CN115537395A - Treatment method for co-culture of liver cancer organoid and TILs (tumor necrosis factor-associated stem cells) and application thereof - Google Patents
Treatment method for co-culture of liver cancer organoid and TILs (tumor necrosis factor-associated stem cells) and application thereof Download PDFInfo
- Publication number
- CN115537395A CN115537395A CN202111297510.6A CN202111297510A CN115537395A CN 115537395 A CN115537395 A CN 115537395A CN 202111297510 A CN202111297510 A CN 202111297510A CN 115537395 A CN115537395 A CN 115537395A
- Authority
- CN
- China
- Prior art keywords
- tils
- cells
- culture
- organoid
- organoids
- 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
- 210000003171 tumor-infiltrating lymphocyte Anatomy 0.000 title claims abstract description 103
- 210000002220 organoid Anatomy 0.000 title claims abstract description 102
- 201000007270 liver cancer Diseases 0.000 title claims abstract description 31
- 208000014018 liver neoplasm Diseases 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000003501 co-culture Methods 0.000 title claims abstract description 24
- 108060008682 Tumor Necrosis Factor Proteins 0.000 title description 3
- 210000000130 stem cell Anatomy 0.000 title description 3
- 102000003390 tumor necrosis factor Human genes 0.000 title description 2
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 50
- 210000001519 tissue Anatomy 0.000 claims abstract description 37
- 238000012258 culturing Methods 0.000 claims abstract description 35
- 238000002156 mixing Methods 0.000 claims abstract description 19
- 230000012010 growth Effects 0.000 claims abstract description 18
- 230000000694 effects Effects 0.000 claims abstract description 11
- 210000002865 immune cell Anatomy 0.000 claims abstract description 6
- 238000000338 in vitro Methods 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 68
- 230000029087 digestion Effects 0.000 claims description 24
- 108010082117 matrigel Proteins 0.000 claims description 21
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 18
- 239000000203 mixture Substances 0.000 claims description 17
- 239000001963 growth medium Substances 0.000 claims description 16
- 239000006285 cell suspension Substances 0.000 claims description 14
- 210000004698 lymphocyte Anatomy 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 13
- 238000010009 beating Methods 0.000 claims description 12
- 238000007664 blowing Methods 0.000 claims description 10
- 230000001079 digestive effect Effects 0.000 claims description 10
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 8
- 102000029816 Collagenase Human genes 0.000 claims description 5
- 108060005980 Collagenase Proteins 0.000 claims description 5
- 108010003272 Hyaluronate lyase Proteins 0.000 claims description 5
- 102000001974 Hyaluronidases Human genes 0.000 claims description 5
- 229960002424 collagenase Drugs 0.000 claims description 5
- 229960002773 hyaluronidase Drugs 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000005520 cutting process Methods 0.000 claims description 4
- 210000000056 organ Anatomy 0.000 claims description 4
- 210000004881 tumor cell Anatomy 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 3
- 210000002808 connective tissue Anatomy 0.000 claims description 3
- 238000000432 density-gradient centrifugation Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 230000001338 necrotic effect Effects 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 239000004017 serum-free culture medium Substances 0.000 claims description 2
- 102000007260 Deoxyribonuclease I Human genes 0.000 claims 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 claims 1
- 239000002054 inoculum Substances 0.000 claims 1
- 239000007788 liquid Substances 0.000 claims 1
- 238000002560 therapeutic procedure Methods 0.000 abstract description 7
- 230000008569 process Effects 0.000 abstract description 5
- 238000009169 immunotherapy Methods 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 3
- 230000003993 interaction Effects 0.000 abstract description 3
- 230000007246 mechanism Effects 0.000 abstract description 3
- 230000001225 therapeutic effect Effects 0.000 abstract description 3
- 238000001727 in vivo Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 15
- 108010002350 Interleukin-2 Proteins 0.000 description 10
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 238000005406 washing Methods 0.000 description 7
- 239000012574 advanced DMEM Substances 0.000 description 6
- 230000010261 cell growth Effects 0.000 description 5
- 238000004020 luminiscence type Methods 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 4
- 210000003743 erythrocyte Anatomy 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000036039 immunity Effects 0.000 description 4
- 210000004985 myeloid-derived suppressor cell Anatomy 0.000 description 4
- 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 4
- 210000004443 dendritic cell Anatomy 0.000 description 3
- 239000012636 effector Substances 0.000 description 3
- 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
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes 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
- 229930182555 Penicillin Natural products 0.000 description 2
- 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 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000001143 conditioned effect Effects 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 229960002518 gentamicin Drugs 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- GHCVXTFBVDVFGE-UHFFFAOYSA-N 4-amino-6-chloro-1,3,5-triazin-2-ol Chemical compound NC1=NC(O)=NC(Cl)=N1 GHCVXTFBVDVFGE-UHFFFAOYSA-N 0.000 description 1
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 1
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 1
- 229940045513 CTLA4 antagonist Drugs 0.000 description 1
- 102000037982 Immune checkpoint proteins Human genes 0.000 description 1
- 108091008036 Immune checkpoint proteins Proteins 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 206010054094 Tumour necrosis Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000005975 antitumor immune response Effects 0.000 description 1
- 238000011398 antitumor immunotherapy Methods 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000003570 cell viability assay Methods 0.000 description 1
- 238000012054 celltiter-glo Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 239000012456 homogeneous solution Substances 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 210000003289 regulatory T cell Anatomy 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- YEENEYXBHNNNGV-XEHWZWQGSA-M sodium;3-acetamido-5-[acetyl(methyl)amino]-2,4,6-triiodobenzoate;(2r,3r,4s,5s,6r)-2-[(2r,3s,4s,5r)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound [Na+].CC(=O)N(C)C1=C(I)C(NC(C)=O)=C(I)C(C([O-])=O)=C1I.O[C@H]1[C@H](O)[C@@H](CO)O[C@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 YEENEYXBHNNNGV-XEHWZWQGSA-M 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/30—Coculture with; Conditioned medium produced by tumour 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to the field of biological medicine, in particular to a treatment method for co-culturing liver cancer organoid and TILs cells, which at least comprises the following steps: s1, organoid culture: taking tumor tissue of liver cancer, culturing in vitro to obtain liver cancer organoid, and amplifying organoid; s2, organoid passage: observing the growth state of the organoids, and carrying out passage on the organoids with good growth vigor; s3, preparing and culturing TILs cells, S4, carrying out immune co-culture: mixing organoids and TILs cells according to a certain proportion for co-culture; the mixing ratio of the organoids to the TILs is 1: (1-120). The invention takes the organoid as a tumor immunotherapy model, establishes a therapy method for co-culturing the liver cancer organoid and the TILs, simulates the interaction between immune cells and tumors in vivo through the co-culture system of the invention, can better reflect the process, curative effect and mechanism of the TILs for treating the tumors, and has important significance for researching or evaluating the therapeutic effect of the TILs.
Description
Technical Field
The invention relates to the field of biological medicines, in particular to IPC (International patent medicine) class number C12N5/09, and more particularly relates to a treatment method for co-culture of liver cancer organs and TILs (dendritic cells).
Background
Tumor Infiltrating Lymphocytes (TILs) are a heterogeneous population of lymphocytes present in tumor tissue and having an anti-tumor effect. TILs include T cells, small numbers of B cells, NK cells, macrophages, dendritic cells, and the like, wherein effector cells include cytotoxic T lymphocytes such as CD8+ T cells, a fraction of CD4+ T cells; and various regulatory lymphocytes such as macrophages, myeloid-derived suppressor cells (MDSC), tregs and the like.
TILs are a group of effector cells with important roles in tumor immunity, and with the continuous and intensive research on the complex roles of TILs in the tumor microenvironment, more new sub-groups of TILs are discovered. TRM and TSCM are a new subset of recently discovered CD8+ T cells with stronger effector functions; MDSC and TAM mainly mediate immunosuppression, so that MDSC and TAM become new targets of immunotherapy and have better treatment effect when combined with treatment modes such as antibody blocking immune checkpoint and the like. The influence of TILs and their subgroups in tumor immunity is the result of mutual balance and adaptation, and simultaneously, the game between the body anti-tumor immune response and the tumor immunity escape is also the result, and the interaction not only influences the generation and subsequent development of tumors, but also plays an important role in the regulation and remodeling of the body immune system. The research on the key action mechanism of TILs and subgroups thereof in tumor immunity has important significance for tumor immunotherapy and improvement of the autoimmune state of organisms.
The primary liver cancer is one of common malignant tumors in China, and as most liver cancer patients have no surgical chance when finding the primary liver cancer and liver cancer cells are insensitive to chemotherapy and radiotherapy, the treatment of the primary liver cancer is always a difficult problem to be solved urgently. The TILs reinfusion is an anti-tumor immunotherapy method developed in recent years, and the difficulty of some technologies and applications exists on how to separate and culture TILs in a tumor microenvironment and expand the TILs to an order of magnitude that can achieve a therapeutic effect, and domestic reports on the aspect are few. Patent CN202110759185.4 discloses a preparation method of TILs cell and application in tumor treatment, which uses tumor infiltrating lymphocyte after transforming TNF-alpha high expression together with prepared CTLA-4 monoclonal antibody, obviously inhibits the proliferation of lung cancer tumor, obtains better effect, but the treatment method of co-culturing TILs cell with organoid is rare.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a treatment method for co-culturing liver cancer organoids and TILs (tumor necrosis factor-like lymphocytes), which at least comprises the following steps:
s1, organoid culture: taking tumor tissue of liver cancer, culturing in vitro to obtain liver cancer organoid, and amplifying organoid;
s2, organoid passage: observing the growth state of the organoids, and carrying out passage on the organoids with good growth vigor;
s3. Preparation and culture of TILs cells
S4, immune co-culture: mixing organoids and TILs cells according to a certain proportion for co-culture;
the mixing ratio of the organoid to the TILs is 1: (1-120).
Preferably, the mixing ratio of the organoid to the TILs cells is 1: (5-100).
More preferably, the organoid and TILs cells are mixed in a ratio of 1:100.
wherein the mixing ratio of organoids and TILs cells specifically refers to the ratio between the number of organoids and the number of TILs cells.
The digestive juice comprises the following components: DMEM/F12+2% penicillin/streptomycin +0.1mg/mL gentamicin +0.6% zymostatin +2mg/mL collagenase A +0.096mg/mL hyaluronidase.
The medium used in organoid and TILs cell cultures according to the invention comprises the following components: advanced DMEM/F12,1% HEPES,1% GlutaMAX,2% B27,100ng/mL A83-01,50ng/mL EGF,100ng/mL of G-G, 500ng/mL of commercial R-spondin1.
In the present invention,% represents a mass-volume percentage unless otherwise specified.
The components of the culture medium used in the subculture of the invention are as follows: conditioned R-spondin1medium from RSPO cells (Curtrex, 3700-100-01), 100ng/mL Nrg1,10mM Y-27632.
The washing solution in the present invention was 1xPBS/5% FCS, and the washing reagents in the present invention were 1xPBS/5% FCS, unless otherwise specified.
In some embodiments, the S1 organoid culture comprises at least the following steps:
1) Collecting tumor tissue of hepatocarcinoma, and cutting into 0.1mm with scissors 3 Adding digestive juice, continuously shaking and incubating for 0.5-1 h at 37 ℃, and manually shaking and mixing once every 15 min;
2) After complete digestion, centrifuging the tissue/cell at 4 ℃ and 1500rpm for 5min, discarding the supernatant, adding 2mg/mL DNasel, and incubating at 37 ℃ for 5min;
3) Washing the tissue/cells with 1xPBS/5% FCS;
4) Adding 0.25% trypsin/EDTA, and incubating at 37 deg.C for 2min;
5) Adding DNasel, and incubating at 37 ℃ for 5min;
6) Centrifuging at 4 deg.C and 1500rpm for 5min, and removing supernatant;
7) Wash tissue/cells with 5% FC/PBS.
8) The cells were lysed by adding red blood cells on ice for 3min.
9) Centrifuging at 4 deg.C and 1500rpm for 5min, and removing supernatant;
10 Fcs) tissue/cells were washed with 1 xPBS/5%.
11 ) treated with erythrocyte lysis buffer, the tissue/cells were filtered through a 40 μm flow tube and washed with 1xPBS/5% FCS to obtain single cell suspensions.
12 Counting the tissues/cells washed for the last time, centrifuging for 5min at 4 ℃ and 1500rpm, and discarding the supernatant;
13 Take matrigel heavy suspension cells on ice, mix cells with matrigel evenly, and take 10. Mu.L of the mixture of cells and matrigel to the center of a 48-well plate to avoid air bubbles. Incubating at 37 ℃ for 5min until matrigel is solidified;
14 250 μ L of organoid culture medium is added into each well and cultured at 37 ℃, and single cells grow into organoids for about 4 to 5 days, and passage and frozen storage are carried out.
Observing the growth state of the organoid under a mirror, and carrying out passage on the organoid with good growth vigor.
In some embodiments, the S2 organoid passaging comprises at least the steps of:
a. using a pipette to suck the culture solution in the hole, and paying attention not to suck matrigel;
b. adding 200 mul tryple into each hole, blowing and beating for many times, putting into an incubator at 37 ℃ for digestion, and digesting the organoid into single cells;
c. after digestion is completed, 200 μ l of Advanced DMEM/F12 diluted tryple is added to each well to terminate digestion;
d. centrifuging the single cells at 4 deg.C and 1500rpm for 5min, and removing the supernatant;
e. taking matrigel to re-suspend the cells on ice, uniformly mixing the cells and the matrigel, transferring 10 mu l of mixed solution of the cells and the matrigel to the center of a 48-well plate to avoid bubbles, incubating at 37 ℃ for 5min, and adding 250 mu l of culture medium to each well for culturing at 37 ℃ after the matrigel is solidified.
In some embodiments, the digestion time in step b is 5-10 min, and if the digestion time is too long or too short, the growth state of the cells is affected, and the blowing process needs to be gentle, otherwise, the state of the cells is also affected.
In some embodiments, the type in the present invention refers to type TM Express enzymes.
In some embodiments, the d step requires care not to aspirate the cells during the discard of the supernatant.
In some embodiments, the preparation of TILs cells in S3 comprises at least the following steps:
(a1) Immediately removing necrotic tissues and connective tissues on the surface of the tumor from the tumor tissues removed by the operation under the aseptic condition, rinsing the tumor tissues by RPMI 1640 containing 1% of antibiotics, cutting the tumor tissues into small pieces, adding digestive juice for digestion, filtering the digested tumor tissues and carrying out centrifugal cleaning;
(a2) Resuspending the pellet centrifuged in step (a 1) with 5% NCS in complete medium RPMI-1640 to obtain a cell suspension, placing equal volumes of 100% and 75% lymphocyte layering solutions in a centrifuge tube in sequence, slowly adding the cell suspension to the uppermost layer, centrifuging with discontinuous density gradient, and collecting tumor cells in the upper layer interface and lymphocytes in the lower layer interface, i.e., TILs.
In some embodiments, the digestive fluid of step (a 1) comprises a mixture of collagenase, hyaluronidase, dnase I.
Preferably, the digestive juice in step (a 1) comprises 0.05% mixed collagenase, 0.001% hyaluronidase, 2mg/mL dnase I.
In some embodiments, the conditions of the digestion in step (a 1): the temperature is 37 ℃ and the time is 2-5h.
Preferably, the conditions of the digestion in step (a 1): the temperature is 37 ℃ and the time is 3-4h.
In some embodiments, the conditions of the discontinuous density gradient centrifugation in step (a 2) are: the rotating speed is 1500-2200r/min, and the time is 15-30min.
Preferably, the conditions of the discontinuous density gradient centrifugation in the step (a 2) are: the rotating speed is 2000r/min, and the time is 20min.
In some embodiments, the 100% and 75% lymphocyte layering solution in step (a 2) is formulated with RPMI-1640 by the following specific formulation method: the 75% lymphocyte layering solution is prepared by adding 25ml RPMI-1640 into 75ml lymphocyte layering solution.
In some embodiments, the lymphocyte layering fluid is lymphoprep (stem cell, CATO 7851).
In some embodiments, the culturing of TILs cells in S3 comprises at least the following steps:
(b1) The prepared TILs cells were washed, centrifuged, and the cell concentration was adjusted to (0.4-0.6). Times.10 by using 20% NCS RPMI-1640 6 Cell suspension per ml;
(b2) Inoculating the cell suspension of step (b 1) to a 24-well culture plate at 1 ml/well, and then culturing.
Preferably, the concentration of the cells adjusted in (b 1) is 0.5X 10 6 One per ml.
In some embodiments, the protocol for culturing in (b 2) comprises the following 3:
the first scheme comprises the following steps: adding IL-2 1000U, setting at 37 ℃ and 5% CO 2 The incubator is used for 3 to 4 weeks, and the freshly prepared RPMI-1640 complete culture solution containing IL-2 is replaced for 3 to 5 days.
Scheme II: adding CD3 McAb1μ g/ml, at 37 ℃ C. 5% CO 2 The culture is carried out in an incubator for 3 to 4 weeks, and the freshly prepared RPMI-1640 complete culture solution containing IL-2 is replaced in 3 to 5 days.
The third scheme is as follows: IL-2 1000U and CD3 McAb 1. Mu.l were added g/ml, 37 ℃,5% CO2 incubator 3-4 weeks, in the 3-5 days between the replacement of freshly prepared IL-2 containing RPMI-1640 complete culture medium.
Cells were counted 1 time every 3 days and passaged periodically.
In some embodiments, the immuno-co-culture in S4 comprises at least the steps of:
(c1) Organoid treatment: taking well-grown tumor organoids, adding tryple for digestion, stopping digestion by a serum-free culture medium after digestion is finished, transferring the organ to a centrifugal tube, and counting;
(c2) And (3) immune cell treatment: gently blowing and beating the TILs cells by using a pipette gun to enable the TILs cells to be re-suspended, transferring the TILs cells into a centrifuge tube, blowing and beating the TILs cells by using the pipette gun to be uniformly mixed, and counting;
(c3) Mixing TILs cells and organoids according to a certain proportion according to counting results, blowing and beating the TILs cells and organoids by using a pipette to uniformly mix the TILs cells and the organoids, centrifuging the mixture, removing supernatant, placing cell precipitates on ice, adding dissolved matrigel, repeatedly blowing and beating the mixture uniformly by using the pipette, then inoculating the mixture into holes of a culture plate, and incubating the culture plate for 3 to 7min at 37 ℃;
(c4) After the incubation is finished, adding the culture medium and then putting the mixture into an incubator for culturing.
In some embodiments, the conditions of digestion in (c 1) are: the temperature is 37 deg.C, and the time is 5-15min, preferably 10min.
In some embodiments, the serum-free medium in (c 1) is Advanced DMEM/F12.
In some embodiments, the conditions of centrifugation in step (c 3) are: the temperature is 3-5 ℃, the rotating speed is 800-1200r/min, and the time is 4-6min.
Preferably, the conditions of the centrifugation in step (c 3) are: the temperature is 4 ℃, the rotating speed is 1000r/min, and the time is 5min.
In some embodiments, the amount of inoculation in step (c 3) is (8-12) ul/well.
Preferably, the amount of inoculation in step (c 3) is 10 ul/well.
In another aspect, the invention provides an application of a treatment method of co-culture of liver cancer organoids and tills cells, which is used for researching or evaluating the effect of the tills cells on treating tumors.
Has the advantages that:
(1) The invention takes organoid as tumor immunotherapy model, establishes the treatment method of co-culture of liver cancer organoid and TILs cell, simulates the interaction of immune cell and tumor in vivo through the co-culture system of the invention, can better reflect the process, curative effect and mechanism of TILs cell for treating tumor, and has important significance for researching or evaluating the therapeutic effect of TILs cell;
(2) The invention successfully cultures and amplifies TILs cells in excised liver cancer tissues by regulating and controlling different TILs cell culture methods to reach 10 10 The magnitude order provides a good foundation for experimental clinical application;
(3) According to the invention, the organoids with a specific proportion are preferably selected and mixed with the TILs for co-culture, so that the TILs in the co-culture process have strong killing power on the hepatoma cells, and the TILs can tend to gather to the tumor organoids and kill the tumor cells within 48 hours.
(4) The invention reduces the experiment period and the experiment cost and improves the success rate.
Drawings
FIG. 1 is a light mirror image of the growth of cells after culturing 5 days for TILs in examples 1-3; wherein FIG. a is a light mirror image of the growth of cells after culturing the TILs cells for 5 days in example 3; FIG. b is a light mirror image of the growth of cells after culturing the TILs cells for 5 days in example 2; FIG. c is a light mirror image of the growth of cells after 5 days of culture of the TILs cells of example 1.
FIG. 2 is a statistical plot of the number of cells grown within 5 days of culture of TILs in examples 1-3.
FIG. 3 shows the tumor organoids killed by TILs after 2h, 24h, and 48h of co-culture of the organoids with TILs in example 1.
FIG. 4 is a statistical chart of cell viability in examples 1 and 4 to7 and comparative example 1.
Detailed Description
Examples
The digestive juice used in the embodiment of the invention comprises the following components: DMEM/F12+2% penicillin/streptomycin +0.1mg/mL gentamicin +0.6% zymostatin +2mg/mL collagenase A +0.096mg/mL hyaluronidase.
The components of the culture medium used for organoid culture and TILs cell culture in the examples of the present invention were as follows: advanced DMEM/F12,1% HEPES,1% GlutaMAX,2% B27,100ng/mL A83-01,50ng/mL EGF,100ng/mL of G-G, 500ng/mL of commercial R-spondin1.
The components of the culture medium used in subculture in the embodiment of the invention are as follows: conditioned R-spondin1medium from RSPO cells (Curtrex, 3700-100-01), 100ng/mL Nrg1,10mM Y-27632.
The washing reagent used in the examples of the present invention was 1xPBS/5% FCS.
Example 1
A treatment method for co-culturing liver cancer organoids and TILs cells comprises the following steps:
s1, organoid culture: taking tumor tissue of liver cancer, culturing in vitro to obtain liver cancer organoid, and amplifying organoid;
s2, organoid passage: observing the growth state of the organoids, and carrying out passage on the organoids with good growth vigor;
s3. Preparation and culture of TILs cells
S4, immune co-culture: organoids were mixed with TILs cells at a ratio of 1:100 mixing and co-culturing;
the S1 organoid culture comprises the following steps:
1) Collecting tumor tissue of hepatocarcinoma, and cutting into 0.1mm with scissors 3 Adding digestive juice, continuously shaking and incubating for 0.5-1 h at 37 ℃, and manually shaking and mixing once every 15 min;
2) After complete digestion, centrifuging the tissue/cell at 4 ℃ and 1500rpm for 5min, discarding the supernatant, adding 2mg/mL DNasel, and incubating at 37 ℃ for 5min;
3) Washing the tissue/cells with 1xPBS/5% FCS;
4) Adding 0.25% trypsin/EDTA, and incubating at 37 deg.C for 2min;
5) Adding DNasel, and incubating at 37 ℃ for 5min;
6) Centrifuging at 4 deg.C and 1500rpm for 5min, and removing supernatant;
7) Wash tissue/cells with 5% FC/PBS.
8) Erythrocytes were added to lyse the cells for 3min on ice.
9) Centrifuging at 4 deg.C and 1500rpm for 5min, and removing supernatant;
10 Washing the tissue/cells with 1xPBS/5% FCS;
11 ) treated with erythrocyte lysis buffer, the tissue/cells were filtered through a 40 μm flow tube and washed with 1xPBS/5% FCS to obtain single cell suspensions.
12 Counting the tissues/cells washed for the last time, centrifuging for 5min at 4 ℃ and 1500rpm, and discarding the supernatant;
13 Take matrigel heavy suspension cells on ice, mix cells with matrigel evenly, and take 10 μ L of the mixture of cells and matrigel to the center of 48-well plate to avoid air bubbles. Incubating at 37 ℃ for 5min until matrigel is solidified;
14 250 μ L of organoid culture medium is added into each well and cultured at 37 ℃, and single cells grow into organoids for about 4 to 5 days, and passage and frozen storage are carried out.
Observing the growth state of the organoid under a mirror, and carrying out passage on the organoid with good growth
The S2 organoid passage comprises the following steps:
a. the culture solution in the hole is sucked away by a pipette gun, and the matrigel is not sucked away;
b. adding 200 μ l of tryple into each hole, beating for multiple times, and digesting into single cell in an incubator at 37 deg.C for 8 min;
c. after digestion is completed, 200 μ l of Advanced DMEM/F12 diluted tryple is added to each well to terminate digestion;
d. centrifuging the single cells at 4 deg.C and 1500rpm for 5min, and removing the supernatant;
e. taking matrigel to re-suspend the cells on ice, uniformly mixing the cells and the matrigel, transferring 10 mu l of mixed solution of the cells and the matrigel to the center of a 48-well plate to avoid bubbles, incubating at 37 ℃ for 5min, and adding 250 mu l of culture medium to each well for culturing at 37 ℃ after the matrigel is solidified.
The preparation of the TILs cells in the S3 comprises the following steps:
(a1) Immediately removing necrotic tissues and connective tissues on the surface of a tumor from the tumor tissue cut by the operation under the aseptic condition, rinsing the tumor tissue by RPMI 1640 containing 1% of antibiotics, shearing the tumor tissue into small blocks of 0.5mm by 1mm, adding digestive juice for digestion, sequentially filtering the tumor tissue by stainless steel nets of 80 meshes and 200 meshes after digesting for 4 hours at 37 ℃, centrifuging the tumor tissue for 8min at the rotating speed of 1500r/min, and cleaning the tumor tissue for 2 times by serum-free RPMI-1640;
(a2) Resuspending the centrifuged pellet of step (a 1) in 5% NCS-containing RPMI-1640 complete medium to obtain a cell suspension, adding 5mL of 100% lymphocyte layering solution to the bottom layer of the test tube, slowly pouring 5mL of 75% lymphocyte layering solution, slowly adding 5mL of cell suspension to the uppermost layer, centrifuging for 20min with a discontinuous density gradient at 2000r/min, and collecting the tumor cells in the upper interface and the lymphocytes in the lower interface, i.e., TILs.
The culture of the TILs in the S3 comprises the following steps:
(b1) Washing the prepared TILs with RPMI-1640 for 2 times, centrifuging at 1500r/min for 8min, adjusting cell concentration to 0.5 × 10 with 20% of RPMI-1640 of NCS 6 Cell suspension per ml;
(b2) The cell suspension in step (b 1) was seeded at 1 ml/well in a 24-well culture plate, and then cultured, and 1 cell was counted every 3 days and passaged periodically.
The culture scheme in the step (b 2) is as follows: adding IL-2 1000U and CD3 McAb1μ g/ml, 37 ℃ C., 5% CO 2 Culturing in incubator for 3 weeks, and replacing freshly prepared complete culture solution of IL-2-containing RPMI-1640 for 4 days
The immune co-culture in S4 comprises the following steps:
(c1) Organoid treatment: adding tryple into well-grown tumor organs, standing at 37 deg.C for digestion for 10min, terminating digestion with serum-free Advanced DMEM/F12, transferring into a centrifuge tube, and counting;
(c2) And (3) immune cell treatment: gently blowing and beating TILs (dendritic cells) by using a 1mL pipette, transferring the TILs into a centrifuge tube after resuspending the TILs, blowing and beating the TILs evenly by using the pipette, and counting;
(c3) According to the counting result, mixing the TILs cells and the organoids according to the proportion of 1;
(c4) After the incubation is finished, adding a conventional culture medium, and then putting the mixture into an incubator for culture, wherein the culture medium is changed every 3-4 days.
Example 2
This example provides a therapeutic method for co-culturing liver cancer organoids and TILs cells, which is implemented in the same manner as example 1, except that the culture scheme in step (b 2) is: addition of CD3 McAb1μ g/ml, at 37 ℃ C. 5% CO 2 Incubate for 3 weeks, during which time the freshly prepared complete culture medium of IL-2-containing RPMI-1640 was replaced for 4 days.
Example 3
This example provides a therapeutic method for co-culturing liver cancer organoids and TILs cells, which is implemented in the same manner as example 1, except that the culture scheme in step (b 2) is as follows: adding IL-2 1000U, setting at 37 deg.C, 5% 2 Incubate for 3 weeks, during which time the freshly prepared complete culture medium of IL-2-containing RPMI-1640 was replaced for 4 days.
Example 4
This example provides a therapeutic method for co-culturing liver cancer organoids and tills cells, which is the same as example 1 except that the organoids and tills cells in S4 are cultured in the ratio of 1:1 mixing and co-culturing.
Example 5
This example provides a therapeutic method for co-culturing liver cancer organoids and tills cells, which is the same as example 1 except that the organoids and tills cells in S4 are cultured in the ratio of 1:5 mixing and co-culturing.
Example 6
This example provides a therapeutic method for co-culturing liver cancer organoids and TILs cells, the specific implementation manner is the same as example 1, except that the ratio of organoids in S4 to TILs cells is 1:10 mixing and co-culturing.
Example 7
This example provides a therapeutic method for co-culturing liver cancer organoids and tills cells, which is the same as example 1 except that the organoids and tills cells in S4 are cultured in the ratio of 1:50 were mixed and co-cultured.
Comparative example 1
The control example 1 refers to the addition of organoids only during co-culture.
Data collection, analysis and sorting
Panoramic images were acquired using the instrument biotekrotation 1 microplate reader. The changes in volume of individual tumor organoids were followed up and compared by taking pictures every 24h, starting from 0h of co-culture and continuing until the end of the experiment.
a. After the instrument is started, the software is opened and the appropriate mode is selected. The cell plate is placed on a slide table. And saving the image after clicking shooting.
b. And adding a ruler to the image and finishing.
c. Images were collected every 24h using BioTek. At least 5 effective samples are randomly selected from each group, and the diameters of the effective samples are measured to evaluate the growth condition of the organoids. The measured data were collated and plotted to obtain growth curves for each group of organoids. The data is processed as follows: relative organoid size = diameter of the end Day of the valid sample experiment/diameter of the valid sample Day 0. The relative growth curves were plotted.
d. Before the completion of the immune co-culture, the number of organoids in the microcarriers of the control group and the treatment group was counted as one of the methods for evaluating the organoid growth status and the effect of immune cell therapy. Organoids >50um in diameter were considered valid samples and counted. After the culture is carried out with the TILs for a period of time, the phenomenon that organoids are wrapped by the TILs and killed to reduce the volume can occur. When the organoid is completely encapsulated by TILs cells and the organoid dies apparently, no statistics are made for them.
Evaluation of Effect
Cell growth status of TILs cell culture
The growth of the cells was observed after 5 days of culture of the TILs cells of examples 1-3, as shown in FIG. 1.
Proliferative Activity of TILs cells
Statistics were made on the number of cells grown within 5 days of culture of the TILs cells of examples 1-3, as shown in FIG. 2.
3. Effect of TILs on tumor organoids in Co-culture
The culture process of TILs cells co-cultured with organoids for 2h, 24h and 48h in example 1 was observed, respectively, and is shown in FIG. 3.
4. Cellular activity
The cell activity was measured using CellTiter-Glo and the results are shown in FIG. 4.
1) Reagent preparation
1. Unfreezing
Buffered and allowed to equilibrate to room temperature before use. For the sake of convenience in the art,
the buffer can be thawed and stored at room temperature for 48 hours prior to use.
2. Lyophilized before use
The matrix was equilibrated to room temperature.
3. Will be of appropriate volume
Buffer bottles (10ml for A grade and 100ml for B grade) were transferred to the flask containing
In the matrix vial, the lyophilized enzyme/substrate mixture is reconstituted. This forms
Reagent。
4. A homogeneous solution was obtained by gentle rotation.
The substrate should be easily brought into solution in less than one minute.
2) Cell viability assay
1. A multi-well opaque plate was prepared using TILs and tumor organoids as culture medium, 100. Mu.l in 96-well plates and 25. Mu.l in 384-well plates.
2. Control wells containing acellular medium were prepared to obtain background luminescence values.
3. And adding the compound to be tested into the experimental well, and culturing according to the culture scheme.
4. The plate and contents were allowed to equilibrate at room temperature for about 30 minutes.
5. Adding a volume equal to the volume of cell culture medium in each well
Reagents (where 100. Mu.l of reagent was added to 100. Mu.l of cell-containing medium in a 96-well plate; 25. Mu.l of reagent was added to 25. Mu.l of cell-containing medium in a 384-well plate).
6. The contents were mixed on an orbital shaker for 2 minutes to induce cell lysis.
7. The well plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal.
8. The luminescence was recorded.
Protocol for generating ATP standards
1. mu.M ATP (100. Mu.l of a 1. Mu.M ATP solution containing 10-10mol ATP) was prepared in the medium.
2. ATP was prepared in 10-fold serial dilutions in culture medium (volumes of 1. Mu.M to 10nM, 100. Mu.l containing 10-10 to 10-12mol ATP).
3. Multi-well plates (25. Mu.l in 384-well plates) were prepared in 100. Mu.l of medium with different concentrations of standard ATP solution.
4. Adding into
The volume of reagent is equal to the volume of standard ATP content per well.
5. The contents were mixed on an orbital shaker for 2 minutes.
6. The plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal.
7. Luminescence was recorded.
Claims (10)
1. A treatment method for co-culturing liver cancer organoids and TILs is characterized by at least comprising the following steps:
s1, organoid culture: taking tumor tissue of liver cancer, culturing in vitro to obtain liver cancer organoid, and amplifying organoid;
s2, organoid passage: observing the growth state of the organoids, and carrying out passage on the organoids with good growth vigor;
s3. Preparation and culture of TILs cells
S4, immune co-culture: mixing organoid and TILs cell in certain proportion for co-culture;
the mixing ratio of the organoids to the TILs is 1: (1-120).
2. The method as claimed in claim 1, wherein the preparation of TILs cells in S3 comprises at least the following steps:
(a1) Immediately removing necrotic tissues and connective tissues on the surface of the tumor from the tumor tissues removed by the operation under the aseptic condition, rinsing the tumor tissues by RPMI 1640, cutting the tumor tissues into small pieces, adding digestive juice for digestion, filtering the digested tumor tissues and carrying out centrifugal cleaning;
(a2) Resuspending the precipitate obtained after centrifugation in the step (a 1) to obtain a cell suspension, sequentially placing 100% and 75% of lymphocyte layering liquid with equal volumes into a centrifuge tube, slowly adding the cell suspension to the uppermost layer, centrifuging by adopting a discontinuous density gradient, and collecting tumor cells on the upper layer interface and lymphocytes on the lower layer interface, namely TILs.
3. The method as claimed in claim 2, wherein the digestive juice of step (a 1) comprises collagenase, hyaluronidase and DNase I.
4. The method of claim 2, wherein the digestion in step (a 1) is performed under conditions selected from the group consisting of: the temperature is 37 ℃ and the time is 2-5h.
5. The method as claimed in claim 2, wherein the non-continuous density gradient centrifugation in step (a 2) is performed under the following conditions: the rotating speed is 1500-2200r/min, and the time is 15-30min.
6. The method as claimed in claim 1, wherein the step of culturing the TILs cells in S3 comprises at least the following steps:
(b1) The prepared TILs cells were washed, centrifuged, and the cell concentration was adjusted to (0.4-0.6). Times.10 by using 20% NCS RPMI-1640 6 Cell suspension per ml;
(b2) Inoculating the cell suspension of step (b 1) to a 24-well culture plate at 1 ml/well, and then culturing.
7. The method as claimed in any one of claims 1 to 6, wherein the step of immuno-cocultivation in S4 comprises at least the following steps:
(c1) Organoid treatment: taking well-grown tumor organoids, adding tryple for digestion, stopping digestion by a serum-free culture medium after digestion is finished, transferring the organ to a centrifugal tube, and counting;
(c2) And (3) immune cell treatment: gently blowing and beating the TILs cells by using a pipette gun, transferring the TILs cells into a centrifuge tube after the TILs cells are resuspended, blowing and beating the TILs uniformly by using the pipette gun, and counting;
(c3) According to counting results, mixing TILs cells and organoids according to a certain proportion, beating the TILs with a pipette to uniformly mix the TILs and the organoids, centrifuging the mixture, removing supernatant, placing cell precipitates on ice, adding dissolved matrigel, repeatedly beating the cells with the pipette to uniformly mix the cells with the matrigel, inoculating the cells into holes of a culture plate, and incubating the cells for 3 to7 minutes at 37 ℃;
(c4) After the incubation is finished, adding the culture medium and then putting the mixture into an incubator for culturing.
8. The method of claim 7, wherein the centrifugation step (c 3) is performed under the conditions of: the temperature is 3-5 ℃, the rotating speed is 800-1200r/min, and the time is 4-6min.
9. The method of claim 7, wherein the amount of inoculum in step (c 3) is (8-12) ul/well.
10. Use of a method of treatment of a liver cancer organoid in co-culture with TILs cells according to any of claims 1-9 for studying or assessing the effect of TILs cells on tumor treatment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111297510.6A CN115537395A (en) | 2021-11-04 | 2021-11-04 | Treatment method for co-culture of liver cancer organoid and TILs (tumor necrosis factor-associated stem cells) and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111297510.6A CN115537395A (en) | 2021-11-04 | 2021-11-04 | Treatment method for co-culture of liver cancer organoid and TILs (tumor necrosis factor-associated stem cells) and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115537395A true CN115537395A (en) | 2022-12-30 |
Family
ID=84723073
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111297510.6A Pending CN115537395A (en) | 2021-11-04 | 2021-11-04 | Treatment method for co-culture of liver cancer organoid and TILs (tumor necrosis factor-associated stem cells) and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115537395A (en) |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017174649A1 (en) * | 2016-04-05 | 2017-10-12 | Fundación Centro Nacional De Investigaciones Oncológicas Carlos Iii | Method for preparing lymphocytes capable of infiltrating a solid tumor, lymphocytes obtainable by said method and uses thereof |
| CN111394299A (en) * | 2020-03-26 | 2020-07-10 | 南京鼓楼医院 | In-vitro construction method and application of liver organoid |
| CN111948392A (en) * | 2020-07-03 | 2020-11-17 | 浙江大学 | Hepatocellular carcinoma PDX model construction method |
| US20200363402A1 (en) * | 2018-02-02 | 2020-11-19 | Wake Forest University Health Sciences | Organoids related to immunotherapy and methods of preparing and using the same |
| CN111989569A (en) * | 2017-12-21 | 2020-11-24 | 荷兰皇家科学院 | Immune cell organoid co-culture |
| CN112143699A (en) * | 2020-09-11 | 2020-12-29 | 上海市第十人民医院 | Method for reconstructing immune microenvironment of colorectal cancer organoid |
| CN112210537A (en) * | 2020-09-28 | 2021-01-12 | 浙江科途医学科技有限公司 | Liver cancer organoid and culture method, culture medium for culture and application thereof |
| CN113106065A (en) * | 2021-03-17 | 2021-07-13 | 上海市第十人民医院 | Colorectal cancer organoid and liver organoid co-culture model and construction method thereof |
| WO2021179354A1 (en) * | 2020-03-10 | 2021-09-16 | 合肥中科普瑞昇生物医药科技有限公司 | Primary liver cancer cell culture medium, primary liver cancer cell culturing method and application thereof |
| CN113481162A (en) * | 2021-07-01 | 2021-10-08 | 丹望医疗科技(上海)有限公司 | Culture medium, method and kit for rapidly culturing tumor organoid |
-
2021
- 2021-11-04 CN CN202111297510.6A patent/CN115537395A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017174649A1 (en) * | 2016-04-05 | 2017-10-12 | Fundación Centro Nacional De Investigaciones Oncológicas Carlos Iii | Method for preparing lymphocytes capable of infiltrating a solid tumor, lymphocytes obtainable by said method and uses thereof |
| CN111989569A (en) * | 2017-12-21 | 2020-11-24 | 荷兰皇家科学院 | Immune cell organoid co-culture |
| US20200363402A1 (en) * | 2018-02-02 | 2020-11-19 | Wake Forest University Health Sciences | Organoids related to immunotherapy and methods of preparing and using the same |
| WO2021179354A1 (en) * | 2020-03-10 | 2021-09-16 | 合肥中科普瑞昇生物医药科技有限公司 | Primary liver cancer cell culture medium, primary liver cancer cell culturing method and application thereof |
| CN111394299A (en) * | 2020-03-26 | 2020-07-10 | 南京鼓楼医院 | In-vitro construction method and application of liver organoid |
| CN111948392A (en) * | 2020-07-03 | 2020-11-17 | 浙江大学 | Hepatocellular carcinoma PDX model construction method |
| CN112143699A (en) * | 2020-09-11 | 2020-12-29 | 上海市第十人民医院 | Method for reconstructing immune microenvironment of colorectal cancer organoid |
| CN112210537A (en) * | 2020-09-28 | 2021-01-12 | 浙江科途医学科技有限公司 | Liver cancer organoid and culture method, culture medium for culture and application thereof |
| CN113106065A (en) * | 2021-03-17 | 2021-07-13 | 上海市第十人民医院 | Colorectal cancer organoid and liver organoid co-culture model and construction method thereof |
| CN113481162A (en) * | 2021-07-01 | 2021-10-08 | 丹望医疗科技(上海)有限公司 | Culture medium, method and kit for rapidly culturing tumor organoid |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111876386B (en) | Breast cancer organoid culture method and co-culture method of tumor-related fibroblasts | |
| CN108504625B (en) | Mouse fibroblast and application thereof | |
| CN112143699A (en) | Method for reconstructing immune microenvironment of colorectal cancer organoid | |
| CN110564687B (en) | Compositions, media, methods and kits for expanding hematopoietic stem cells | |
| Wickström et al. | Expansion of tumor-infiltrating lymphocytes from melanoma tumors | |
| CN112662631B (en) | CAR-T cell perfusion culture method | |
| CN108192865B (en) | NK cell in-vitro amplification method and kit used for same | |
| CN114209814A (en) | Application of TNFSF15 protein in promoting differentiation of bone marrow stem cells into macrophages and amplification | |
| CN111548994B (en) | Cell culture medium and method for culturing NK cells by using same | |
| CN115537395A (en) | Treatment method for co-culture of liver cancer organoid and TILs (tumor necrosis factor-associated stem cells) and application thereof | |
| CN112048474B (en) | Method for enhancing hematopoietic stem cell transplantation capability | |
| CN110669732A (en) | Use of compositions for reprogramming hematopoietic progenitor cells to hematopoietic stem cells | |
| CN112608899B (en) | Application of serum-free culture medium in culturing spheroids of cancer tissue origin | |
| CN114672457A (en) | T lymphocyte derived from tumor tissue and having tumor specific killing effect, preparation method thereof and cell preparation | |
| CN115161282A (en) | Mouse brain microvascular endothelial cell and pericyte combined extraction and culture method | |
| CN115094022A (en) | Construction method of lung cancer fibroblast and lung cancer organoid co-culture model | |
| CN114752626A (en) | Reversible immortalized II-type alveolar epithelial cell and construction and application thereof | |
| CN114058584A (en) | Preparation method of natural killer cells for clinical use | |
| CN109954139B (en) | Application of matrine as immunoadjuvant in preparation of gastric cancer dendritic cell vaccine | |
| CN104498435A (en) | In-vitro cell culture method for primary B cell acute lymphoblastic leukemia | |
| CN115521898A (en) | Immune cell treatment method for co-culture of liver cancer organs and NK cells and application | |
| CN115521912A (en) | Immune cell treatment method by co-culture of organoid and T cell and application | |
| CN116103234B (en) | Preparation method and application of novel DC-like cells | |
| CN112961827B (en) | Application of forskolin in T cell culture | |
| CN111333698B (en) | Breast cancer target antigen combination, CTL cell cultured by breast cancer target antigen combination stimulation and application thereof |
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 |