CN114317439B - Method for culturing tumor organoids - Google Patents
Method for culturing tumor organoids Download PDFInfo
- Publication number
- CN114317439B CN114317439B CN202111590436.7A CN202111590436A CN114317439B CN 114317439 B CN114317439 B CN 114317439B CN 202111590436 A CN202111590436 A CN 202111590436A CN 114317439 B CN114317439 B CN 114317439B
- Authority
- CN
- China
- Prior art keywords
- hydrogel
- organoid
- cancer
- tumor
- culturing
- 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 68
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 59
- 238000012258 culturing Methods 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 22
- 239000000017 hydrogel Substances 0.000 claims abstract description 75
- 201000011510 cancer Diseases 0.000 claims abstract description 22
- 241001494479 Pecora Species 0.000 claims abstract description 21
- 210000001519 tissue Anatomy 0.000 claims abstract description 18
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 17
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 16
- 210000000981 epithelium Anatomy 0.000 claims abstract description 13
- 239000011159 matrix material Substances 0.000 claims abstract description 13
- 239000000701 coagulant Substances 0.000 claims abstract description 9
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 27
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 27
- 239000001110 calcium chloride Substances 0.000 claims description 26
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 26
- 210000004027 cell Anatomy 0.000 claims description 17
- 238000004113 cell culture Methods 0.000 claims description 17
- 210000004881 tumor cell Anatomy 0.000 claims description 14
- 238000000338 in vitro Methods 0.000 claims description 9
- 230000036210 malignancy Effects 0.000 claims description 9
- 208000005243 Chondrosarcoma Diseases 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 206010005003 Bladder cancer Diseases 0.000 claims description 5
- 206010006187 Breast cancer Diseases 0.000 claims description 5
- 208000026310 Breast neoplasm Diseases 0.000 claims description 5
- 206010009944 Colon cancer Diseases 0.000 claims description 5
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 5
- 208000008839 Kidney Neoplasms Diseases 0.000 claims description 5
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 5
- 206010033128 Ovarian cancer Diseases 0.000 claims description 5
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 5
- 206010038389 Renal cancer Diseases 0.000 claims description 5
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 5
- 206010017758 gastric cancer Diseases 0.000 claims description 5
- 201000010982 kidney cancer Diseases 0.000 claims description 5
- 201000007270 liver cancer Diseases 0.000 claims description 5
- 208000014018 liver neoplasm Diseases 0.000 claims description 5
- 201000005202 lung cancer Diseases 0.000 claims description 5
- 208000020816 lung neoplasm Diseases 0.000 claims description 5
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 5
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 5
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 4
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 4
- 102000004142 Trypsin Human genes 0.000 claims description 4
- 108090000631 Trypsin Proteins 0.000 claims description 4
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 4
- 230000001613 neoplastic effect Effects 0.000 claims description 4
- 201000002528 pancreatic cancer Diseases 0.000 claims description 4
- 201000011549 stomach cancer Diseases 0.000 claims description 4
- 239000012588 trypsin Substances 0.000 claims description 4
- 208000035269 cancer or benign tumor Diseases 0.000 claims 2
- 241000124008 Mammalia Species 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 4
- 238000001879 gelation Methods 0.000 abstract description 2
- 230000035699 permeability Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 33
- 210000002381 plasma Anatomy 0.000 description 27
- 235000011148 calcium chloride Nutrition 0.000 description 20
- 108010082117 matrigel Proteins 0.000 description 15
- 235000018102 proteins Nutrition 0.000 description 14
- 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 description 14
- 229960005322 streptomycin Drugs 0.000 description 9
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 7
- 101800003838 Epidermal growth factor Proteins 0.000 description 7
- 229940116977 epidermal growth factor Drugs 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 7
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 description 6
- 150000003839 salts Chemical class 0.000 description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- JWDFQMWEFLOOED-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(pyridin-2-yldisulfanyl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSC1=CC=CC=N1 JWDFQMWEFLOOED-UHFFFAOYSA-N 0.000 description 4
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 4
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 4
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 4
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 4
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 4
- 229930182555 Penicillin Natural products 0.000 description 4
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 4
- 229960004308 acetylcysteine Drugs 0.000 description 4
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 4
- 229960003942 amphotericin b Drugs 0.000 description 4
- 239000003636 conditioned culture medium Substances 0.000 description 4
- 229940049954 penicillin Drugs 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- TVZRAEYQIKYCPH-UHFFFAOYSA-N 3-(trimethylsilyl)propane-1-sulfonic acid Chemical compound C[Si](C)(C)CCCS(O)(=O)=O TVZRAEYQIKYCPH-UHFFFAOYSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 3
- ZKQDCIXGCQPQNV-UHFFFAOYSA-N Calcium hypochlorite Chemical compound [Ca+2].Cl[O-].Cl[O-] ZKQDCIXGCQPQNV-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 3
- 206010053567 Coagulopathies Diseases 0.000 description 3
- 102000004864 Fibroblast growth factor 10 Human genes 0.000 description 3
- 108090001047 Fibroblast growth factor 10 Proteins 0.000 description 3
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 description 3
- 101000652736 Homo sapiens Transgelin Proteins 0.000 description 3
- 102100031013 Transgelin Human genes 0.000 description 3
- NKWPZUCBCARRDP-UHFFFAOYSA-L calcium bicarbonate Chemical compound [Ca+2].OC([O-])=O.OC([O-])=O NKWPZUCBCARRDP-UHFFFAOYSA-L 0.000 description 3
- 229910000020 calcium bicarbonate Inorganic materials 0.000 description 3
- YYRMJZQKEFZXMX-UHFFFAOYSA-L calcium bis(dihydrogenphosphate) Chemical compound [Ca+2].OP(O)([O-])=O.OP(O)([O-])=O YYRMJZQKEFZXMX-UHFFFAOYSA-L 0.000 description 3
- 229960002713 calcium chloride Drugs 0.000 description 3
- 239000004227 calcium gluconate Substances 0.000 description 3
- 229960004494 calcium gluconate Drugs 0.000 description 3
- 235000013927 calcium gluconate Nutrition 0.000 description 3
- 229910001424 calcium ion Inorganic materials 0.000 description 3
- 229910000389 calcium phosphate Inorganic materials 0.000 description 3
- NEEHYRZPVYRGPP-UHFFFAOYSA-L calcium;2,3,4,5,6-pentahydroxyhexanoate Chemical compound [Ca+2].OCC(O)C(O)C(O)C(O)C([O-])=O.OCC(O)C(O)C(O)C(O)C([O-])=O NEEHYRZPVYRGPP-UHFFFAOYSA-L 0.000 description 3
- JXRVKYBCWUJJBP-UHFFFAOYSA-L calcium;hydrogen sulfate Chemical compound [Ca+2].OS([O-])(=O)=O.OS([O-])(=O)=O JXRVKYBCWUJJBP-UHFFFAOYSA-L 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000035602 clotting Effects 0.000 description 3
- 239000012531 culture fluid Substances 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 235000019691 monocalcium phosphate Nutrition 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 description 3
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 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
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 102000016911 Deoxyribonucleases Human genes 0.000 description 2
- 108010053770 Deoxyribonucleases Proteins 0.000 description 2
- 102400000921 Gastrin Human genes 0.000 description 2
- 108010052343 Gastrins Proteins 0.000 description 2
- 229930182566 Gentamicin Natural products 0.000 description 2
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 239000012981 Hank's balanced salt solution Substances 0.000 description 2
- 239000012574 advanced DMEM Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- AOXOCDRNSPFDPE-UKEONUMOSA-N chembl413654 Chemical compound C([C@H](C(=O)NCC(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@H](CCSC)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](C)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]1N(CCC1)C(=O)CNC(=O)[C@@H](N)CCC(O)=O)C1=CC=C(O)C=C1 AOXOCDRNSPFDPE-UKEONUMOSA-N 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- NZNMSOFKMUBTKW-UHFFFAOYSA-N cyclohexanecarboxylic acid Chemical compound OC(=O)C1CCCCC1 NZNMSOFKMUBTKW-UHFFFAOYSA-N 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- ASXBYYWOLISCLQ-HZYVHMACSA-N dihydrostreptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](CO)(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 ASXBYYWOLISCLQ-HZYVHMACSA-N 0.000 description 2
- 108010007093 dispase Proteins 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 229960002518 gentamicin Drugs 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000003211 malignant effect Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 102000045246 noggin Human genes 0.000 description 2
- 108700007229 noggin Proteins 0.000 description 2
- 239000003805 procoagulant Substances 0.000 description 2
- STWNGMSGPBZFMX-UHFFFAOYSA-N pyridine-3-carboxamide Chemical compound NC(=O)C1=CC=CN=C1.NC(=O)C1=CC=CN=C1 STWNGMSGPBZFMX-UHFFFAOYSA-N 0.000 description 2
- LLXVXPPXELIDGQ-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(2,5-dioxopyrrol-1-yl)benzoate Chemical compound C=1C=CC(N2C(C=CC2=O)=O)=CC=1C(=O)ON1C(=O)CCC1=O LLXVXPPXELIDGQ-UHFFFAOYSA-N 0.000 description 1
- FSILHPZFNRDTOR-RZVRUWJTSA-N (2R)-2-acetamido-3-sulfanylpropanoic acid Chemical compound CC(=O)N[C@@H](CS)C(O)=O.CC(=O)N[C@@H](CS)C(O)=O FSILHPZFNRDTOR-RZVRUWJTSA-N 0.000 description 1
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108091016585 CD44 antigen Proteins 0.000 description 1
- 208000010667 Carcinoma of liver and intrahepatic biliary tract Diseases 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 108010042086 Collagen Type IV Proteins 0.000 description 1
- 102000004266 Collagen Type IV Human genes 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 102000018386 EGF Family of Proteins Human genes 0.000 description 1
- 108010066486 EGF Family of Proteins Proteins 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 1
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 1
- 102000003972 Fibroblast growth factor 7 Human genes 0.000 description 1
- 108090000385 Fibroblast growth factor 7 Proteins 0.000 description 1
- 102000006395 Globulins Human genes 0.000 description 1
- 108010044091 Globulins Proteins 0.000 description 1
- 102000008055 Heparan Sulfate Proteoglycans Human genes 0.000 description 1
- 229920002971 Heparan sulfate Polymers 0.000 description 1
- 206010073069 Hepatic cancer Diseases 0.000 description 1
- 101000851176 Homo sapiens Pro-epidermal growth factor Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 108010085895 Laminin Proteins 0.000 description 1
- 102000007547 Laminin Human genes 0.000 description 1
- 102100037369 Nidogen-1 Human genes 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 108090000054 Syndecan-2 Proteins 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000003470 adrenal cortex hormone Substances 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- VZFUCHSFHOYXIS-UHFFFAOYSA-N cycloheptane carboxylic acid Natural products OC(=O)C1CCCCCC1 VZFUCHSFHOYXIS-UHFFFAOYSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 208000021045 exocrine pancreatic carcinoma Diseases 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 229940098448 fibroblast growth factor 7 Drugs 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 208000010749 gastric carcinoma Diseases 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 108010084091 heregulin beta1 Proteins 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 201000002250 liver carcinoma Diseases 0.000 description 1
- 239000002075 main ingredient Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- GVUGOAYIVIDWIO-UFWWTJHBSA-N nepidermin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C(C)C)C(C)C)C1=CC=C(O)C=C1 GVUGOAYIVIDWIO-UFWWTJHBSA-N 0.000 description 1
- 108010008217 nidogen Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- YNCMLFHHXWETLD-UHFFFAOYSA-N pyocyanin Chemical compound CN1C2=CC=CC=C2N=C2C1=CC=CC2=O YNCMLFHHXWETLD-UHFFFAOYSA-N 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 201000000498 stomach carcinoma Diseases 0.000 description 1
- TYFQFVWCELRYAO-UHFFFAOYSA-L suberate(2-) Chemical compound [O-]C(=O)CCCCCCC([O-])=O TYFQFVWCELRYAO-UHFFFAOYSA-L 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Medicinal Preparation (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for culturing tumor organoids. The invention provides a hydrogel for culturing tumor organoids, which is formed by taking isolated sheep plasma or other mammal plasma or human plasma as a matrix and adding a coagulant and a protein cross-linking agent. The hydrogel has the advantages of low cost, simple preparation, loose structure, moderate strength, good water permeability, complete gelation in a short time, and good cell biocompatibility, and is suitable for culturing malignant tumors and organoids from epithelial tissues and mesenchymal tissues.
Description
Technical Field
The invention relates to the biomedical field, in particular to a method for culturing tumor organoids.
Background
The tumor organoid is a three-dimensional cell complex with a certain space structure formed by in vitro culture of primary tumor cells, has similar structural characteristics and functional characteristics as those of the source tumor tissue, and can be stably amplified in an in vitro three-dimensional culture system. The tumor organoid provides an ideal platform for preclinical drug screening, drug safety evaluation, personalized treatment and basic research of tumors because the phenotype and genotype of the tumor tissue are maintained.
The current method for culturing tumor organoids is to use Matrigel as a scaffold for in vitro three-dimensional culture. Matrigel is a soluble extracellular matrix extracted from mouse sarcoma, and contains laminin, type IV collagen, entactin protein, heparan sulfate proteoglycan, etc. as main ingredients. Matrigel, although achieving successful culture of a variety of tumor organoids, is unsuitable for use as a matrix gel for large-scale culture of tumor organoids due to its high cost; secondly, matrigel is gelled at 22-37 ℃ and needs to be operated in a low-temperature environment, so that improper Matrigel gelation is easy to cause inoculation failure; in addition, after Matrigel and tumor cells are mixed, the Matrigel can be completely gelled after being stood for 30 minutes in a cell culture box at 37 ℃, and a large number of tumor cells are settled at the bottom of a culture dish due to the action of gravity in the process, so that the three-dimensional support effect of Matrigel is lost; finally, matrigel is currently only suitable for culturing epithelial tissue derived malignant organoids, which is not reported for culturing mesenchymal tissue derived malignant organoids.
Disclosure of Invention
In order to overcome the defects of Matrigel, the invention uses sheep plasma as a matrix material to prepare the tumor organoid three-dimensional culture hydrogel.
In a first aspect, the invention claims a hydrogel for culturing a tumor organoid.
The hydrogel for culturing tumor organoids is prepared by taking isolated sheep plasma or other mammal plasma or human plasma as a matrix and adding a coagulant and a protein crosslinking agent.
Wherein the coagulant may be any one or more of inorganic salts or/and organic salts containing calcium ions.
Further, the accelerator may be selected from any one or more of the following: calcium chloride, calcium gluconate, calcium biphosphate, calcium nitrate, calcium bicarbonate, calcium bisulfate, calcium hypochlorite, and the like.
In a specific embodiment of the invention, the setting accelerator is calcium chloride.
Wherein, the protein cross-linking agent can be any one or any plurality of existing protein cross-linking agents. Such as may be selected from any one or more of the following: glutaraldehyde, EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride), SMCC (N-hydroxysuccinimide ester of 4- (N-maleimidomethyl) cyclohexane carboxylic acid), DSS (bissuccinimidyl suberate), DST (bissuccinimidyl tartrate), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), SPDP (succinimidyl 3- (2-pyridyldithio) -propionate), and the like.
In a specific embodiment of the invention, the protein cross-linking agent is glutaraldehyde.
In the specific embodiment of the invention, the hydrogel is formed by taking isolated sheep plasma as a matrix and adding calcium chloride and glutaraldehyde; wherein the concentration of calcium chloride in the hydrogel is 3-6mmol/L (such as 4.5 mmol/L), and the volume percentage of glutaraldehyde in the hydrogel is 0.1-0.5% (such as 0.25%).
Wherein said calcium chloride and said glutaraldehyde are added in the form of a 100 x stock solution.
In a second aspect, the invention claims a method of preparing a hydrogel for culturing a tumor organoid.
The method for preparing the hydrogel for culturing the tumor organoids, which is claimed by the invention, can comprise the following steps: the hydrogel is formed by taking isolated sheep plasma or other mammal plasma or human plasma as a matrix and adding a coagulant and a protein cross-linking agent.
Wherein the coagulant may be any one or more of inorganic salts or/and organic salts containing calcium ions.
Further, the accelerator may be selected from any one or more of the following: calcium chloride, calcium gluconate, calcium biphosphate, calcium nitrate, calcium bicarbonate, calcium bisulfate, calcium hypochlorite, and the like.
In a specific embodiment of the invention, the setting accelerator is calcium chloride.
Wherein, the protein cross-linking agent can be any one or any plurality of existing protein cross-linking agents. Such as may be selected from any one or more of the following: glutaraldehyde, EDC, SMCC, DSS, DST, MBS, SPDP, etc.
In a specific embodiment of the invention, the protein cross-linking agent is glutaraldehyde.
In a specific embodiment of the invention, the method comprises the steps of: taking isolated sheep plasma as a matrix, and adding calcium chloride and glutaraldehyde to form the hydrogel; wherein the concentration of calcium chloride in the hydrogel is 3-6mmol/L (such as 4.5 mmol/L), and the volume percentage of glutaraldehyde in the hydrogel is 0.1-0.5% (such as 0.25%).
Wherein said calcium chloride and said glutaraldehyde are added in the form of a 100 x stock solution.
In a third aspect, the invention claims a method of culturing a neoplastic organoid.
The method for culturing the tumor organoids, which is a method for culturing the tumor organoids in vitro, can comprise the following steps:
(A1) Mixing tumor cells to be cultured with matrix to give final cell concentration of 5×10 3 /mL; the matrix is in vitro sheep plasma or other mammal plasma or human plasma;
(A2) Adding a coagulant and a protein cross-linking agent into the (A1), and mixing to obtain a hydrogel solution containing tumor cells;
(A3) Adding the hydrogel solution containing tumor cells to the bottom of a cell culture container, and standing in a cell culture box at 37 ℃ until the hydrogel solution is coagulated;
(A4) Adding a tumor organoid culture solution to the hydrogel of the step (A3), and culturing to obtain the tumor organoid.
Wherein the coagulant may be any one or more of inorganic salts or/and organic salts containing calcium ions.
Further, the accelerator may be selected from any one or more of the following: calcium chloride, calcium gluconate, calcium biphosphate, calcium nitrate, calcium bicarbonate, calcium bisulfate, calcium hypochlorite, and the like.
In a specific embodiment of the invention, the setting accelerator is calcium chloride. The concentration of the calcium chloride in the hydrogel solution is 3-6mmol/L (e.g., 4.5 mmol/L).
Wherein, the protein cross-linking agent can be any one or any plurality of existing protein cross-linking agents. Such as may be selected from any one or more of the following: glutaraldehyde, EDC, SMCC, DSS, DST, MBS, SPDP, etc.
In a specific embodiment of the invention, the protein cross-linking agent is glutaraldehyde. The glutaraldehyde is present in the hydrogel solution in a volume percentage of 0.1-0.5% (e.g., 0.25%).
Wherein said calcium chloride and said glutaraldehyde are added in the form of a 100 x stock solution.
In the step (A3), the hydrogel solution containing tumor cells is added to the bottom of a cell culture container, and the cell culture container is kept stand in a flat state at 37 ℃ for 5-10min (e.g. 8 min).
In the step (A4), the volume of the culture solution for the tumor organoids is preferably such that the culture solution completely covers the surface of the hydrogel.
In step (A4), the culture was conducted at 37℃with 5% CO 2 Culturing under the condition, and replacing fresh tumor organoid culture solution every 3-4 days.
The method may further comprise the step of dissolving the hydrogel after absorbing the culture solution on the hydrogel after adding 0.125-1.25g/L of trypsin solution at 37℃for 30 minutes after the step (A4).
In a fourth aspect, the invention claims also any one of the following products or applications:
p1, a kit for culturing a tumor organoid, consisting of a hydrogel as described in the first aspect above and a tumor organoid culture fluid.
Wherein, the tumor organoid culture solution can be the existing tumor organoid culture solution commonly used in the field.
Use of a hydrogel as described in the first aspect hereinbefore in the culture (in vitro culture) of a tumour organoid;
the use of said packages in P3, P1 for culturing (in vitro culture) tumor organoids;
use of a hydrogel as described in the first aspect hereinbefore in the culture (in vitro culture) of normal tissue organoids.
In the above aspects, the tumor organoid may be an organoid of an epithelial tissue-derived malignancy or an organoid of a mesenchymal tissue-derived malignancy.
Further, the epithelial tissue-derived malignancy may be selected from any one of the following: lung cancer, breast cancer, stomach cancer, colorectal cancer, bladder cancer, kidney cancer, liver cancer, ovarian cancer, and pancreatic cancer; the mesenchymal tissue-derived malignancy may be selected from any one of: chondrosarcoma and stromal tumor.
The hydrogel of the invention has the following characteristics:
1. sheep blood plasma is cheap and easy to obtain, and the culture cost is greatly reduced.
2. The combination of procoagulant and crosslinking reagent in low concentration results in the formation of crosslinked protein of dominant protein (albumin and globulin) in plasma and the synergistic formation of porous network with fibrin produced by procoagulant reaction. The prepared hydrogel has moderate strength and good water permeability, and is beneficial to infiltration of cell culture solution.
3. The operation is carried out at normal temperature, the tumor cells and the hydrogel are completely gelled after being mixed and placed for 5-10 minutes at 37 ℃, and the tumor cells are uniformly distributed on each layer of the hydrogel and are in effective contact with the reticular structure.
4. The cell biocompatibility is better. The inherent amino acids, vitamins, inorganic substances, lipid substances, nucleic acid derivatives and the like in the blood plasma are substances necessary for cell growth, and can provide various hormones (such as insulin, adrenocortical hormone, steroid hormone and the like) and growth factors (such as fibroblast growth factors, epidermal growth factors, platelet growth factors and the like) to promote the growth of cells together.
5. Is suitable for culturing malignant tumor organoids of epithelial tissue source and mesenchymal tissue source.
Drawings
FIG. 1 shows the general morphology of hydrogels.
Fig. 2 is a hydrogel microstructure.
FIG. 3 shows the results of organoid culture of an epithelial tissue derived malignancy.
FIG. 4 shows results of a mesenchymal tissue derived malignant tumor organoid culture.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The results shown in the following examples are all results verified by repeated experiments.
Example 1 optimization of hydrogel preparation method
Different concentrations of calcium chloride and glutaraldehyde were added to sheep plasma (zhengzhou nine-Dragon biol Co., ltd.). Two-dimensionally culturing HepG2 cells (ATCC, USA) of human liver cancer to logarithmic phase, and then culturing with trypsin in an amount of 0.25% (% expressed as g/100 mL)(Sigma) digestion, obtaining single cell suspensions at a final concentration of 5X 10 3 mixing/mL with above groups (sheep plasma added with calcium chloride and glutaraldehyde with different concentrations), spreading on the bottom of cell culture plate hole, and observing clotting time of each group. A DMEM broth (Thermo scientific) containing 10% (v/v) fetal bovine serum (Thermo Scientific) and 1% (m/v) cyan/streptomycin was added over the gel with clotting time of 30 minutes or less. At 37℃with 5% CO 2 After 3 days of culture under the condition, the cell states of each group were observed. As a result, it was found that the plasma can form a hydrogel faster after 3-6mmol/L of calcium chloride and 0.1-0.5% (v/v) glutaraldehyde are added to sheep plasma, the clotting time is 5-10min, and each group of cells is in good state, and HepG2 cells can generate spherical three-dimensional structures in the gel. In which sheep plasma is preferably supplemented with 4.5mmol/L calcium chloride and 0.25% (v/v) glutaraldehyde as the optimal combination.
Example 2 preparation of hydrogel
1. Preparing 100 Xcalcium chloride storage liquid
Anhydrous calcium chloride 3.3294-6.6588 g is weighed and dissolved in 100ml deionized water, the concentration of the calcium chloride is 300-600mmol/L, and the solution is filtered and sterilized by a filter membrane with the pore diameter of 0.22 mu m.
2. Preparing 100 Xglutaraldehyde storage liquid
10-50 mL glutaraldehyde is measured, deionized water is added to 100mL, the glutaraldehyde concentration is 10-50% (v/v), and filtration sterilization is performed by using a filter membrane with a pore size of 0.22 μm.
3. Sheep plasma: zhengzhou Jiulong biological products Inc.
4. Preparation of three-dimensional cell culture scaffolds
980. Mu.L of sheep plasma was thoroughly mixed with 10. Mu.L of 100 Xcalcium chloride stock (450 mmol/L) and 10. Mu.L of 100 Xglutaraldehyde stock (25%,% expressed as volume%) and added to the wells of the cell culture plate, and left to stand flat at 37℃for 8min until the plasma coagulated into a jelly.
Fig. 1 and 2 show the appearance and structure of the prepared hydrogels under an optical microscope, respectively. The hydrogel is visible under the mirror to form a network structure inside.
Example 3 cultivation of tumor organoids
This example examined the organoid culture effects of various malignant tumors of epithelial origin (lung cancer, breast cancer, stomach cancer, colorectal cancer, bladder cancer, kidney cancer, liver cancer, ovarian cancer and pancreatic cancer) and malignant tumors of mesenchymal origin (chondrosarcoma and interstitial tumor).
1. Human tumor tissue samples were washed three times with pre-chilled Hank's balanced salt solution (containing 200U/mL penicillin, 200mg/mL streptomycin, and 0.5mg/mL amphotericin B, all available from Sigma).
2. Sterile scissors cut the tumor tissue under test to about 0.5mm 3 Is a small block of (a). The minced tissue was transferred to a 15mL centrifuge tube and about 10mL of pre-chilled Hank's balanced salt solution was added. The mixture was aspirated up and down with a 10mL pipette several times.
3. The tube was allowed to stand for 2 to 3 minutes to settle the tissue mass to the bottom of the tube, and about 7.5mL of supernatant was aspirated off. The washing step was repeated once.
4. After centrifugation at 200g for 5 minutes, about 10mL of pre-warmed tissue digest (formulation: DMEM/F12 broth containing 0.001% DNase (Sigma), 1mg/mL collagenase/dispase (Roche), 200U/mL penicillin, 200mg/mL streptomycin, and 0.5mg/mL amphotericin B) was added to a 100mm dish after all supernatant was aspirated. Digestion is carried out at 37℃for 45-60 minutes. During which the oscillation occurs several times.
5. The suspension was repeatedly aspirated several times with a 10mL pipette and filtered through a cell filter with a 70 μm pore size. The filtrate was centrifuged at 200g for 5 minutes.
6. Cell viability was assessed by 0.4% trypan blue staining, with viable cell ratios above 95%. Cell numbers were counted with a cytometer.
7. At normal temperature, adding sheep plasma with a certain volume according to the cell number to make the final cell concentration 5×10 3 And (3) adding a certain volume of 100 Xcalcium chloride storage solution and 100 Xglutaraldehyde storage solution into the solution to prepare the three-dimensional culture hydrogel solution, wherein the final concentration of glutaraldehyde is 0.25% (v/v), and the final concentration of calcium chloride is 4.5mmol/L. After fully and evenly mixing, the three-dimensional culture hydrogel solution is flatly paved at the bottom of a cell culture plate hole, and a cell culture box is flatly paved and kept stand for 8min at 37 ℃. The volume of the three-dimensional culture hydrogel solution added to each well of the cell culture plates of different specifications is shown in Table 1. Meanwhile, matrigel hydrogel is usedBD BioCoat) was used as a control to culture cells at the same final concentration for the scaffolds.
TABLE 1 volume of hydrogel solution for three-dimensional culture added per well of cell culture plates of different formats
8. After the hydrogel is solidified, adding corresponding tumor organoid culture solution above, and adding the culture solution to the volume so that the culture solution completely covers the surface of the hydrogel. 37 ℃ and 5% CO 2 Culturing under the condition, and replacing fresh organoid culture solution every 3-4 days. The components of the various tumor organoids were as follows:
lung cancer, bladder cancer, kidney cancer, chondrosarcoma, and interstitial tumor organoid culture fluid: DMEM/F12 (Thermo Scientific) was supplemented with 20ng/mL bFGF (basic fibroblast growth factor) (Invitrogen), 50ng/mL human EGF (epidermal growth factor) (Invitrogen), 1 XN 2 (Invitrogen), 1 XB 27 (Invitrogen), 10. Mu. MROCK inhibitor (Enzo Life Sciences) and 1 XPenicillin/Streptomycin (Gibco). The concentration of each of the above substances is the final concentration in the culture solution.
Breast, ovarian and cervical cancer organoids: advanced DMEM/F12 (Gibco) was added with 1X Glutamax (Gibco), 10mM HEPES (Gibco), 1X Penicillin/Streptomycin (Penicillin-Streptomycin) (Gibco), 50. Mu.g/mL Primocin (Gibco), 1 XB 27 (Invitrogen), 5mM Nicotinamide (Nicotinamide) (Sigma), 1.25mM N-acetylcysteine (acetyl cysteine) (Sigma), 250ng/mL R-spondin3 (Invitrogen), 5nM Heregulin beta-1 (Invitrogen), 100ng/mL Noggin (PeproTech), 20ng/mL FGF-10 (fibroblast growth factor-10) (PeproTeFGF), 5/mL-7 (fibroblast growth factor-7) (Invitrogen), 5ng/mL EGF (epidermal growth factor) (Invitrogen), nM A83-01 (Invitrogen), 500202190 nM (Invitrogen), and 5nM (SBR-M Y). The concentration of each of the above substances is the final concentration in the culture solution.
Gastric, liver and pancreatic carcinoma organoids: DMEM/F12 (Thermo Scientific) was supplemented with 10mM HEPES (Gibco), 1 XL-glutamine (L-glutamine) (Thermo Scientific), 1 XPenicillin/Streptomycin (Penicillin-Streptomycin) (Gibco), 1 XN 2 (Invitrogen), 1 XB 27 (Invitrogen), 1mM N-acetylcysteine (Acetylcysteine) (Sigma), 10mM Nicotinamide (Nicotinamide) (Sigma), 50ng/mL EGF (epidermal growth factor) (Invitrogen), 100ng/mL Noggin (PeproTech), 30% (v/v) R-spondin conditioned media (R-phosphondin conditioned medium), 30% (v/v) wnt conditioned media (FGF conditioned medium), 200/mL 10 (fibroblast growth factor-10) (PeproTech), 1nM gamin (Gastrin) (Sigma), 10mM Y-27632 (Sigma), 1 Xlight genentin B/gentamicin (Gibcin) (Gibco). The concentration of each of the above substances is the final concentration in the culture solution.
Colorectal cancer organoid culture fluid: advanced DMEM/F12 (Gibco) was supplemented with 10nM gateway (Gastrin) (Sigma), 1 XB 27 (Invitrogen), 1 XN 2 (Invitrogen), 1mM N-acetyl cysteine (acetyl cysteine) (Sigma), 1X glutaMAX (Thermo Scientific), 5% FBS (fetal bovine serum) (Gibco), 100. Mu.g/mL genetamicin (gentamicin) (Solarbio), 1.25. Mu.g/mL amphotericin B (amphotericin B) (Gibco), 100. Mu.g/mL primocin (Invivogen), 10. Mu.M SB202190 (Sigma), 10mM Y-27632 (Sigma), 50ng/mL EGF (epidermal growth factor) (Invitrogen), 5ng/mL bFGF (basic fibroblast growth factor) (Invitrogen). The concentration of each of the above substances is the final concentration in the culture solution.
9. After 3 weeks of culture, the results are shown in FIGS. 3 and 4. It can be seen that various types of primary tumor cells form three-dimensional structures with different morphologies and different sizes in the hydrogel. The volumes of the malignant tumor organoids derived from the epithelial tissues of the two groups (the hydrogel group prepared by sheep plasma and the Matrigel hydrogel group) are measured by NoviSight 3D analysis software, and the results are shown in the table 2, and the average organoid volumes of the hydrogel group prepared by sheep plasma and the Matrigel hydrogel group are found to be not obviously different after statistics, so that the culture effect of Matrigel hydrogel can be achieved when the malignant tumor organoids derived from the epithelial tissues are cultured by using the hydrogel prepared by sheep plasma. The mesenchymal tissue-derived malignant tumors (chondrosarcoma and interstitial tumor) were not seen to form three-dimensional structures after three weeks of culture with Matrigel hydrogel, and the cells remained in a single cell state.
TABLE 2 comparison of the results of organoid culture of malignant tumors derived from epithelial tissue
10. Subculture: after 3-4 weeks of culture, the upper organoid culture solution on the hydrogel is removed. For a 6 well cell culture plate, 2mL of 0.025% (% expressed as g/100 ml) trypsin (Sigma) was added to each well, the hydrogel was blown off by repeated blow-suction with a pipette, digested for 30 minutes at 37℃and shaken several times during which time the hydrogel was completely digested to a solution, thereby releasing the organoids.
11. After centrifugation at 200g for 5 minutes, all supernatants were blotted off and added to digests (formulation: DMEM/F12 broth containing 0.001% DNase (Sigma), 1mg/mL collagenase/dispase (Roche), 200U/mL penicillin, 200mg/mL streptomycin) in 100mm dishes. Digestion is carried out at 37℃for 45-60 minutes. During which the tumor organoid is digested into a single cell suspension by shaking several times.
12. After centrifugation at 200g for 5 minutes, all supernatants were blotted off and the cells were subcultured at a ratio of 1 to 3. The operation method is shown in the steps 7 to 9.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (11)
1. A hydrogel for culturing a neoplastic organoid, characterized by: the hydrogel is formed by taking isolated sheep plasma as a matrix and adding calcium chloride and glutaraldehyde; wherein the concentration of calcium chloride in the hydrogel is 3-6mmol/L, and the volume percentage of glutaraldehyde in the hydrogel is 0.1-0.5%.
2. A method of preparing a hydrogel for culturing a tumor organoid comprising the steps of: taking isolated sheep plasma as a matrix, and adding calcium chloride and glutaraldehyde to form the hydrogel; wherein the concentration of calcium chloride in the hydrogel is 3-6mmol/L, and the volume percentage of glutaraldehyde in the hydrogel is 0.1-0.5%.
3. A method of culturing a neoplastic organoid comprising the steps of:
(A1) Mixing tumor cells to be cultured with matrix to give final cell concentration of 5×10 3 /mL; the matrix is in-vitro sheep plasma;
(A2) Adding a coagulant and a protein cross-linking agent into the (A1), and mixing to obtain a hydrogel solution containing tumor cells;
the coagulant is calcium chloride; the concentration of the calcium chloride in the hydrogel solution is 3-6mmol/L;
the protein cross-linking agent is glutaraldehyde; the glutaraldehyde accounts for 0.1-0.5% of the hydrogel solution by volume;
(A3) Adding the hydrogel solution containing tumor cells to the bottom of a cell culture container, and standing in a 37 ℃ incubator until the hydrogel solution is coagulated;
(A4) Adding a tumor organoid culture solution to the hydrogel of the step (A3), and culturing to obtain the tumor organoid.
4. A method according to claim 3, characterized in that:
in the step (A3), the hydrogel solution containing tumor cells is added to the bottom of a cell culture container, and the cell culture container is placed in a flat state for 5-10min at 37 ℃.
5. The method according to claim 4, wherein: the method further comprises the step of dissolving the hydrogel after absorbing the culture solution on the hydrogel after adding 0.125-1.25g/L of trypsin solution at 37 ℃ for 30 minutes after the step (A4).
6. The method according to any one of claims 3-5, wherein: the tumor organoid is an organoid of an epithelial tissue-derived malignancy or an organoid of a mesenchymal tissue-derived malignancy.
7. The method according to claim 6, wherein: the malignant tumor derived from the epithelial tissue is selected from any one of the following: lung cancer, breast cancer, stomach cancer, colorectal cancer, bladder cancer, kidney cancer, liver cancer, ovarian cancer, and pancreatic cancer; the malignant tumor derived from the mesenchymal tissue is selected from any one of the following: chondrosarcoma and stromal tumor.
8. A kit for culturing a neoplasm organoid, comprising the hydrogel of claim 1 and a neoplasm organoid culture.
9. Use of the hydrogel of claim 1 or the kit of claim 8 for culturing a neoplastic organoid.
10. The use according to claim 9, characterized in that: the tumor organoid is an organoid of an epithelial tissue-derived malignancy or an organoid of a mesenchymal tissue-derived malignancy.
11. The use according to claim 10, characterized in that: the malignant tumor derived from the epithelial tissue is selected from any one of the following: lung cancer, breast cancer, stomach cancer, colorectal cancer, bladder cancer, kidney cancer, liver cancer, ovarian cancer, and pancreatic cancer; the malignant tumor derived from the mesenchymal tissue is selected from any one of the following: chondrosarcoma and stromal tumor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111590436.7A CN114317439B (en) | 2021-12-23 | 2021-12-23 | Method for culturing tumor organoids |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111590436.7A CN114317439B (en) | 2021-12-23 | 2021-12-23 | Method for culturing tumor organoids |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114317439A CN114317439A (en) | 2022-04-12 |
| CN114317439B true CN114317439B (en) | 2024-04-16 |
Family
ID=81054187
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111590436.7A Active CN114317439B (en) | 2021-12-23 | 2021-12-23 | Method for culturing tumor organoids |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114317439B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114736869B (en) * | 2022-04-20 | 2024-05-28 | 上海交通大学医学院附属第九人民医院 | Mucous membrane melanoma 3D organoid and culture method and application thereof |
| CN114921413B (en) * | 2022-05-31 | 2023-03-24 | 创芯国际生物科技(广州)有限公司 | Osteosarcoma organoid culture medium and culture method |
| CN115094041B (en) * | 2022-08-25 | 2022-12-09 | 杭州艾名医学科技有限公司 | Stomach cancer organoid culture medium and culture method |
| CN115820560B (en) * | 2023-01-09 | 2023-06-09 | 山东第一医科大学附属肿瘤医院(山东省肿瘤防治研究院、山东省肿瘤医院) | Construction method and application of recurrent respiratory papilloma disease organoid |
| CN116836934B (en) * | 2023-08-31 | 2023-11-24 | 北京大橡科技有限公司 | Osteosarcoma organoid culture solution, culture reagent combination and culture method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106178110A (en) * | 2015-05-04 | 2016-12-07 | 清华大学 | Ice glue three-dimensional structure, its preparation method and application |
| CN106916781A (en) * | 2015-12-25 | 2017-07-04 | 清华大学 | A kind of construction method of external 3 D human body hepatic tissue and its application |
| CN110914408A (en) * | 2017-06-09 | 2020-03-24 | 儿童医院医学中心 | Liver organoid compositions and methods of making and using same |
| CN112940304A (en) * | 2021-03-16 | 2021-06-11 | 杭州基智生物科技有限公司 | Three-dimensional cell culture scaffold, fibroblast gel and preparation method thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1747264B1 (en) * | 2004-05-21 | 2012-09-12 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Multicellular tissue and organ culture systems |
| EP2854883B1 (en) * | 2012-05-17 | 2019-06-12 | Cartiheal (2009) Ltd | Biomatrix hydrogels and methods of use thereof |
-
2021
- 2021-12-23 CN CN202111590436.7A patent/CN114317439B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106178110A (en) * | 2015-05-04 | 2016-12-07 | 清华大学 | Ice glue three-dimensional structure, its preparation method and application |
| CN106916781A (en) * | 2015-12-25 | 2017-07-04 | 清华大学 | A kind of construction method of external 3 D human body hepatic tissue and its application |
| CN110914408A (en) * | 2017-06-09 | 2020-03-24 | 儿童医院医学中心 | Liver organoid compositions and methods of making and using same |
| CN112940304A (en) * | 2021-03-16 | 2021-06-11 | 杭州基智生物科技有限公司 | Three-dimensional cell culture scaffold, fibroblast gel and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114317439A (en) | 2022-04-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114317439B (en) | Method for culturing tumor organoids | |
| Kim et al. | Hydrogels with an embossed surface: an all-in-one platform for mass production and culture of human adipose-derived stem cell spheroids | |
| Skardal et al. | The generation of 3-D tissue models based on hyaluronan hydrogel-coated microcarriers within a rotating wall vessel bioreactor | |
| KR102345330B1 (en) | A composition for culturing brain organoid based on decellularized brain matrix and the method for preparing thereof | |
| CN114231490B (en) | Method for culturing tumor organoids by permeable hydrogel scaffold | |
| Frampton et al. | Fabrication and optimization of alginate hydrogel constructs for use in 3D neural cell culture | |
| Krontiras et al. | Adipogenic differentiation of stem cells in three‐dimensional porous bacterial nanocellulose scaffolds | |
| Muhammad et al. | Micro-and nano-topography to enhance proliferation and sustain functional markers of donor-derived primary human corneal endothelial cells | |
| Wang et al. | The effect of topology of chitosan biomaterials on the differentiation and proliferation of neural stem cells | |
| JP6826244B1 (en) | Culture materials and their uses | |
| Chen et al. | Biomaterial-assisted scalable cell production for cell therapy | |
| CN112111444B (en) | Method for reprogramming umbilical cord mesenchymal stem cells into liver cells and prepared liver organoid | |
| Kuo et al. | Material-driven differentiation of induced pluripotent stem cells in neuron growth factor-grafted poly (ε-caprolactone)-poly (β-hydroxybutyrate) scaffolds | |
| Belli et al. | Towards a 3D culture of mouse ovarian follicles | |
| US20130295637A1 (en) | Fibrous substrates for cell propagation and differentiation | |
| CN109790515B (en) | Method for producing cellular tissue and porous membrane | |
| Kuo et al. | Neuronal differentiation of induced pluripotent stem cells in hybrid polyester scaffolds with heparinized surface | |
| WO1988008448A2 (en) | Cell culture processes, materials and products | |
| Calejo et al. | Co-culture of human induced pluripotent stem cell-derived retinal pigment epithelial cells and endothelial cells on double collagen-coated honeycomb films | |
| WO2005014774A1 (en) | Carrier for culturing animal cell, and method for culturing or transplanting animal cell using said carrier for culture | |
| Åstrand et al. | Assembly of FN-silk with laminin-521 to integrate hPSCs into a three-dimensional culture for neural differentiation | |
| Fannon et al. | A fiber alginate co-culture platform for the differentiation of mESC and modeling of the neural tube | |
| Lee et al. | Functional Skeletal Muscle Regeneration Using Muscle Mimetic Tissue Fabricated by Microvalve‐Assisted Coaxial 3D Bioprinting | |
| CN114231491B (en) | Method for preparing hydrogel personalized culture tumor organoids by using autologous chest and ascites of tumor patient | |
| CN115305233A (en) | Preparation and application of daidzein and apigenin composite agarose-collagen hydrogel three-dimensional culture stem cells and extracellular vesicles |
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 |