CN117247903A - Stomach cancer 3D organoid and application thereof - Google Patents
Stomach cancer 3D organoid and application thereof Download PDFInfo
- Publication number
- CN117247903A CN117247903A CN202311032985.1A CN202311032985A CN117247903A CN 117247903 A CN117247903 A CN 117247903A CN 202311032985 A CN202311032985 A CN 202311032985A CN 117247903 A CN117247903 A CN 117247903A
- Authority
- CN
- China
- Prior art keywords
- gastric cancer
- organoid
- organoids
- cell
- culture
- 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 212
- 208000005718 Stomach Neoplasms Diseases 0.000 title claims abstract description 140
- 206010017758 gastric cancer Diseases 0.000 title claims abstract description 140
- 201000011549 stomach cancer Diseases 0.000 title claims abstract description 140
- 239000003814 drug Substances 0.000 claims abstract description 64
- 238000000034 method Methods 0.000 claims abstract description 33
- 210000001519 tissue Anatomy 0.000 claims abstract description 32
- 239000000203 mixture Substances 0.000 claims abstract description 17
- 239000006143 cell culture medium Substances 0.000 claims abstract description 14
- 238000011534 incubation Methods 0.000 claims abstract description 13
- 239000000654 additive Substances 0.000 claims abstract description 12
- 230000000996 additive effect Effects 0.000 claims abstract description 12
- 238000001914 filtration Methods 0.000 claims abstract description 12
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims abstract description 12
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 11
- 239000007640 basal medium Substances 0.000 claims abstract description 11
- 108010082117 matrigel Proteins 0.000 claims abstract description 10
- 102000029816 Collagenase Human genes 0.000 claims abstract description 8
- 108060005980 Collagenase Proteins 0.000 claims abstract description 8
- 229960002424 collagenase Drugs 0.000 claims abstract description 8
- 239000012913 medium supplement Substances 0.000 claims abstract description 8
- 230000001079 digestive effect Effects 0.000 claims abstract description 7
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims abstract description 7
- 238000002156 mixing Methods 0.000 claims abstract description 7
- 102100028412 Fibroblast growth factor 10 Human genes 0.000 claims abstract description 6
- 101000917237 Homo sapiens Fibroblast growth factor 10 Proteins 0.000 claims abstract description 6
- 102000004895 Lipoproteins Human genes 0.000 claims abstract description 6
- 108090001030 Lipoproteins Proteins 0.000 claims abstract description 6
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229960000890 hydrocortisone Drugs 0.000 claims abstract description 6
- 239000012577 media supplement Substances 0.000 claims abstract description 4
- 230000001376 precipitating effect Effects 0.000 claims abstract description 4
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 claims abstract description 4
- 229940079593 drug Drugs 0.000 claims description 55
- 238000012258 culturing Methods 0.000 claims description 23
- 238000000338 in vitro Methods 0.000 claims description 16
- 230000029087 digestion Effects 0.000 claims description 8
- 238000011282 treatment Methods 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 7
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 6
- 239000012091 fetal bovine serum Substances 0.000 claims description 6
- 239000003112 inhibitor Substances 0.000 claims description 6
- 239000003761 preservation solution Substances 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
- 235000009754 Vitis X bourquina Nutrition 0.000 claims description 5
- 235000012333 Vitis X labruscana Nutrition 0.000 claims description 5
- 235000014787 Vitis vinifera Nutrition 0.000 claims description 5
- 230000001788 irregular Effects 0.000 claims description 5
- 239000008188 pellet Substances 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 4
- 229960003942 amphotericin b Drugs 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 238000001179 sorption measurement Methods 0.000 claims description 4
- 108090000145 Bacillolysin Proteins 0.000 claims description 3
- 102100021022 Gastrin Human genes 0.000 claims description 3
- 108010052343 Gastrins Proteins 0.000 claims description 3
- 102000035092 Neutral proteases Human genes 0.000 claims description 3
- 108091005507 Neutral proteases Proteins 0.000 claims description 3
- 102000004142 Trypsin Human genes 0.000 claims description 3
- 108090000631 Trypsin Proteins 0.000 claims description 3
- 108010007093 dispase Proteins 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 229960005322 streptomycin Drugs 0.000 claims description 3
- 239000012588 trypsin Substances 0.000 claims description 3
- AOXOCDRNSPFDPE-UKEONUMOSA-N chembl413654 Chemical compound C([C@H](C(=O)NCC(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@H](CCSC)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](C)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]1N(CCC1)C(=O)CNC(=O)[C@@H](N)CCC(O)=O)C1=CC=C(O)C=C1 AOXOCDRNSPFDPE-UKEONUMOSA-N 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 240000006365 Vitis vinifera Species 0.000 claims 1
- 150000003180 prostaglandins Chemical class 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 16
- 238000010276 construction Methods 0.000 abstract description 14
- 238000011084 recovery Methods 0.000 abstract description 4
- 238000011109 contamination Methods 0.000 abstract description 3
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 238000012136 culture method Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 47
- 206010028980 Neoplasm Diseases 0.000 description 38
- 238000012360 testing method Methods 0.000 description 38
- 239000008055 phosphate buffer solution Substances 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 238000011160 research Methods 0.000 description 9
- 238000001514 detection method Methods 0.000 description 8
- 238000007877 drug screening Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 238000009472 formulation Methods 0.000 description 7
- 239000003550 marker Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- 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 6
- 238000007710 freezing Methods 0.000 description 6
- 230000008014 freezing Effects 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 239000000427 antigen Substances 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000003255 drug test Methods 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 108060000903 Beta-catenin Proteins 0.000 description 4
- 102000015735 Beta-catenin Human genes 0.000 description 4
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 4
- 241000219095 Vitis Species 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 239000003102 growth factor Substances 0.000 description 4
- 238000010166 immunofluorescence Methods 0.000 description 4
- 238000011532 immunohistochemical staining Methods 0.000 description 4
- 230000000877 morphologic effect Effects 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 238000010257 thawing Methods 0.000 description 4
- 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 3
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 3
- -1 ProstaglandinE2 prostaglandin Chemical class 0.000 description 3
- 239000003855 balanced salt solution Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000009509 drug development Methods 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000012827 research and development Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 239000001993 wax Substances 0.000 description 3
- 238000012605 2D cell culture Methods 0.000 description 2
- 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 description 2
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 2
- 206010064571 Gene mutation Diseases 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
- GWVPFVHTQIDCFF-UHFFFAOYSA-J O.O.O.O.O.O.O.O.O.O.O.O.O.O.[Zn++].[Zn++].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O Chemical compound O.O.O.O.O.O.O.O.O.O.O.O.O.O.[Zn++].[Zn++].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O GWVPFVHTQIDCFF-UHFFFAOYSA-J 0.000 description 2
- 230000004156 Wnt signaling pathway Effects 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000021164 cell adhesion Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000005138 cryopreservation Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229960002986 dinoprostone Drugs 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 238000002575 gastroscopy Methods 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 238000010874 in vitro model Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000002547 new drug Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 108700025694 p53 Genes Proteins 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- WGCXTGBZBFBQPP-UHFFFAOYSA-N prostaglandin E2 methyl ester Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(=O)OC WGCXTGBZBFBQPP-UHFFFAOYSA-N 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- 238000005563 spheronization Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 238000011269 treatment regimen Methods 0.000 description 2
- 239000000439 tumor marker Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 201000003883 Cystic fibrosis Diseases 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 208000002699 Digestive System Neoplasms Diseases 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 1
- 208000005016 Intestinal Neoplasms Diseases 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- 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 description 1
- 206010038111 Recurrent cancer Diseases 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 238000011226 adjuvant chemotherapy Methods 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- ULXXDDBFHOBEHA-CWDCEQMOSA-N afatinib Chemical compound N1=CN=C2C=C(O[C@@H]3COCC3)C(NC(=O)/C=C/CN(C)C)=CC2=C1NC1=CC=C(F)C(Cl)=C1 ULXXDDBFHOBEHA-CWDCEQMOSA-N 0.000 description 1
- 229960001686 afatinib Drugs 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- XKRFYHLGVUSROY-UHFFFAOYSA-N argon Substances [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000007012 clinical effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010219 correlation analysis Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000007405 data analysis Methods 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
- 238000003745 diagnosis Methods 0.000 description 1
- 238000001861 endoscopic biopsy Methods 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- 238000013210 evaluation model Methods 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000013095 identification testing Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 201000002313 intestinal cancer Diseases 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000002906 medical waste Substances 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000001778 pluripotent stem cell Anatomy 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0679—Cells of the gastro-intestinal tract
-
- 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
-
- 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
- G01N33/5076—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 involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
-
- 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
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/22—Zinc; Zn chelators
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/119—Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins 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
- 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
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Oncology (AREA)
- Gastroenterology & Hepatology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The application relates to the technical field of organoid culture, in particular to a gastric cancer 3D organoid and application thereof. The application provides a gastric cancer 3D organoid culture method, which comprises the following steps: 1) Digesting the sheared gastric cancer tissues with cell digestive juice, centrifuging, precipitating and filtering, and collecting cells; 2) Mixing the cells in the step 1) with matrigel, placing the mixture on a culture plate for incubation, and then adding a cell culture medium for culture to obtain a gastric cancer 3D organoid; the components of the cell culture medium include: DMEM/F12 basal medium and medium supplements; the media supplement includes a combination of one or more of an N2 serum-free additive, a B-27 serum-free additive, a glutamine substitute, hydrocortisone, a lipoprotein, zinc sulfate heptahydrate, FGF10, and collagenase. The method can lead the success rate of gastric cancer 3D organoid culture to be 80%, the recovery success rate to be 100%, the microbial contamination rate to be 0% and the overall construction success rate to be more than 90%; and the sensitivity degree of the gastric cancer 3D organoid model and the medicine is more than 70%.
Description
Technical Field
The application relates to the technical field of organoid culture, in particular to a gastric cancer 3D organoid and application thereof.
Background
According to the report of world health organization, the incidence of gastric cancer is at the 5 th place of the global incidence of malignant tumor, the death rate is higher at the 3 rd place, and the incidence is next to lung cancer and liver cancer. The new occurrence of gastric cancer is about 100 ten thousand every year worldwide, and more than 40% of gastric cancer is occupied in China.
Because of the poor disease screening system, once gastric cancer is found, most of the gastric cancer is in middle and late stages, surgery is the first choice for treating cancer, and adjuvant chemotherapy is one of the important treatment means. For unresectable progressive gastric cancer, recurrent cancer, chemotherapy remains difficult to cure completely, with the aim of improving clinical symptoms, delaying recurrent time or prolonging survival. There is still a lack of specific chemotherapeutic drugs in the market, and many drugs fail to enter clinical stage two and three due to the lack of an excellent in vitro drug screening model. Therefore, an efficient medicine sieve model needs to appear, new medicine development is assisted, and organoids are expected to appear to solve the problem.
Organoids are tissue analogs with a spatial structure formed by 3D culture in vitro using adult stem cells or pluripotent stem cells. Compared with 2D cultured cells, organoids can simulate tissue structure and function in vivo to the greatest extent and can be stably subcultured for a long period of time. Until now, organoid models of intestine, liver, stomach, lung, breast, pancreas, prostate, brain, esophagus, blood vessels, etc. have been built worldwide. Similar to normal tissue, tissue-specific tumor organoids can also be obtained by 3D culture methods, with a genetic background that is highly consistent with the genetic background of the tumor tissue from which it was derived. Genetic mutation in the stomach organoid is highly matched with mutation in a corresponding tumor tissue specimen, is consistent with the conventional large-scale gastric cancer mutation analysis result, and can well simulate in-situ tumor in aspects of gene expression profile, protein mass spectrum, pathological morphology, tumor heterogeneity and the like.
Several studies have shown that organoid responses to drugs can be used to predict clinical efficacy of drugs. For example, a study on cystic fibrosis showed that its organoid drug response was consistent with published clinical trial results; among them, for 3 cases of rare mutant patients, the patients with good reaction of the organoid drugs obtained better results after administration, and the patients with poor organoid reaction had poor clinical effects. Recent research in Science journal in 2018 shows that the response of tumor organoids to drugs is highly consistent with the clinical efficacy of patients with metastatic gastrointestinal tumors, which suggests 88% positive predictive value and 100% negative predictive value, and provides a powerful support for the in vitro drug screening model of tumor organoids as accurate medical treatment.
Meanwhile, new england journal, newjm, 2019, first demonstrated great potential of organoids in drug development and tumor therapy in a review format. Compared with the traditional 2D cell culture and animal model, the in vitro model of the three-dimensional structure avoids the complexity of animal experiments, can show the structure and the behavior of organs from which the three-dimensional structure is derived, can keep the stability of genome, and can be brought into full play by combining with the imaging technology of the front edge. The report of NEJM has pushed organoids to a completely new focus.
The above fully illustrates that organoids are expected to become excellent in vitro models for replacing cell lines and PDX, and can be applied to research and development of tumor medicaments and guiding clinical medication in the future. Based on the genetic background that tumor organoids (PDTOs) are highly consistent with in-situ tumors, and have absolute advantages in cost and time relative to PDX models, PDTO models are becoming a new generation of functional platforms for drug enterprise new drug development and accurate medical treatment of tumor patients.
In 2015, hans Clevers of netherlands scientists first published success in building intestinal cancer organoids biological libraries in the journal of Cell, and illustrate the application of tumor organoids-high flux screening, accurate medicine, and exploration of tumor targets (biomarks). The scientific team led by the method is currently utilizing tumor organoids to screen medicines in large scale and high flux. After that, researchers have explored various kinds of tumor organoids, and have been successful in establishing organoids such as colorectal cancer, breast cancer, pancreatic cancer, gastric cancer, liver cancer, bladder cancer, lung cancer, ovarian cancer, etc., and organoid establishment rates have been different depending on laboratory techniques and tumor types, but are generally better. In 2017, pa μLi et al were published above Cancer Discovery and set forth the great potential of tumor organoids as a functional screening platform for accurate medical treatment, and successfully performed matching analysis on 4 tumor patients, the authors finally suggested that the drug screening function of tumor organoids provided a new possibility of treatment regimen selection for advanced malignant tumor patients for whom no clinical treatment regimen was available. 2 months 2018, science is a milestone article which reveals that the tumor organoids can predict the prognosis of treatment of tumor patients, the positive prediction value is 88%, and the negative prediction value is 100%, which provides basis for clinical large-scale guiding research, and the organoids are further in the accurate treatment direction of tumors.
Recent two years of domestic scientific research institutions begin to enter organoid research, but high-quality achievements have not been published, and more medical institutions begin to pay attention to the functions and potential of tumor organoids. Some businesses in the business arts have focused on tumor organoids. For example, beijing-department-Tu-medical science, guangzhou's core-creating international development is earlier and more mature. Other companies such as Rhin Shaanxi, hangzhou Union medical, phenoki, kun love, etc. are also invested in organoid industry, but most are still in the beginning. Some pharmaceutical enterprises are concerned about tumor organoids, such as Ming Kangde, which also hope to establish tumor organoid departments, and promote drug development, but progress slowly.
Along with the great reports of International journal of top Science, nature and Cell, the exposure rate of organoids is increased year by year, the heat of organoids is also increased linearly, and more scientific research institutions and companies in China begin to pay attention to organoid industry, so that the application prospect of organoids in the fields of drug research and development is commonly seen by the same. The earlier the field is entered, the greater the potential commercial value that will be brought to the expert in the field. Organoids are not commercialized products, and natural quality standards of organoids are not recognized, so that organoid library construction is completed as soon as possible, and the quality standards of the organoids are established, so that the organoids are significant.
In the prior art, the related stomach cancer organoid culture technology has the problems of low construction success rate, high preparation and culture cost, single morphology and the like, and the correlation between the stomach cancer 3D organoid and the clinical medicine effectiveness lacks further research and needs to be solved.
By exploring a more efficient construction and culture flow of gastric cancer organoids, the cost can be effectively reduced, the commercialized price of gastric cancer 3D organoids tends to the level of 2D gastric cancer cells, and large-scale and multi-batch preparation is realized. In addition, as the physiological forms of the three-dimensional cell culture are greatly different from those of the 2D cells widely used at present, the traditional 2D cell application means are not necessarily suitable for the application of the organoids, and the organoids need to be purposefully applied to in-vitro efficacy evaluation tests of different tested drugs according to different types of the gastric cancer organoids so as to summarize the application methods and characteristics of the gastric cancer organoids in various forms.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, an object of the present application is to provide a gastric cancer 3D organoid and application thereof for solving the problems in the prior art.
To achieve the above and other related objects, a first aspect of the present application provides a method for culturing a gastric cancer 3D organoid, comprising the steps of:
1) Digesting gastric cancer tissues with cell digestive juice, centrifuging, precipitating and filtering, and collecting cells;
2) Mixing the cells in the step 1) with matrigel, placing the mixture on a culture plate for incubation, and then adding a cell culture medium for culture to obtain a gastric cancer 3D organoid;
the components of the cell culture medium include: DMEM/F12 basal medium and medium supplements; the media supplement includes a combination of one or more of an N2 serum-free additive, a B-27 serum-free additive, a glutamine substitute, hydrocortisone, a lipoprotein, zinc sulfate heptahydrate, FGF10, and collagenase.
The second aspect of the present application provides a gastric cancer 3D organoid obtained by culturing the gastric cancer 3D organoid according to the above-described method.
A third aspect of the present application provides a method for culturing the gastric cancer 3D organoids described above or an application of the gastric cancer 3D organoids described above in preparing an in vitro drug sieve model of gastric cancer.
Compared with the prior art, the beneficial effects of this application are:
1. the method for constructing the gastric cancer organoids can enable the success rate of gastric cancer 3D organoids to be 80%, the recovery success rate to be 100%, the microbial contamination rate to be 0%, and the overall construction success rate to be more than 90%.
2. The gastric cancer 3D organoids provided by the application comprise five forms, and can be applied to in vitro tests. On one hand, the gastric cancer 3D organoids provided by the application construct a more ideal test model for developing specific medicines; on the other hand, the method realizes more accurate disease prediction and diagnosis and provides medication guidance for clinical personalized control and therapy. The research results improve the vigilance of gastric cancer high incidence groups to diseases, and pertinently adjust the strategies of preventing and treating gastric cancer, thus having important clinical significance.
3. The gastric cancer 3D organoids with different forms are suitable for different kinds of medicines and different test purposes, the gastric cancer 3D organoid model and the medicine sensitivity degree are more than 70%, and the gastric cancer 3D organoids have statistical significance.
Drawings
FIG. 1 is a diagram showing analysis of gastric cancer organoid library status.
Fig. 2 is a sample of a gastric cancer 3D organoid. Wherein, FIG. 2A is a sample plot of early and late passages, and FIG. 2B is a sample plot of organoid subtypes and organoids after 6 days of resuscitation.
FIG. 3 is a graph of marker expression identified by immunofluorescence.
Fig. 4 is a 3D organoid diagram of gastric cancer of different morphology. Wherein, fig. 4A and 4F are irregular official cavities, fig. 4E is regular official cavities, fig. 4B is solid sphere, fig. 4C is grape cluster, and fig. 4D is mixed type.
FIG. 5 is a drug sensitivity test. Wherein, FIG. 5A is a concentration gradient design, FIG. 5B is a multiple organoid typing vs single drug test, and FIG. 5C is a single organoid typing vs multi drug test.
Fig. 6 is a sample detection flow of embodiment 1.
Detailed Description
In order to make the objects, technical solutions and advantageous effects of the present application clearer, the present application is further described below with reference to examples. It should be understood that the examples are presented by way of illustration only and are not intended to limit the scope of the application. The test methods used in the following examples are conventional, unless otherwise indicated, and other advantages and effects of the present application will be readily apparent to those skilled in the art from the disclosure herein.
The inventor of the present application has found gastric cancer 3D organoids and applications thereof through a great deal of research and study, and completed the present application on the basis of this.
In one aspect, the present application provides a method for culturing a gastric cancer 3D organoid, comprising the steps of:
1) Digesting gastric cancer tissues with cell digestive juice, centrifuging, precipitating and filtering, and collecting cells;
2) Mixing the cells in the step 1) with matrigel, placing the mixture on a culture plate for incubation, and then adding a cell culture medium for culture to obtain a gastric cancer 3D organoid;
the components of the cell culture medium include: DMEM/F12 basal medium and medium supplements; the media supplement includes a combination of one or more of an N2 serum-free additive, a B-27 serum-free additive, a glutamine substitute, hydrocortisone, a lipoprotein, zinc sulfate heptahydrate, FGF10, and collagenase.
According to the method for constructing the gastric cancer organoids, the success rate of gastric cancer 3D organoids is 80%, the recovery success rate is 100%, the microbial contamination rate is 0%, and the overall construction success rate is more than 90%.
In the method for culturing gastric cancer 3D organoids provided in the present application, step 1) means that gastric cancer tissues are digested with a cell digestive juice, and cells are collected after centrifugation, precipitation and filtration. Wherein the gastric cancer tissue is obtained by fresh sampling and then cold chain transportation in tissue preservation liquid. Further, the tissue preservation solution comprises the following components: 90-100% (v/v) of fetal bovine serum, 9-15% (v/v) of ethylenediamine tetraacetic acid, 8-16 mu mol/L Y-27632 inhibitor, 100-150U/ml of green streptomycin and 100-150U/ml of gentamicin-amphotericin B mixed solution. In a preferred embodiment of the present application, the tissue preservation fluid comprises the following components: 90% (v/v) FBS fetal bovine serum, 9% (v/v) EDTA ethylenediamine tetraacetic acid, 8-16 mu mol/L Y-27632 inhibitor, 100U/ml green streptomycin, 100U/ml gentamycin-amphotericin B mixed solution. The tissue preservation solution has the functions of: in the construction process of gastric cancer organoids, the high activity of tissues is maintained, and the tissues are prevented from being polluted.
In step 1), the components of the cell digestive juice include: 4-8mg/mL of Dispase II neutral protease (for cell separation), 10ng/mL ProstaglandinE2 prostaglandin, 4-8mg/mL TrypLE trypsin (for cell separation), 10ng/mL Gastin Gastrin. The temperature of digestion was 37 ℃. The digestion time is 1-2 h; specifically, the time period may be 1 to 1.2 hours, 1.2 to 1.5 hours, or 1.5 to 2 hours.
In step 1), the digestion further comprises an intermediate treatment step: the digested supernatant was transferred and the pellet was resuspended in PBS.
In the step 1), the filtering comprises the filtering by a cell screen, and further, the particle size of the cell screen is 50-200 mu m; specifically, it may be 50 to 100. Mu.m, 100 to 120. Mu.m, 120 to 200. Mu.m, or the like.
In step 1), the filtration step further comprises a centrifugation step, and the cell pellet is collected for mixing with the matrigel in step 2).
In the method for culturing the gastric cancer 3D organoids, the step 2) is to mix the cells in the step 1) with matrigel, place the mixture on a culture plate for incubation, and then add a cell culture medium for culturing to obtain the gastric cancer 3D organoids. Wherein the culture plate is a low adsorption plate; further, the low adsorption plate is a 24-well plate. In step 2), the incubation temperature was 37 ℃. The incubation time is 10-20min; specifically, it may be 10 to 12 minutes, 12 to 15 minutes, 15 to 20 minutes, or the like.
In step 2), the volume ratio of DMEM/F12 basal medium is 90% (v/v) based on the total volume of the cell culture medium in some embodiments. The volume of the medium supplement was 10% (v/v) based on the total volume of the cell culture medium. The DMEM/F12 basal medium is prepared by using 90% (v/v) 1X stock solution and 10% (v/v) DPBS balanced salt solution.
In step 2), in one embodiment of the present application, the cell culture medium consists of the following components: 90% (v/v) DMEM/F12 (1X) basal medium for formulation of 90% (v/v) basal medium, 10% (v/v) DPBS balanced salt solution (for washing cells and formulation of medium), N2 serum-free additive for formulation of 10% (v/v) medium supplement, B-27 (50X) serum-free additive, glutaMAX Supplement glutamine substitute, hydrocortisone, lipoprotein, zinc sulfate heptahydrate zinc sulfate heptahydrate, FGF10 cell growth factor, collagenase Type I/IV collagenase (for 3D spheronization of cells). The present application found that good organoid culture was not achieved using only the existing media. Thus, the media used in this application are reagents that are selected and proportioned by purchasing various brands of basal media and cell growth factors, and then by themselves.
In the step 2), the culturing time is at least one week, and further, 7-14 d.
In step 2), the size of the gastric cancer 3D organoids is between hundred nanometers and millimeter in diameter; further, the diameter of the gastric cancer 3D organoid is 100 nm-10 mm.
In another aspect, the present application provides a gastric cancer 3D organoid obtained by culturing the gastric cancer 3D organoid according to the above-described method. Gastric cancer 3D organoids include at least one of the following 5 types of typing: irregular official cavities, regular official cavities, solid spheres, grape strings and mixtures. The gastric cancer 3D organoid provided by the application has the characteristics of easiness in freezing and thawing, suitability for long-term culture, morphological stability and gene stability, and good passage performance and subtype culture. The results provide a good technical basis for automatic mass production of the gastric cancer 3D organoid model in the future.
The application completes immunofluorescence identification test of gastric cancer organoids, expresses and summarizes various gastric cancer tumor markers through immunofluorescence staining method, and provides a visual and clear means for gastric cancer pathological morphology research. The application concludes five kinds of tumor organoids of irregular official cavity, regular official cavity, solid sphere, grape cluster and mixed type through experience of multiple organoid construction, the above forms comprise all kinds of tumor organoids at present, and the organoid typing is analyzed by combining operation records of organoid construction and is caused by the difference of operation methods of different batches. Therefore, the best operation method is found through the result, and the unified standard for constructing gastric cancer organoids is facilitated to be established.
In another aspect, the present application provides a method for culturing the gastric cancer 3D organoids described above or an application of the gastric cancer 3D organoids described above in preparing an in vitro drug sieve model of gastric cancer. The gastric cancer 3D organoids with different forms are suitable for different kinds of medicines and different test purposes, and the gastric cancer 3D organoid model and the medicine sensitivity degree are more than 70%.
In the aspect of drug sensitivity detection, the application completes the concentration gradient test of gastric cancer 3D organoids, the test of multiple sample vs single drugs and single sample vs multiple drugs. The concentration gradient test is to add inhibitors with different concentrations for organoid culture, and detect the growth condition of organoids by using high performance liquid chromatography, and find that the organoids die all 6 days after 20uM action, and design the concentration gradient of 3D organoids aiming at gastric cancer: 0uM,2uM,5uM,10uM,20uM,50uM; the test of multiple sample vs single drugs is to culture 5 types of gastric cancer 3D organoids and the same test drug respectively on the basis of gastric cancer 3D organoid typing analysis so as to obtain the conclusion that the reactivity difference of different types of organoids to the same drug is obvious, so that it can be inferred that detection of multiple sample vs single drugs is necessary before using the gastric cancer 3D organoids as an in-vitro drug screening model so as to select the optimal organoid typing aiming at the test drug; the single sample vs multi-drug test is to culture the same typed gastric cancer 3D organoid and different tested drugs to finally obtain the obvious sensitivity difference of the same organoid to different drugs, which indicates that the gastric cancer 3D organoid model can be well applied to in vitro drug screening and evaluation tests.
The present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.
Example 1
(1) Sample of
The gastric cancer specimen is sourced from the people's hospitals in Putuo district of Zhoushan, 10 cases of samples are taken, the flow is shown in figure 6, wherein the gastroscopy link is mainly used for guaranteeing screening of non-gastric cancer patients, and only gastric cancer patients are reserved as volunteers of the project. For the same patient's tissue, gastroscopy and blood sample collection are performed simultaneously.
Sample requirements:
surgical sample: the total sample amount is more than 0.5g, the size of about 3 soybean grains is about 3, and the tumor proportion is more than 50%;
puncturing a sample: the total length of the sample is more than 3cm, and the tumor proportion is more than 50%;
endoscopic biopsy tissue: more than 3 blocks are clamped, the total amount is more than or equal to 0.5g, and the tumor proportion is more than 50%;
* The whole sampling process needs to be paid attention to aseptic operation.
And (5) placing the gastric cancer sample in tissue preservation solution for cold chain transportation.
(2) Clinical specimen handing over and handling
To create a more complete Organoid Biobank (Organoid Biobank), the integrity of the Biobank information is guaranteed, the value of the Biobank is increased, and the specimens are recorded according to the specimen information delivery table as shown in table 1:
TABLE 1
The sample was received and the processing steps as in table 2 were taken:
TABLE 2
(3) Reagent selection of gastric cancer organoids
Organoid culture is one of the difficulties, and good results cannot be achieved by using only the existing culture medium. Therefore, the culture medium used in the project is prepared by purchasing basic culture medium and cell growth factors of various brands and then screening and proportioning the reagents by self. The "Homemade" words in the following tables are formulation reagents.
The specific formula of the Homemade culture medium is as follows: 90% (v/v) DMEM/F12 (1X) basal medium for formulation of 90% (v/v) basal medium, 10% (v/v) DPBS balanced salt solution (for washing cells and formulation of medium), N2 serum-free additive for formulation of 10% (v/v) medium supplement, B-27 (50X) serum-free additive, glutaMAX Supplement glutamine substitute, hydrocortisone, lipoprotein, zinc sulfate heptahydrate zinc sulfate heptahydrate, FGF10 cell growth factor, collagenase Type I/IV collagenase (for 3D spheronization of cells).
The specific formula of the Homemode tissue digestion solution is as follows: 4-8mg/mL Dispase II neutral protease (for cell separation), 10ng/mL ProstaglandinE2 prostaglandin, 4-8mg/mLTrypLE trypsin (for cell separation), 10ng/mL Gastrin Gastrin
The Homemode tissue preservation solution comprises the following specific formula: 90% (v/v) FBS fetal bovine serum, 9% (v/v) EDTA ethylenediamine tetraacetic acid, 8-16. Mu. Mol/L Y-27632 inhibitor, 100U/ml penicillin, 100U/ml gentamicin-amphotericin B mixed solution.
(4) Tumor cell isolation and culture
i, after the specimen is transported to a laboratory by a cold chain, discarding tissue preservation solution, and placing the specimen into a sterile 100mm culture dish;
ii washing the tissue with PBS buffer;
iii the tissue was cut into small pieces and transferred to a 50mL centrifuge tube containing Homemade tissue digests;
iv digestion for 1-2 hours at 37 ℃;
v preparing a 24-well culture plate;
vi, transferring the supernatant to a new 50mL centrifuge tube after digestion, and uniformly treating medical wastes after the experiment is finished;
adding 30mL of PBS (phosphate buffer solution) into the precipitate obtained in the previous step, centrifuging at 4 ℃ for 5min, and collecting cells;
viii discarding supernatant, adding PBS for resuspension, filtering with 100 μm cell screen, and collecting cells with 50ml centrifuge tube;
centrifuging at 300g for 5min at 4 ℃ to collect cells;
taking matrigel to resuspend cells and fully mixing to obtain cell suspension;
xi adding the cell suspension to a 24-well culture plate;
the xii puts the plates into a 37 ℃ incubator for incubation for 10-20min;
xiii plate was removed and 500 μl homemap medium was added;
placing xiv into an incubator for culturing, and observing the growth condition of organoids and the presence of pollution the next day.
(5) Frozen stock of stomach cancer organoid
i, observing the organoids cultured in the step (4) by a microscope, and freezing and preserving the organoids when the organoids grow to about 0.4 mm;
ii discarding the old medium and washing with PBS once;
iii adding Organoid Harvesting Solution and incubating for 30min;
iv according to Organoid Harvesting Solution: DMEM is 1: adding DMEM into the mixture for cleaning for 2 times according to the volume ratio of 10;
v centrifuging 300g,4 ℃ for 5min;
vi adding conventional cell cryopreservation liquid, and sub-packaging into cryopreservation tubes;
vii, placing the freezing tube into a freezing box pre-cooled at 4 ℃ and transferring the freezing tube into a liquid nitrogen tank the next day overnight at-80 ℃;
after viii one week the organoids were resuscitated and the recovery efficiency was determined.
The resuscitation process is as follows:
(1) 10ml were taken in a 15ml centrifuge tube.
(2) The frozen tumor organoid cells were taken out of the liquid nitrogen tank and rapidly thawed in a 37 ℃ water bath.
(3) In the water bath thawing process, the freezing tube is gently shaken to ensure complete thawing of the frozen stock solution within 1-2 minutes.
(4) The lysed organoid cells were quickly transferred to a 15ml sterile centrifuge tube, gently pipetted 3 times, centrifuged at 300g for 3 minutes, and the supernatant removed and the organoid cell pellet collected. Adding proper amount of gastric cancer organoid buffer A, re-suspending, transferring into a 1.5ml centrifuge tube, and centrifuging for 5min at 300 g.
(5) The matrigel was resuspended, 20 μl matrigel per well was spread in 24 control plates, and placed in an incubator for 10min with 500 μl gastric cancer organoid medium.
The resuscitation process is described in the literature: [1] corning Organoids for Disease modeling. Nov.11,2019 [2]Lee,J.et al.Nat Commun 11,4283 (2020) [3]SCIENCING.Types of Sea Sponges.2019 ] [4]Wilson,H.V.Development of sponges from dissociated tissue cells ] [5]Sato,T.et al.Nature 459,262-265 (2009) [6] corr co C et al am J Physiol Cell physiol.2020jul 1;319 (1) C151-C165.
(6) Quality control method
Class i organ morphology identification
The organoid morphology was observed with a 10-fold eyepiece (Gastric Cancer Organoids 10 x) and the results are shown in fig. 4.
Immunohistochemical staining (IHC) is performed as follows:
i, when the organoids in the step (4) grow to the size of 0.2mm, the organoids can be used for IHC identification;
ii taking out the 24-well plate, taking out a few of the 24-well plate, and sucking out the culture medium;
iii 500uL of freshly prepared 4% PFA was added and fixed for 30min;
iv washing off the fixing solution by PBS after the fixing is finished;
v transferring organoids to a tissue embedding cassette;
vi is put into a dehydrator for dehydration overnight: soaking 70% ethanol-95% ethanol-absolute ethanol-xylene-paraffin wax;
taking out the dehydrated sample in the next day, and putting the sample into an embedding machine to prepare a wax block;
viii slicing work can be performed after the wax block is solidified;
ix wax block, fixed to Leica microtome;
x continuous slices with slice thickness of 5um;
xi, placing the cut slices in a slice baking machine at 37 ℃ for more than 4 hours;
xii preparation of immunohistochemical staining;
xiii dewaxing and rehydration: 3 times of dimethylbenzene, namely 2 times of absolute ethyl alcohol, 2 times of 95% ethyl alcohol and 2 times of 70% ethyl alcohol, sequentially passing through an xiv cylinder for 5min for one time;
xv, placing the sodium citrate antigen retrieval liquid into a steamer to be preheated for 30min;
after the xvi dewaxing is finished, PBS is washed for 2 times, and the time is 5 min/time;
xvii, putting the flakes into a sodium citrate antigen retrieval liquid for 20min;
xviii is left at room temperature for 30min;
xix PBST wash three times; 5 min/time;
after xx 3% hydrogen peroxide is added, RT is carried out for 10min;
xxi PBST is washed three times for 5 min/time;
xxii 10% donkey serum RT incubation for 1h;
xxiii 1 incubation against RT for 2 hours or overnight at 4 ℃;
xxiv taking out the flakes, and washing with PBST three times for 5 min/time;
xxv incubation of 2 antibodies, RT 30min;
xxvi PBST washes three times, 5 min/time;
xxvii, developing under an optical microscope;
after the end of xxviii color development, ddH 2 O washing twice, hematoxylin counterstaining;
dehydrating and sealing after xxix;
relevant markers were recorded under an xxx microscope.
The reagent information used is as in table 4:
TABLE 4 Table 4
In the table above: HE is hematoxylin/eosin staining, belonging to pathology/morphology; CEA is carcinoembryonic antigen, a relatively common tumor marker in gastric cancer; p53 is also a marker closely related to gastric cancer occurrence, and p53 gene mutation is more common in gastric cancer patients; ck20 gastrointestinal epithelial markers; ki67 is a marker of cell proliferation; beta-catenin (i.e., beta-catenin) is an important molecule of wnt signaling pathway, associated with cell adhesion.
ii PI staining assay Activity (Using conventional methods)
1. Collecting small parts of organoids, centrifuging for 5min at 500-1000 r/min, and discarding culture solution.
2.3ml PBS was washed 1 time.
3. The PBS was removed by centrifugation, and the mixture was fixed with ice-chilled 70% ethanol at 4℃for 1-2 hours.
4. The fixative was removed by centrifugation and 3ml PBS was resuspended for 5min.
The filter is carried out for 1 time by a 5.400 mesh screen, the centrifugation is carried out for 5min at 500-1000 r/min, and PBS is discarded.
6. The mixture was dyed with 1ml of PI dye solution at 4℃for 30min in the dark.
7. Flow cytometer detection: the PI single staining method is based on the principle that fluorescence is excited by argon ions, so that the decrease of cell activity can lead to difficult staining, namely, scattered light is reduced, and the activity is detected and calculated more accurately by means of a flow cytometer instead of manual analysis.
iii detection of the rate of resurrection (calculated by conventional means using a cell counter and a cell counter plate)
1. Sample loading: samples were loaded onto the cell counter plate of a cell counter, commonly used by my company as a counting plate of the Nexcelom and CountStar brand.
2. Optical detection: the cytometer detects and counts organoid living cells optically.
3. Data analysis: the cytometer converts the detected data into a number of cells and calculates the ratio of living cells.
iv potential biological infection detection: HIV-1/PCR; HBV/PCR; HCV/PCR; TP/PCR; EBV/PCR; CMV/PCR; mycoplasma DNA/PCR; bacteria, fungi/PCR. And detecting by a third party agency.
As shown in FIG. 1, the success rate of culturing gastric cancer 3D organoids is 80%, the success rate of resuscitation is 100%, the microorganism pollution rate is 0%, and the overall construction success rate is more than 90%. Wherein 80% success rate means that 8 out of 10 samples were constructed successfully, and the overall construction rate was (80% + 100%)/2.
(7) Marker expression condition for gastric cancer organoid immunofluorescence identification
Known antibodies or antigen molecules are labeled with fluorescein by conventional immunofluorescence labeling techniques, and when reacting with the corresponding antigen or antibody, a certain amount of fluorescein is carried on the formed complex, and the antigen-antibody binding site which emits fluorescence can be seen under a fluorescence microscope, so that the antigen or antibody is detected.
The results are shown in FIG. 3.
(8) Typing and morphological analysis of gastric cancer 3D organoids
Until now, organoids were only cultivated in a laboratory environment, which is highly dependent on the skill of the relevant technician and results in a small number of organoids. Organoids are not suitable for high throughput drug testing. Furthermore, since they rely on culture skills, there is little standardization between different culture processes, which means that studies on different batches of organoids may not be directly compared. Thus, there is a need to produce a large number of organoids and to produce organoids with consistent morphology and function. This would allow for a wider use of organoids in drug testing and increase the comparability of test results.
According to the method, accumulated experience and test data of multiple organoid constructions are used for classifying the forms of the gastric cancer 3D organoids, the size, the form and the test details of the 3D organoids constructed each time are recorded, and factors which possibly influence the organoid forms in the culture process are analyzed. The results are shown in FIG. 4.
(9) Gastric cancer 3D organoid drug sensitivity test
In vitro drug susceptibility testing refers to testing the sensitivity of organisms or pathogens to a drug to provide relevant data for in vivo animal tests and clinical tests, and help new drug research and development institutions to more accurately screen out highly sensitive drug to be used as a therapeutic drug or to formulate a more reasonable combined drug regimen. The drug susceptibility test of gastric cancer 3D organoids mainly comprises the following aspects:
a) Concentration gradient: the morphological difference between the gastric cancer 3D organoid model and 2D cell culture is very large, and the design of concentration gradient for in vitro drug evaluation of the 2D cells is not suitable for carrying. Therefore, it is necessary to redesign the concentration gradient suitable for gastric cancer 3D organoids. Concentration gradient test organoid culture was performed by adding inhibitors at different concentrations, using high performance liquid chromatography to examine organoid growth, and found that organoids all died 6 days after 20uM action, and concentration gradients for gastric cancer 3D organoids were designed: 0uM,2uM,5uM,10uM,20uM,50uM.
b) Multiple sample vs single drug: based on the analysis of the stomach cancer 3D organoids, respectively culturing 5 stomach cancer 3D organoids with the same test agent to obtain a conclusion that the reactivity difference of different organoids to the same drug is obvious; moreover, the test has very close relation with the above-mentioned gastric cancer 3D organoid polymorphism analysis. The different gastric cancer organoids are of different properties, and it can be concluded from the test results that it is necessary to perform a test of a plurality of sample vs single agents to select the optimal organoid typing for the test agent before using the gastric cancer 3D organoids as an in vitro test agent evaluation model.
c) Single sample vs multidrug: and selecting different tested medicines to treat the same type of gastric cancer 3D organoids, verifying the sensitivity reaction gap of a single type of gastric cancer organoids to different medicines, thereby verifying the effectiveness of the medicines, and screening out the high-sensitivity medicines in the medicines. The gastric cancer 3D organoids of the same type are cultured with different test drugs, and the obvious sensibility difference of the same type organoids to different drugs is finally obtained, which indicates that the gastric cancer 3D organoid model can be well applied to in vitro drug screening and evaluation tests.
(10) Correlation analysis of gastric cancer 3D organoids and drug sensitivity
The gastric cancer 3D organoid drug sensitivity test result is verified by adopting a double-side test mode, the correlation between the gastric cancer 3D organoid model and the drug sensitivity degree is more than 70%, and the P value is less than 0.05, which is considered to have statistical significance.
The results are shown in FIG. 5.
Example 2
Experimental results and analysis
1. Culture and construction of gastric cancer 3D organoids
As shown in FIG. 1, the success rate of culturing gastric cancer 3D organoids is 80%, the success rate of resuscitation is 100%, the microorganism pollution rate is 0%, and the overall construction success rate is more than 90%.
As shown in figure 2, the cultured organoids have the characteristics of easy freeze thawing, suitability for long-term culture, morphological stability and gene stability, and good passage performance.
2. Marker expression for immunofluorescence identification
As shown in fig. 3: HE is hematoxylin/eosin staining, belonging to pathology/morphology; CEA is carcinoembryonic antigen, a more common tumor marker in gastric cancer; p53 is also a marker closely related to gastric cancer occurrence, and p53 gene mutation is more common in gastric cancer patients; ck20 gastrointestinal epithelial markers; ki67 is a marker of cell proliferation; beta-catenin (i.e., beta-catenin) is an important molecule of wnt signaling pathway, associated with cell adhesion.
Through multiple organoid build experiments, gastric cancer 3D organoids were typed as shown in fig. 4: 1. irregular cavity (A & F)
2. Regular official cavity (E) 3, solid sphere (B) 4, grape cluster (C) 5, and mixed type (D). The above 5 morphologies contained essentially all types of tumor organoids.
3. Drug sensitivity detection
As shown in fig. 5A, afatinib was used for the organoids and concentration gradients were set to design a concentration gradient suitable for gastric cancer 3D organoids: the organoids all died 6 days after 0um,2um,5um,10um,20um,50um,20 um.
As shown in fig. 5B, the difference in responsiveness of the different typed organoids to the same drug was evident. When using a tumor 3D organoid as an in vitro drug screening model, it is necessary to first perform a test of a plurality of sample vs. single drugs to select the best organoid typing from among those that are appropriate for the given test drug.
As shown in FIG. 5C, the sensitivity difference of the same organoid type to different drugs is obvious, and all 5 types of gastric cancer 3D organoids can be used as an excellent in vitro medicine sieve model.
The drug sensitivity test adopts a double-side test mode to verify the test result, and achieves the effects: correlation of gastric cancer 3D organoid model with drug sensitivity is greater than 70% and P values less than 0.05 would be considered statistically significant.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the application. Modifications and variations may be made to the above-described embodiments by those of ordinary skill in the art without departing from the spirit and scope of the present application. Accordingly, it is intended that all equivalent modifications and variations which can be accomplished by persons skilled in the art without departing from the spirit and technical spirit of the present disclosure shall be covered by the claims of this application.
Claims (10)
1. A method for culturing a gastric cancer 3D organoid, comprising the steps of:
1) Digesting gastric cancer tissues with cell digestive juice, centrifuging, precipitating and filtering, and collecting cells;
2) Mixing the cells in the step 1) with matrigel, placing the mixture on a culture plate for incubation, and then adding a cell culture medium for culture to obtain a gastric cancer 3D organoid;
the components of the cell culture medium include: DMEM/F12 basal medium and medium supplements; the media supplement includes a combination of one or more of an N2 serum-free additive, a B-27 serum-free additive, a glutamine substitute, hydrocortisone, a lipoprotein, zinc sulfate heptahydrate, FGF10, and collagenase.
2. The method for culturing a gastric cancer 3D organoid according to claim 1,
in the step 1), the gastric cancer tissue is obtained by fresh sampling and then cold chain transportation in tissue preservation liquid; preferably, the tissue preservation solution comprises the following components: 90-100% (v/v) of fetal bovine serum, 9-15% (v/v) of ethylenediamine tetraacetic acid, 8-16 mu mol/L Y-27632 inhibitor, 100-150U/ml of green streptomycin and 100-150U/ml of gentamicin-amphotericin B mixed solution.
3. The method for culturing a gastric cancer 3D organoid according to claim 1,
in step 1), the components of the cell digestive juice comprise: 4-8mg/mL of Dispase II neutral protease, 10ng/mL of prostaglandin, 4-8mg/mL of TrypLE trypsin and 10ng/mL of gastrin.
4. The method for culturing a gastric cancer 3D organoid according to claim 1, wherein in step 1), the temperature of digestion is 37 ℃; the digestion time is 1-2 h.
5. The method for culturing a gastric cancer 3D organoid according to claim 1, wherein in step 1), the step of post-digestion further comprises an intermediate treatment step of: the digested supernatant was transferred and the pellet was resuspended in PBS.
6. The method for culturing a gastric cancer 3D organoid according to claim 1, wherein in step 1), the filtration comprises filtration with a cell sieve, further wherein the cell sieve has a particle size of 50 to 200 μm;
and/or, in step 1), a centrifugation step is further included after filtration, and the cell pellet is collected for mixing with the matrigel in step 2).
7. The method for culturing a gastric cancer 3D organoid according to claim 1, wherein in step 2), the culture plate is a low adsorption plate; further, the low adsorption plate is a 24-hole plate;
and/or, in step 2), the temperature of the incubation is 37 ℃; the incubation time is 10-20min;
and/or, in step 2), the volume ratio of the DMEM/F12 basal medium is 90% (v/v) based on the total volume of the cell culture medium;
and/or, in step 2), the volume of the medium supplement is 10% (v/v) based on the total volume of the cell culture medium;
and/or, in the step 2), the culture time is 7-14 d;
and/or in the step 2), the size of the gastric cancer 3D organoid is 100 nm-10 mm in diameter.
8. A gastric cancer 3D organoid obtained by culturing the gastric cancer 3D organoid according to any one of claims 1 to 7.
9. The gastric cancer 3D organoid of claim 8, wherein the gastric cancer 3D organoid comprises at least one of the following 5 types: irregular official cavities, regular official cavities, solid spheres, grape strings and mixtures.
10. The method of culturing gastric cancer 3D organoids according to any one of claims 1 to 7 or the use of gastric cancer 3D organoids according to any one of claims 8 to 9 for the preparation of an in vitro drug sieve model of gastric cancer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311032985.1A CN117247903A (en) | 2023-08-16 | 2023-08-16 | Stomach cancer 3D organoid and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311032985.1A CN117247903A (en) | 2023-08-16 | 2023-08-16 | Stomach cancer 3D organoid and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117247903A true CN117247903A (en) | 2023-12-19 |
Family
ID=89125500
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311032985.1A Pending CN117247903A (en) | 2023-08-16 | 2023-08-16 | Stomach cancer 3D organoid and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117247903A (en) |
-
2023
- 2023-08-16 CN CN202311032985.1A patent/CN117247903A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2021208130A1 (en) | Culture medium for esophageal squamous cell carcinoma epithelial cells, culture method, and application thereof | |
| US12038432B2 (en) | Organoids related to immunotherapy and methods of preparing and using the same | |
| US20240228921A1 (en) | Method and device for detecting circulating tumor cell | |
| WO2021088119A1 (en) | Primary breast epithelial cell culture medium, culture method, and use thereof | |
| US20110195413A1 (en) | Integrated Method for Enriching and Detecting Rare Cells from Biological Body Fluid Sample | |
| CN109563486A (en) | For making the diagnostic method of the specific Treatment decsion of patient in cancer is nursed | |
| US20080008999A1 (en) | Process for detecting the existence of mesenchymal chrondrosarcoma | |
| Zhang et al. | A pre‑clinical model combining cryopreservation technique with precision‑cut slice culture method to assess the in vitro drug response of hepatocellular carcinoma | |
| CN113755441A (en) | Culture method of lung cancer micro-tumor cell model | |
| US8192949B2 (en) | Microaggregates including endothelial cells | |
| CN117247903A (en) | Stomach cancer 3D organoid and application thereof | |
| CN114958753B (en) | Culture medium, culture method and identification method of tongue cancer organoids | |
| US20040175707A1 (en) | Methods for modeling infectious disease and chemosensitivity in cultured cells and tissues | |
| CN115820560B (en) | Construction method and application of recurrent respiratory papilloma disease organoid | |
| WO2019002456A1 (en) | Methods for identifying agents which induce (re) differentiation in un- or dedifferentiated solid tumor cells | |
| RU2819362C1 (en) | Culture medium for primary epithelial cells of mammary gland, method of cultivation and use thereof | |
| CN114891735B (en) | Mammary gland epithelial organoid model construction method | |
| CN113969262B (en) | Culture medium for lung cancer epithelial cells, culture method and application thereof | |
| CN114736866A (en) | Culture medium and culture method for bile duct cancer organoid | |
| CN114164250A (en) | Individual drug sensitivity detection method for tumor tissue homogenate serum-replacement micro tissue block | |
| CN117987367A (en) | Preparation method and application of pancreatic cancer liver metastasis source organoid | |
| CN114634964A (en) | Method for screening breast cancer targeted drugs | |
| CN118441008A (en) | Drug sensitivity detection method for bone tumor organoids | |
| CN116478922A (en) | Preparation method of cord blood stem cells and stem cell bank prepared by preparation method | |
| CN116036070A (en) | Application of 9' -salvianolic acid B monomethyl ester in preparation of medicines for treating breast cancer tumor cell proliferation, migration and invasion |
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 |