 Penicillin Natural products 0.000 claims description 3
- 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 3
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 claims description 3
- 210000000577 adipose tissue Anatomy 0.000 claims description 3
- 230000001338 necrotic effect Effects 0.000 claims description 3
- 102000045246 noggin Human genes 0.000 claims description 3
- 108700007229 noggin Proteins 0.000 claims description 3
- 239000008188 pellet Substances 0.000 claims description 3
- 229940049954 penicillin Drugs 0.000 claims description 3
- 238000011536 re-plating Methods 0.000 claims description 3
- 239000011550 stock solution Substances 0.000 claims description 3
- 229960005322 streptomycin Drugs 0.000 claims description 3
- 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 2
- 108090001047 Fibroblast growth factor 10 Proteins 0.000 claims description 2
- 102100028412 Fibroblast growth factor 10 Human genes 0.000 claims description 2
- 101000825954 Homo sapiens R-spondin-1 Proteins 0.000 claims description 2
- 108090000556 Neuregulin-1 Proteins 0.000 claims description 2
- 102000048238 Neuregulin-1 Human genes 0.000 claims description 2
- 102100022762 R-spondin-1 Human genes 0.000 claims description 2
- 102100022766 R-spondin-3 Human genes 0.000 claims description 2
- 229960004308 acetylcysteine Drugs 0.000 claims description 2
- 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 2
- 239000002244 precipitate Substances 0.000 claims description 2
- 239000012487 rinsing solution Substances 0.000 claims description 2
- 238000010008 shearing Methods 0.000 claims description 2
- 230000007480 spreading Effects 0.000 claims description 2
- 230000012010 growth Effects 0.000 abstract description 11
- 108010035532 Collagen Proteins 0.000 abstract description 4
- 102000008186 Collagen Human genes 0.000 abstract description 4
- 238000004113 cell culture Methods 0.000 abstract description 4
- 229920001436 collagen Polymers 0.000 abstract description 4
- 150000001875 compounds Chemical class 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 42
- 230000000052 comparative effect Effects 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 13
- 239000000523 sample Substances 0.000 description 6
- 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 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000012188 paraffin wax Substances 0.000 description 4
- 238000007747 plating Methods 0.000 description 4
- 230000003833 cell viability Effects 0.000 description 3
- 239000000306 component Substances 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 102000006495 integrins Human genes 0.000 description 3
- 108010044426 integrins Proteins 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 239000008399 tap water Substances 0.000 description 3
- 230000035899 viability Effects 0.000 description 3
- 239000008096 xylene Substances 0.000 description 3
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 2
- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 description 2
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 101001060261 Homo sapiens Fibroblast growth factor 7 Proteins 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- 102100039037 Vascular endothelial growth factor A Human genes 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000000481 breast Anatomy 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 210000001671 embryonic stem cell Anatomy 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 102000057239 human FGF7 Human genes 0.000 description 2
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 2
- 230000036210 malignancy Effects 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 101000917237 Homo sapiens Fibroblast growth factor 10 Proteins 0.000 description 1
- 101000851176 Homo sapiens Pro-epidermal growth factor Proteins 0.000 description 1
- 101001109800 Homo sapiens Pro-neuregulin-1, membrane-bound isoform Proteins 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 101710098940 Pro-epidermal growth factor Proteins 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000011226 adjuvant chemotherapy Methods 0.000 description 1
- 239000012574 advanced DMEM Substances 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 108010072041 arginyl-glycyl-aspartic acid Proteins 0.000 description 1
- 210000002469 basement membrane Anatomy 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000005907 cancer growth Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 210000003027 ear inner Anatomy 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- DZGCGKFAPXFTNM-UHFFFAOYSA-N ethanol;hydron;chloride Chemical compound Cl.CCO DZGCGKFAPXFTNM-UHFFFAOYSA-N 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 229960002743 glutamine Drugs 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 238000001794 hormone therapy Methods 0.000 description 1
- 102000057243 human FGF10 Human genes 0.000 description 1
- 102000055650 human NRG1 Human genes 0.000 description 1
- 102000054144 human RSPO3 Human genes 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 238000010309 melting process Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- GVUGOAYIVIDWIO-UFWWTJHBSA-N nepidermin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C(C)C)C(C)C)C1=CC=C(O)C=C1 GVUGOAYIVIDWIO-UFWWTJHBSA-N 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000008672 reprogramming Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 210000001525 retina Anatomy 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 210000005005 sentinel lymph node Anatomy 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 239000001993 wax Substances 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
-
- 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
- C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
-
- 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
- C12N2501/155—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
- C12N2501/10—Growth factors
- C12N2501/165—Vascular endothelial growth factor [VEGF]
-
- 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
- C12N2501/60—Transcription factors
- C12N2501/603—Oct-3/4
-
- 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
- C12N2501/999—Small molecules not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
Abstract
The invention relates to the technical field of cell culture, and discloses a special culture medium for breast cancer organoids and a culture method, wherein the culture medium comprises the following components: reduced serum DMEM, 10 mM HEPES, 2 mM Glutamax, 50 XN-21, 50 XB-27, 1% antibiotics, cell growth factors, small molecule compounds, 0.1 mg/mL type IV collagen, 0.01 mg/mL Oct4 protein, and 0.02 mg/mL FOXM1 protein. The special culture medium for the breast cancer organoids provided by the invention simulates the growth environment close to the body by improving the cell growth factors, small molecular compounds, culture factors and other essential elements, and especially the added IV-type collagen, oct4 protein and FOXM1 protein not only promote the growth of the breast cancer organoids, but also improve the success rate of the culture of the organoids.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a special culture medium for breast cancer organoids and a culture method.
Background
Organoids (organoids) are miniature organs produced in vitro by culturing cells with multipotency for 3 days. The organoids have a similar micro-anatomical structure to the original tissue, are capable of self-renewal and self-organization, and reproduce a part of the function of the corresponding organ, providing a highly physiologically relevant system. And the organoid culture needs few tissues, has short culture period and high success rate, and can avoid the problems of long modeling period, long culture time, high cost and the like of an animal model. The organoid technology has great application potential in the biomedical transformation research fields of disease research, drug screening, drug toxicity and toxicological response, gene and cell therapy and the like, and organoids are evaluated as Preclinical models of human disease, a human disease preclinical model in journal of new England medicine in 2019.
Organoid sources can be derived from stem cells of the original tissue, embryonic Stem Cells (ESCs), induced Pluripotent Stem Cells (iPSCs) and established cell lines and organ explants comprising a variety of cell types. However, due to ethical issues and acquisition difficulties, ipscs derived primarily from primitive tissues or from somatic reprogramming are now being studied and clinically used. The first successful culture established intestinal organoids from the professor team of Hans Clevers, the netherlands scientist in 2009, which underwent tremendous development. At present, organoid culture has been applied to organoid research models of a variety of normal or tumor tissues including esophagus, intestine, stomach, liver, pancreas, lung, breast, kidney, prostate, bladder, brain, thyroid, inner ear, retina, heart, and ovary.
Breast Cancer (BC) is a malignancy that occurs in the ductal epithelium or lobule of the female Breast, and is the most common cause of cancer and cancer death among women worldwide, and recent national cancer statistics published by the national cancer center in month 1 of 2019 show that Breast cancer is still the malignancy of female at position 1, and the number of people who develop it annually is about 30.4 ten thousand. In recent years, the incidence of breast cancer has been continuously rising, and the quality of daily life and life safety of females are seriously threatened. Currently, the treatment and prognosis regimen for breast cancer is largely dependent on the staging of the breast cancer. The characteristics, invasion range and infiltration degree of the tumor need to be accurately evaluated before operation. The key information provided by clinical staging determines the surgical mode, whether adjuvant chemotherapy or hormone therapy, radiation therapy, sentinel lymph node biopsy, axillary lymph node cleaning, etc. are required before and after surgery. According to the stage and the parting of the breast cancer, the high-risk recurrence factor of an individual is considered, the most effective treatment scheme is designed, a certain treatment effect can be achieved, but the residual focus is extremely easy to cause the recurrence of the local breast cancer, so that the overall survival rate of patients is not high. While recent advances in organoid culture provide new opportunities for establishing and analyzing patient samples, allowing malignant cells to reproduce under conditions similar to the three-dimensional growth of breast tumors, they have proven to be effective in maintaining the heterogeneity of primary samples and are becoming new models for further characterizing breast cancer molecules.
The choice of medium is an important factor in the course of breast cancer organoids. Traditional culture media are not stable in organoids due to lack of necessary factors for organoid growth and differentiation. Organoid media are more complex and specific than traditional cell culture media in order to provide a physiological environment that is closer to the organoids to better maintain and study organoid structure and function. Different organoid media require modification of the appropriate formulation depending on the particular organoid type to obtain optimal experimental results.
The application publication No. CN114634909A discloses a culture medium and a culture method for breast cancer organoids, wherein the culture medium contains recombinant human R-Spondin 3 protein, recombinant human Heregulin beta-1 protein, FGF-7, recombinant human KGF/FGF-7 factor, recombinant human FGF-10 factor, recombinant human EGF factor, recombinant human Noggin protein, A83-01, Y-27632, SB202190, B27, N-acetyl-L-cysteine, nicotinamide, HEPES buffer solution, penicillin, streptomycin, glutamine supplement and Advanced DMEM/F12. Although the culture medium and the culture method can shorten the culture period of organoids, long-term stable culture cannot be achieved.
Disclosure of Invention
The invention aims to provide a special culture medium for breast cancer organoids, which improves the success rate of organoid culture by improving the essential factors such as cell growth factors, small molecular compounds, culture factors and the like to simulate the growth environment close to the body.
In order to achieve the above object, the present invention provides a special culture medium for breast cancer organoids, comprising: reduced serum DMEM, 10 mM HEPES, 2 mM Glutamax, 1 XN-21, 1 XB-27, 1% antibiotics, cell growth factors, small molecule compounds, 0.1 mg/mL type IV collagen, 0.01 mg/mL Oct4 protein, and 0.02 mg/mL FOXM1 protein.
Preferably, the antibiotic is penicillin-streptomycin-amphotericin B mixed solution; the concentration of penicillin in the penicillin-streptomycin-amphotericin B mixed solution is 10000 mug/mL, the concentration of streptomycin is 10000 mug/mL, and the concentration of amphotericin B is 25 mug/mL.
Preferably, the cell growth factor is a mixture of 5 ng/mL EGF, 100 μg/mL Noggin, 100 μg/mL R-Spondin 1, 100 μg/mL R-Spondin 3, 10 nM Neuregulin 1, 20 ng/mL FGF-10, 5 ng/mL FGF-7.
Preferably, the small molecule compound is a mixture of 5. Mu. M Y-27632, 10 mM Nicotinamide, 1.25 mM N-Acetylcysteine, 500 nM A83-01, 1. Mu.M SB 202190.
The invention also provides a breast cancer organoid culture method, which comprises the following steps:
placing a fresh breast cancer tissue sample into a precooled tissue preservation solution for pretreatment;
adding tissue enzymolysis liquid into the pretreated fresh breast cancer tissue to digest the cancer tissue, oscillating at 37 ℃ for digestion for 10-20 min, and adding three-volume tissue cleaning liquid;
filtering with 100 μm sieve, collecting filtrate, centrifuging at 300 g for 3-5 min, and removing supernatant;
adding matrigel with the volume of 25 times of tissue into the centrifugally collected tissue, carrying out plate paving after resuspension, photographing, tracking and positioning;
placing the paved plates into a 37 ℃ incubator for 10-15 min to form gel, adding a special culture medium for breast cancer organoids, enabling the culture medium to be free of cells, continuously culturing in the 37 ℃ incubator, replacing the organoids culture medium for 1 time every 2-3 days, and culturing the cells on matrigel for 30 days to obtain organoid-matrigel mixture;
taking down the organoid-matrigel mixture by using a cell scraper to carry out organoid passage to obtain organoid cells, and adding the organoid cells into the frozen stock solution for freezing and preserving according to the density of 500/mL to obtain frozen organoid cells;
and taking out the frozen organoid cells from the liquid nitrogen tank for organoid resuscitation.
Preferably, the specific method for pretreatment is as follows: placing fresh breast cancer tissue sample into precooled tissue preservation solution, removing adipose tissue and necrotic tumor tissue, selecting only living tumor tissue, transferring the tissue sample into centrifuge tube, rinsing the tissue sample with 4 ℃ precooled PBS solution until the rinsing solution is in a clear liquid state, transferring the rinsed tissue into a culture dish, and shearing until the rinsed tissue reaches 1mm 3 Is included in the block of tissue.
Preferably, the specific method for the organoid passage is as follows:
transferring the organoid-matrigel mixture into 15 mL conical tube, standing for 3 min, centrifuging for 5 min at 300 g, and removing supernatant; adding 10 times of the volume of the organoid digestive juice, blowing and mixing uniformly by a pipetting gun, and then placing into a 37 ℃ water bath for 12 min; adding organoid cleaning liquid, filtering with 100 μm sieve, centrifuging for 3-5 min at 300 g, and removing supernatant; re-plating with matrigel and resuspension.
Preferably, the specific method for the organoid resuscitation comprises the following steps: taking out frozen organoid cells from a liquid nitrogen tank, rapidly placing the frozen organoid cells in a water bath kettle at 37 ℃ for thawing, and shaking a freezing pipe in the thawing process; transferring the thawed organoid cells to a 15 mL conical tube, blowing with a pipette 3 times, centrifuging at 300 g for 3 min, removing the supernatant and collecting organoid cell pellets; adding organoid cleaning liquid into organoid cell sediment, re-suspending, transferring into a centrifuge tube, centrifuging for 5 min at 300 and g to remove supernatant, and collecting tissue sediment;
adding matrigel to the centrifugally collected tissue precipitate for resuspension to obtain a tissue-matrigel mixed solution, spreading 20 mu L of the tissue-matrigel mixed solution in a 24-pore plate, placing in a 37 ℃ incubator for 10 min, adding 1.5 mL of breast cancer organoid special culture medium, and culturing for 30 days.
The invention has the beneficial effects that:
the IV type collagen is a main component of a cell basement membrane, and the IV type collagen is added into a culture medium, so that the matrigel can be assisted to simulate the supporting effect of an extracellular matrix in a body, the physiological environment of breast cancer cells in the body can be better simulated, and the attachment and growth of organoid cells can be facilitated.
Arg-Gly-Asp (RGD) short peptide motif, CB3 fragment and the like in type IV collagen can be combined with extracellular integrin and non-integrin receptors and participate in cell signal transduction. The invention adds IV type collagen into the culture medium, which can combine with the integrin of cancer cells, thereby activating the signal transduction pathway in the cancer cells, promoting the proliferation, growth and migration of the cancer cells, and being beneficial to promoting the growth of breast cancer organoids and gradually forming specific structures and tissues.
Oct4 is a transcription factor of the POU family, whose main function is to bind to tenna containing an octamer motif (ATGCAAAT), transcriptionally activate expression of its downstream target gene, play a key role in embryonic development and maintenance of stem cell multipotency, and promote renewal and differentiation of tumor stem cells. The FOXM1 can be combined with a promoter of a vascular endothelial growth factor, and can regulate and control the expression of the vascular endothelial growth factor at a transcription level, so that the generation process of tumor blood vessels can be effectively promoted, and the generation and growth of tumor tissues can be further promoted. According to the invention, the Oct4 protein and the FOXM1 protein are added into the culture medium, so that the transcription level of related genes for proliferation and growth of cancer cells is synergistically improved, and the growth of breast cancer organoids is promoted.
The special culture medium for the breast cancer organoids provided by the invention contains a plurality of specific culture factors, and simultaneously has cell factors and small molecular compounds required by the organoids, so that the microenvironment of breast cancer cells in a human body can be greatly simulated, and the growth and differentiation of the organoids are promoted.
The special culture medium for breast cancer organoids effectively reduces the complexity of organoid culture, simplifies the original preparation process of tens of reagents and tens of reagents into a culture medium finished product which is convenient to take at any time, greatly simplifies the operation flow, and simultaneously reduces the pollution and the loss possibly occurring in the original preparation process.
Drawings
FIG. 1 is a morphology of primary breast cancer organoids cultured in example 1 under an optical microscope;
FIG. 2 is a comparison of H & E staining of breast cancer tissue (left) and breast cancer organoids (right) after passage of example 1;
FIG. 3 is a graph showing the cell viability of organoids of example 1 and comparative examples 1-4 after different passage times after plating for 14 days.
Detailed Description
The invention is further described below with reference to the accompanying drawings. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.
Example 1
A breast cancer organoid culture method comprising the steps of:
(1) A breast cancer organoid dedicated medium was prepared according to the components in table 1.
TABLE 1 Special Medium Components for Breast cancer organoids
(2) Breast cancer organoid primary cell culture
Fresh breast cancer tissue samples were placed in pre-chilled tissue preservation fluid (abs 9474, shanghai Biotechnology Co., ltd.) and adipose tissue and necrotic tumor tissue (usually yellow or white) were removed, and only viable tumor tissue (usually pink) was selected.
Transferring the pretreated tissue sample into a 50 mL centrifuge tube, adding 15 mL of 4 ℃ precooled PBS solution to rinse the tissue sample for several times, and removing blood and fragments. This step is repeated until the rinse solution is in a clear state (centrifuge tube replacement is required each time).
III. Transfer the tissue to Petri dishes and shear it to about 1mm 3 Adding tissue enzymolysis solution (abs 9482, abbe) for digestion, shaking at 37deg.C for 20 min, adding three times volume of tissue washing solution, and stopping digestion.
Filtering with 100 μm sieve, observing small amount of filtrate under a mirror to obtain obvious tissue block, collecting filtrate, centrifuging at 300 g for 5 min, removing supernatant, and collecting tissue.
And V. Adding matrigel (next san Biotech (Shanghai) Co., ltd., 40183ES 08) with 25 times of tissue volume to the collected tissue for resuspension, and performing plating (operation at 4 ℃) for photographing tracking positioning.
Placing the paved plates into a 37 ℃ incubator for 15 min to form gel, adding 1.5 mL breast cancer organoid culture medium prepared in the step (1), making the culture medium not pass through cells, continuously culturing in the incubator, changing 1 organoid culture medium every 2-3 days, growing cells on matrigel, and culturing for 30 days to obtain organoid-matrigel mixture. The morphological structure of the organoids was observed under an optical microscope, and as shown in fig. 1, it can be seen that the formed breast cancer organoids exhibited typical three-dimensional spheres and grew well.
(3) Breast cancer organoid passaging
The organoid-matrigel mixture of step (2) was removed using a cell scraper and transferred to a 15 mL conical tube, allowed to stand for 3 min, centrifuged at 300 g for 5 min to remove the supernatant, and the pellet was collected.
And II, adding a 10-time volume of organoid digestive juice (41421 ES30 of the Hitachi Biotechnology Co., ltd.) into the sediment after the centrifugation in the step I, gently blowing a plurality of times by a pipette for mixing, and then putting into a water bath at 37 ℃ for 12 min to digest matrigel.
III, adding an organoid wash (PRS-TCR-1, popular biological medicine Co., ltd.) in an equal volume to the digestive juice, stopping digestion, filtering with a 100 μm sieve, centrifuging for 5 min at 300 g, removing the supernatant, and collecting the tissue.
And V, re-suspending by using matrigel, re-plating, adding matrigel with the volume of 25 times of tissue volume into the collected tissue for re-suspending, and performing plating (operation at 4 ℃) photographing, tracking and positioning.
Placing the paved plates into a 37 ℃ incubator for 15 min to form gel, adding the breast cancer organoid culture medium prepared in the step (1) of 1.5 and mL, continuously culturing in the incubator, and replacing the organoid culture medium for 1 time every 2-3 days, and culturing for 30 days to obtain organoid cell culture solution.
And VII, centrifuging the culture solution 300 g obtained in the step VI for 3 min, removing the supernatant, adding the organoid frozen stock solution (41422) into the culture solution at a density of 500 pieces/mL, and freezing to obtain frozen organoid cells.
(4) Breast cancer organoid resuscitation
And I, taking out frozen organoid cells from a liquid nitrogen tank, rapidly placing the frozen organoid cells in a water bath kettle at 37 ℃ for melting, and slightly shaking a freezing tube to accelerate the melting in the water bath melting process.
Transferring the melted organoid cells to a 15 mL conical tube, lightly blowing for 3 times by using a pipette, centrifuging for 3 min at 300 g, removing supernatant and collecting organoid cell sediment, adding 10 mL organoid cleaning liquid into the organoid cell sediment for resuspension, transferring into a 1.5 mL centrifuge tube, centrifuging for 5 min at 300 g, and collecting tissue sediment.
And III, re-suspending by using matrigel, namely adding matrigel with the volume of 25 times of tissue volume into the collected tissue sediment for re-suspending to obtain a tissue-matrigel mixed solution, paving 20 mu L of the tissue-matrigel mixed solution in a 24-pore plate, placing the 24-pore plate in a 37 ℃ incubator for 10 min, adding 1.5 and mL of the breast cancer organoid culture medium prepared in the step (1), replacing 1 organoid culture medium every 2-3 days, and culturing for 30 days.
Comparative example 1
The method for culturing breast cancer organoids is different from example 1 in that the culture medium dedicated to breast cancer organoids prepared in step (1) does not contain type IV collagen.
Comparative example 2
A method of culturing a breast cancer organoid, which differs from example 1 in that the breast cancer organoid-specific medium prepared in step (1) does not contain Oct4 protein.
Comparative example 3
A method of culturing a breast cancer organoid, which differs from example 1 in that the breast cancer organoid-specific medium prepared in step (1) does not contain foxM1 protein.
Comparative example 4
The method for culturing breast cancer organoids is different from example 1 in that the culture medium dedicated to breast cancer organoids prepared in step (1) does not contain type IV collagen, oct4 protein and FOXM1 protein.
Test 1
Taking a proper amount of the breast cancer tissue of the example 1 and the breast cancer organoids after passage respectively into a 15 mL centrifuge tube, standing, sucking and discarding the culture medium under negative pressure, adding 5 mL of DPBS for washing, centrifuging for 3 min at 300 g, discarding the DPBS, adding 5 mL of 4% PFA solution into the centrifuge tube, and standing on a shaking table at room temperature for 30 min.
After the fixation was completed, the 4% pfa solution was discarded, and dehydration embedding was performed in the following order: 75% alcohol 4 h-85% alcohol 2 h-90% alcohol 2 h-95% alcohol 1 h-absolute alcohol 30 min-xylene 10 min-wax 1 h.
And (3) placing the dehydrated and embedded tissues into a grinding tool containing paraffin, adjusting the positions of the tissues, and cooling the tissues on a cooling table of a paraffin embedding machine.
The paraffin block is fixed on a paraffin slicer for slicing, the slice is closely fixed on a slide, fished out and put into a 60 ℃ oven for baking 2 h.
Dewaxing and rehydrating the baked slices according to the following sequence: xylene 20 min-absolute ethanol 10 min-95% alcohol 3 min-80% alcohol 3 min-75% alcohol 3 min-tap water.
The dewaxed, rehydrated sections were placed horizontally in a wet box and stained in the following order: hematoxylin 5 min-tap water flushing 5 min-1% ethanol hydrochloride 1 s-tap water reverse blue 10 min-eosin 5 min-95% ethanol 5 min.
Dewaxing and rehydration are carried out according to the following sequence: 3 min of 95% alcohol, 10 min of absolute alcohol and 20 min of xylene.
The sealing was performed using neutral resin and cover glass, and the results were observed under a microscope, as shown in fig. 2, and it can be seen that breast cancer organoids formed cell clusters and had a high morphological consistency with breast cancer cells.
Test 2
Grinding 4 g trypan blue with a small amount of distilled water, adding double distilled water to a volume of 100 mL, filtering with filter paper to obtain 4% trypan blue mother liquor, and adding 90 mL PBS to 10 mL trypan blue mother liquor to dilute to obtain 0.4% trypan blue solution.
Organoid culture solutions (passage number 0 is primary cultured organoid cells) of example 1 and comparative examples 1 to 4 were taken 2 mL after different passage numbers of 14 days of plating culture, respectively, and 100-fold dilution was performed with PBS to obtain cell suspensions.
The cell suspension was combined with 0.4% trypan blue solution at 9:1, and uniformly mixing the components.
As a result of calculation of the cell viability using a cell counter, as shown in FIG. 3, the cell viability of the primary organoid cells cultured in example 1 was almost unchanged from that of the primary organoid cells cultured in comparative examples 1 to 4, but the primary organoid viability cultured in example 1 was higher than that of the primary organoid cells cultured in comparative examples 1 to 4 after one passage, and the primary organoid viability cultured in example 1 was also decreased after 3 passages, but the magnitude of decrease was smaller than that of the primary organoid viability cultured in comparative examples 1 to 4. Compared with comparative example 4 in which no type IV collagen, oct4 protein and FOXM1 protein were added to the medium, only type IV collagen was not added to the medium used in comparative example 1, only Oct4 protein was not added to the medium used in comparative example 2, only FOXM1 protein was not added to the medium used in comparative example 3, and the resulting breast cancer organoid culture effect was better than that of comparative example 4, so that both type IV collagen and Oct4 protein and FOXM1 protein promoted growth effectively for breast cancer organoids, and when these three proteins were simultaneously added to the medium, a more synergistic effect was exerted, which was optimal for the breast cancer organoid culture effect.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and variations could be made by those skilled in the art without departing from the technical principles of the present invention, and such modifications and variations should also be regarded as being within the scope of the invention.
Claims (5)
1. A breast cancer organoid dedicated medium comprising: reduced serum DMEM, 10 mM HEPES, 2 mM Glutamax, 1 xn-21, 1 xb-27, 1% antibiotics, cell growth factors, small molecule compounds, 0.1 mg/mL type IV collagen, 0.01 mg/mL Oct4 protein, and 0.02 mg/mL FOXM1 protein; the antibiotics are penicillin-streptomycin-amphotericin B mixed solution; penicillin concentration in the penicillin-streptomycin-amphotericin B mixed solution is 10000 mug/mL, streptomycin concentration is 10000 mug/mL, amphotericin B concentration is 25 mug/mL; the cell growth factors are a mixture of 5 ng/mL EGF, 100 mug/mL Noggin, 100 mug/mL R-Spondin 1, 100 mug/mL R-Spondin 3, 10 nM Neuregulin 1, 20 ng/mL FGF-10, 5 ng/mL FGF-7; the small molecule compound was a mixture of 5. Mu. M Y-27632, 10 mM Nicotinamide, 1.25 mM N-Acetylcysteine, 500 nM A83-01, 1. Mu.M SB 202190.
2. A method of breast cancer organoid culture comprising:
placing a fresh breast cancer tissue sample into a precooled tissue preservation solution for pretreatment;
adding tissue enzymolysis liquid into the pretreated fresh breast cancer tissue to digest the cancer tissue, oscillating at 37 ℃ for digestion for 10-20 min, and adding three-volume tissue cleaning liquid;
filtering with 100 μm sieve, collecting filtrate, centrifuging at 300 g for 3-5 min, and removing supernatant;
adding matrigel with the volume of 25 times of tissue into the centrifugally collected tissue, carrying out plate paving after resuspension, photographing, tracking and positioning;
placing the paved plates into a 37 ℃ incubator for 10-15 min to form gel, adding the special culture medium for breast cancer organoids according to claim 1, enabling the culture medium to be free of cells, continuously culturing in the 37 ℃ incubator, replacing 1 organoid culture medium every 2-3 days, growing cells on matrigel, and culturing for 30 days to obtain organoid-matrigel mixture;
taking down the organoid-matrigel mixture by using a cell scraper to carry out organoid passage to obtain organoid cells, and adding the organoid cells into the frozen stock solution for freezing and preserving according to the density of 500/mL to obtain frozen organoid cells;
and taking out the frozen organoid cells for organoid resuscitation.
3. The method for culturing breast cancer organoids according to claim 2, wherein the specific method for pretreatment is as follows: placing fresh breast cancer tissue sample into precooled tissue preservation solution, removing adipose tissue and necrotic tumor tissue, selecting only living tumor tissue, transferring the tissue sample into centrifuge tube, rinsing the tissue sample with 4 ℃ precooled PBS solution until the rinsing solution is in a clear liquid state, transferring the rinsed tissue into a culture dish, and shearing until the rinsed tissue reaches 1mm 3 Is included in the block of tissue.
4. The method for culturing breast cancer organoids according to claim 2, wherein the organoids are passaged by the following specific methods:
transferring the organoid-matrigel mixture into 15 mL conical tube, standing for 3 min, centrifuging for 5 min at 300 g, and removing supernatant;
adding 10 times of the volume of the organoid digestive juice, blowing and mixing uniformly by a pipetting gun, and then placing into a 37 ℃ water bath for 12 min;
adding organoid cleaning liquid, filtering with 100 μm sieve, centrifuging for 3-5 min at 300 g, and removing supernatant;
re-plating with matrigel and resuspension.
5. The method for culturing breast cancer organoids according to claim 2, wherein the specific method for organoid resuscitation is as follows:
taking out frozen organoid cells from a liquid nitrogen tank, and placing the frozen organoid cells in a water bath at 37 ℃ for thawing, and shaking a freezing tube in the thawing process;
transferring the thawed organoid cells to a 15 mL conical tube, blowing with a pipette 3 times, centrifuging at 300 g for 3 min, removing the supernatant and collecting organoid cell pellets;
adding organoid cleaning liquid into organoid cell sediment, re-suspending, transferring into a centrifuge tube, centrifuging for 5 min at 300 and g to remove supernatant, and collecting tissue sediment;
adding matrigel to the centrifugally collected tissue precipitate for resuspension to obtain a tissue-matrigel mixed solution, spreading 20 mu L of the tissue-matrigel mixed solution in a 24-pore plate, placing in a 37 ℃ incubator for 10 min, adding 1.5 mL of the breast cancer organoid special culture medium according to claim 1, and culturing for 30 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311578139.XA CN117286108B (en) | 2023-11-24 | 2023-11-24 | Special culture medium for breast cancer organoids and culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311578139.XA CN117286108B (en) | 2023-11-24 | 2023-11-24 | Special culture medium for breast cancer organoids and culture method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117286108A CN117286108A (en) | 2023-12-26 |
| CN117286108B true CN117286108B (en) | 2024-03-01 |
Family
ID=89252064
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311578139.XA Active CN117286108B (en) | 2023-11-24 | 2023-11-24 | Special culture medium for breast cancer organoids and culture method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117286108B (en) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010096627A1 (en) * | 2009-02-20 | 2010-08-26 | Lisanti Michael P | Therapeutics and methods for treating neoplastic diseases comprising determining the level of caveolin-1 and/or caveolin-2 in a stromal cell sample |
| CN110494555A (en) * | 2017-03-24 | 2019-11-22 | 纽约市哥伦比亚大学理事会 | The generation of lung bud organoid with branched structure and its purposes for being used for lung disease modeling |
| CN110582564A (en) * | 2015-09-15 | 2019-12-17 | 新加坡科技研究局 | derivation of liver organoids from human pluripotent stem cells |
| CN111565798A (en) * | 2017-10-10 | 2020-08-21 | 儿童医院医学中心 | Esophageal tissue and/or organoid compositions and methods of making same |
| CN111876386A (en) * | 2020-08-10 | 2020-11-03 | 上海市第一人民医院 | Method for culturing breast cancer organoid and co-culturing tumor-associated fibroblast |
| WO2021113328A1 (en) * | 2019-12-03 | 2021-06-10 | Neuvogen, Inc. | Tumor cell vaccines |
| WO2021222700A1 (en) * | 2020-04-30 | 2021-11-04 | University Of Maryland, Baltimore | Compositions and methods for treating cancer with andrographolide and melatonin combination therapy |
| CN114317443A (en) * | 2022-03-10 | 2022-04-12 | 北京大橡科技有限公司 | Breast cancer organoid culture solution, and culture reagent combination and culture method thereof |
| CN114402072A (en) * | 2019-08-07 | 2022-04-26 | 艾比克斯生物科学有限责任公司 | Use of PCBP1 in generating induced pluripotent stem cells and inhibiting tumorigenesis |
| CN114634909A (en) * | 2020-12-15 | 2022-06-17 | 四川药之助科技有限公司 | Culture medium and culture method for breast cancer organoid culture |
| CN116234924A (en) * | 2020-07-30 | 2023-06-06 | 国立研究开发法人理化学研究所 | Bladder organoid and method for producing same |
-
2023
- 2023-11-24 CN CN202311578139.XA patent/CN117286108B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010096627A1 (en) * | 2009-02-20 | 2010-08-26 | Lisanti Michael P | Therapeutics and methods for treating neoplastic diseases comprising determining the level of caveolin-1 and/or caveolin-2 in a stromal cell sample |
| CN110582564A (en) * | 2015-09-15 | 2019-12-17 | 新加坡科技研究局 | derivation of liver organoids from human pluripotent stem cells |
| CN110494555A (en) * | 2017-03-24 | 2019-11-22 | 纽约市哥伦比亚大学理事会 | The generation of lung bud organoid with branched structure and its purposes for being used for lung disease modeling |
| CN111565798A (en) * | 2017-10-10 | 2020-08-21 | 儿童医院医学中心 | Esophageal tissue and/or organoid compositions and methods of making same |
| CN114402072A (en) * | 2019-08-07 | 2022-04-26 | 艾比克斯生物科学有限责任公司 | Use of PCBP1 in generating induced pluripotent stem cells and inhibiting tumorigenesis |
| WO2021113328A1 (en) * | 2019-12-03 | 2021-06-10 | Neuvogen, Inc. | Tumor cell vaccines |
| WO2021222700A1 (en) * | 2020-04-30 | 2021-11-04 | University Of Maryland, Baltimore | Compositions and methods for treating cancer with andrographolide and melatonin combination therapy |
| CN116234924A (en) * | 2020-07-30 | 2023-06-06 | 国立研究开发法人理化学研究所 | Bladder organoid and method for producing same |
| CN111876386A (en) * | 2020-08-10 | 2020-11-03 | 上海市第一人民医院 | Method for culturing breast cancer organoid and co-culturing tumor-associated fibroblast |
| CN114634909A (en) * | 2020-12-15 | 2022-06-17 | 四川药之助科技有限公司 | Culture medium and culture method for breast cancer organoid culture |
| CN114317443A (en) * | 2022-03-10 | 2022-04-12 | 北京大橡科技有限公司 | Breast cancer organoid culture solution, and culture reagent combination and culture method thereof |
Non-Patent Citations (3)
| Title |
|---|
| FOXM1 facilitates breast cancer cell stemness and migration in YAP1-dependent manner;Sun等;Archives of Biochemistry and Biophysics;第685卷;108349 * |
| PRMT7与HSPA5相互作用促进肺癌转移的研究;程德志;徐朝娜;张翔;郑亮承;张信波;谢德耀;;实用药物与临床(08);14-17 * |
| 去泛素化酶UCHL1促进乳腺癌化疗耐药作用与机制研究;缪祥;顾玉婷;张雁云;;现代免疫学(06);78-81 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117286108A (en) | 2023-12-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2023274338A1 (en) | Culture medium, method, and reagent kit for rapidly culturing tumor organoids | |
| Zhau et al. | Establishment of a three-dimensional human prostate organoid coculture under microgravity-simulated conditions: evaluation of androgen-induced growth and PSA expression | |
| CN114317443B (en) | Breast cancer organoid culture solution, and culture reagent combination and culture method thereof | |
| CN111411083B (en) | Culture medium and culture method for stomach cancer organoid | |
| CN114292816B (en) | Lung cancer organoid culture solution, and culture reagent combination and culture method thereof | |
| CN112111444B (en) | Method for reprogramming umbilical cord mesenchymal stem cells into liver cells and prepared liver organoid | |
| US20190264179A1 (en) | Serum-free medium inducing differentiation of umbilical cord mesenchymal stem cell into insulin-secretion-like cell and preparation method and use thereof | |
| EP2146728A2 (en) | Progenitor cells from urine and methods for using the same | |
| CN112266898A (en) | Culture method and culture solution for colorectal cancer organoid | |
| CN113423816A (en) | Three-dimensional culture method for large-scale preparation of stem cells | |
| CN104988110A (en) | Method for transforming umbilical cord mesenchymal stem cells into islet cells | |
| CN114752565B (en) | Retina organoid with immune cells and construction method thereof | |
| CN112852709B (en) | Method for culturing mouse lung organoid | |
| CN116836934B (en) | Osteosarcoma organoid culture solution, culture reagent combination and culture method | |
| CN113943755B (en) | Method for constructing in-situ primary esophageal cancer animal model | |
| CN117286108B (en) | Special culture medium for breast cancer organoids and culture method | |
| CN117004572A (en) | Construction method and application of patient-derived transplanted tumor organoid model PDXO | |
| CN115094022B (en) | Construction method of lung cancer fibroblast and lung cancer organoid co-culture model | |
| CN107058225B (en) | Compound induction culture medium and method for inducing umbilical cord mesenchymal stem cells into neuron-like cells by adopting culture medium | |
| CN112592883B (en) | Mouse pancreas organoid culture medium and application thereof | |
| RU2272638C1 (en) | Biotransplant, method for its preparing and method for treatment of chronic hepatitis and liver cirrhosis (variants) | |
| CN113957035B (en) | Duck embryo primary liver cell separation culture method | |
| CN117050934B (en) | Preparation method of mouse prostate organoid and primary in situ prostate cancer animal model | |
| CN115181730B (en) | Gastric stromal tumor organoid culture medium and culture method | |
| Huang et al. | Progress and perspective of organoid technology in breast cancer research |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |