CN114214284B - Method for culturing kidney tumor organoids - Google Patents
Method for culturing kidney tumor organoids Download PDFInfo
- Publication number
- CN114214284B CN114214284B CN202111678865.XA CN202111678865A CN114214284B CN 114214284 B CN114214284 B CN 114214284B CN 202111678865 A CN202111678865 A CN 202111678865A CN 114214284 B CN114214284 B CN 114214284B
- Authority
- CN
- China
- Prior art keywords
- kidney
- culture
- extracellular matrix
- kidney tumor
- tumor organoid
- 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
- 208000008839 Kidney Neoplasms Diseases 0.000 title claims abstract description 106
- 210000002220 organoid Anatomy 0.000 title claims abstract description 101
- 238000000034 method Methods 0.000 title claims abstract description 25
- 238000012258 culturing Methods 0.000 title claims abstract description 21
- 210000003734 kidney Anatomy 0.000 claims abstract description 65
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims abstract description 62
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims abstract description 62
- 210000002744 extracellular matrix Anatomy 0.000 claims abstract description 61
- 238000007877 drug screening Methods 0.000 claims abstract description 14
- 238000004113 cell culture Methods 0.000 claims abstract description 6
- 239000002981 blocking agent Substances 0.000 claims abstract description 5
- 238000012136 culture method Methods 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 30
- 206010038389 Renal cancer Diseases 0.000 claims description 25
- 201000010982 kidney cancer Diseases 0.000 claims description 24
- 102000057297 Pepsin A Human genes 0.000 claims description 6
- 108090000284 Pepsin A Proteins 0.000 claims description 6
- 229940111202 pepsin Drugs 0.000 claims description 6
- 229920001992 poloxamer 407 Polymers 0.000 claims description 6
- 230000010412 perfusion Effects 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 238000004108 freeze drying Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 16
- 229940079593 drug Drugs 0.000 abstract description 12
- 239000002245 particle Substances 0.000 abstract description 7
- 238000002493 microarray Methods 0.000 description 24
- 206010028980 Neoplasm Diseases 0.000 description 23
- 239000004205 dimethyl polysiloxane Substances 0.000 description 12
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 12
- 238000005516 engineering process Methods 0.000 description 9
- 108010082117 matrigel Proteins 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 208000006265 Renal cell carcinoma Diseases 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 238000007639 printing Methods 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 239000002147 L01XE04 - Sunitinib Substances 0.000 description 4
- 229920004890 Triton X-100 Polymers 0.000 description 4
- 239000013504 Triton X-100 Substances 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 229940057950 sodium laureth sulfate Drugs 0.000 description 4
- SXHLENDCVBIJFO-UHFFFAOYSA-M sodium;2-[2-(2-dodecoxyethoxy)ethoxy]ethyl sulfate Chemical compound [Na+].CCCCCCCCCCCCOCCOCCOCCOS([O-])(=O)=O SXHLENDCVBIJFO-UHFFFAOYSA-M 0.000 description 4
- 229960001796 sunitinib Drugs 0.000 description 4
- WINHZLLDWRZWRT-ATVHPVEESA-N sunitinib Chemical compound CCN(CC)CCNC(=O)C1=C(C)NC(\C=C/2C3=CC(F)=CC=C3NC\2=O)=C1C WINHZLLDWRZWRT-ATVHPVEESA-N 0.000 description 4
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 238000000465 moulding Methods 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 229910052710 silicon Inorganic materials 0.000 description 3
- 239000010703 silicon Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- HKVAMNSJSFKALM-GKUWKFKPSA-N Everolimus Chemical compound C1C[C@@H](OCCO)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 HKVAMNSJSFKALM-GKUWKFKPSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 210000004504 adult stem cell Anatomy 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000001671 embryonic stem cell Anatomy 0.000 description 2
- 238000005530 etching Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 229960005167 everolimus Drugs 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 238000010874 in vitro model Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- ZAHRKKWIAAJSAO-UHFFFAOYSA-N rapamycin Natural products COCC(O)C(=C/C(C)C(=O)CC(OC(=O)C1CCCCN1C(=O)C(=O)C2(O)OC(CC(OC)C(=CC=CC=CC(C)CC(C)C(=O)C)C)CCC2C)C(C)CC3CCC(O)C(C3)OC)C ZAHRKKWIAAJSAO-UHFFFAOYSA-N 0.000 description 2
- 210000002254 renal artery Anatomy 0.000 description 2
- 229960002930 sirolimus Drugs 0.000 description 2
- QFJCIRLUMZQUOT-HPLJOQBZSA-N sirolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 QFJCIRLUMZQUOT-HPLJOQBZSA-N 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 208000029584 urinary system neoplasm Diseases 0.000 description 2
- 208000008035 Back Pain Diseases 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 102000004266 Collagen Type IV Human genes 0.000 description 1
- 108010042086 Collagen Type IV Proteins 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 108010085895 Laminin Proteins 0.000 description 1
- 208000008930 Low Back Pain Diseases 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- -1 Polydimethylsiloxane Polymers 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000012137 double-staining Methods 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 208000006750 hematuria Diseases 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000002601 intratumoral effect Effects 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 229960002378 oftasceine Drugs 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 229920000570 polyether Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 208000019465 refractory cytopenia of childhood Diseases 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 201000010174 renal carcinoma Diseases 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 210000001635 urinary tract Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/08—Flask, bottle or test tube
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/10—Petri dish
-
- 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
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Sustainable Development (AREA)
- Clinical Laboratory Science (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Oncology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for culturing kidney tumor organoids, belonging to the organoid culture field. The invention provides a kidney tumor organoid culture container, which is a cell culture container with a kidney extracellular matrix imprinting block array on the inner bottom surface; the area of the bottom surface not covered by the extracellular matrix of the kidney is blocked by a blocking agent; the kidney extracellular matrix imprinting block array consists of circular lattice points with the diameter of 50-200 mu m, and the distance between each two circular lattice points is 25-300 mu m. The invention also provides a culture method of the kidney tumor organoids, which shortens the culture time, simplifies the culture mode, effectively controls the particle size and arrangement of the kidney tumor organoids, and improves the repeatability and stability of drug screening. The cultured kidney tumor organoids are used for drug screening, so that the method has higher efficiency and safety, and can be used for personalized treatment and guiding clinical medication.
Description
Technical Field
The invention belongs to the field of organoid culture, and particularly relates to a method for culturing kidney tumor organoids.
Background
Urinary system tumors are one of the most common malignant tumors in the world, including prostate cancer, bladder cancer and renal cell carcinoma. Renal cell carcinoma (renal cell carcinoma, RCC) is abbreviated as renal carcinoma, and has the third most serious incidence among urinary system tumors. Renal cancer is a malignant tumor that originates from the urinary tract epithelial system of the kidney parenchyma, severely affects the renal function of the patient, and can metastasize to the outside of the kidney with the progression of the condition. At present, the pathogenesis and progress of kidney cancer is not completely clear, and is mostly related to genetics, environment, related chronic medical history and bad life habit. About 55% of patients with kidney cancer have no typical early manifestation, and more symptomatic patients are found in lumbago and hematuria than in healthy physical examination, and fewer patients are diagnosed due to the fact that the abdomen can touch the tumor. At present, the treatment measures about the kidney cancer mainly comprise comprehensive treatment including operation, radiotherapy and chemotherapy and targeted treatment. With the development of accurate medicine, tumor treatment has entered the individualization age: firstly, using high-flux gene sequencing to find out tumor related mutation and searching a drug target; and secondly, the sensitivity of the targeted drug is tested by using a tumor model, so that personalized treatment is realized. However, when kidney cancer is diagnosed, it is often too late, and it is important how to efficiently and briefly build an in vitro model capable of reflecting the heterogeneity of patients. To solve the above problems, researchers have focused on organoid technology.
Organoid technology is the most rapidly developing in vitro culture technology in the last decade, and has been honored as one of the most important scientific advances in 2013. The technology can induce and differentiate in vitro into functional cell clusters with the basic characteristics of organs derived from the cells, the embryonic stem cells and the adult stem cells by virtue of the tissues, the embryonic stem cells and the adult stem cells, and provides a new in vitro model for simulating living organs. In modern biomedical research, the most commonly used cell culture and animal models only partially simulate in-vivo environments, and cannot fully show the real situation of a research target in a human body. Researchers have therefore given great attention to 3D culture techniques, which they wish to better mimic the microenvironment of in vivo cell growth, especially tumor cells, based on the material structure basis that they retain the cell microenvironment. The human tumor organoids (PDO) retain individual tumor heterogeneity, and the screening of targeted drugs by PDO guides clinical drugs, thus showing great value in personalized treatment. The stability and fidelity of tumor organoids as drug screening models can be divided into three aspects. First, the tumor organoids retain the heterogeneity of the primary tumor during long-term culture, wherein the results of different generations of tumor organoids on anti-tumor drug screening are substantially consistent. Second, the results of drug screening of the tumor organoids are consistent with clinical medication of patients, which indicates that the tumor organoids have great potential value for accurate treatment. Thirdly, the tumor organoid can be regarded as a 'cell line' in the long-term culture process, and the same tumor organoid cell line is subjected to drug screening, so that the same tumor organoid has consistent drug screening results.
The tumor organoids can be passaged for a long time without losing or changing genetic information during subculture. The heterogeneity between intratumoral and external is preserved. At present, colorectal cancer, non-small cell lung cancer, breast cancer and pancreatic cancer tumor organoid models have been widely applied to drug evaluation, biomarker identification, biological research and personalized treatment. Patent CN113186165A, CN111607495A discloses a method for culturing kidney tumor organoids, which is mainly obtained by mixing kidney cancer cells or kidney cancer tissues with matrigel, or wrapping the cells or tissues with matrigel and then culturing the cells or tissues in a specific culture medium. However, the existing method for culturing kidney tumor organoids has the defects of complicated operation, higher cost, small scale, different organoids with different particle sizes, different planes, difficult analysis and observation, and the like, and cannot be used for large-scale mass production. Meanwhile, the existing method for culturing kidney tumor organoids has long culture period and may cause delay of patient's illness.
Therefore, the method for quickly searching the kidney tumor organoids has the advantages of being quick, simple and convenient to operate and low in cost, being uniform in culture size, growing on the same plane and being convenient to analyze and observe, and achieving the quick large-scale culture of the kidney tumor organoids has important significance.
Disclosure of Invention
The invention aims to provide a method for culturing kidney tumor organoids.
The invention provides a kidney tumor organoid culture container, which is a cell culture container with a kidney extracellular matrix imprinting block array on the inner bottom surface;
the area of the bottom surface not covered by the extracellular matrix of the kidney is blocked by a blocking agent;
the kidney extracellular matrix imprinting block array consists of circular lattice points with the diameter of 50-200 mu m, and the distance between each two circular lattice points is 25-300 mu m.
Further, the kidney extracellular matrix blot block array is composed of circular lattice points with a diameter of 100 μm, and the interval between each circular lattice point is 50 μm.
Further, the area of the kidney extracellular matrix blotting block array is 1-10 cm 2 ；
Preferably, the area of the kidney extracellular matrix blotting block array is 4cm 2 。
Further, the container is a culture dish, a culture box, a culture plate or a culture bottle;
and/or the blocking agent is Pluronic F-127;
preferably, the mass fraction of Pluronic F-127 is 1-5%.
Further, the kidney extracellular matrix is porcine kidney extracellular matrix;
preferably, the preparation method of the pig kidney extracellular matrix comprises the following steps:
(1) Preparing a pig whole kidney decellularized scaffold by adopting a Triton-SLES-Triton perfusion method;
(2) The obtained decellularized kidney stent is obtained by freeze-drying and crushing the decellularized kidney stent and then carrying out enzymolysis by pepsin.
Preferably, the Triton-SLES-Triton infusion method is to take fresh pig kidney, cannulate renal artery, freeze and thaw the kidney after removing blood, infuse with 1% Triton X-100 for 3 hours, then 1% sodium laureth sulfate (SLES) for 6 hours, infuse with 1% Triton X-100 for 3 hours, and finally infuse with PBS for 2 hours to balance the decellularized kidney, thus obtaining the decellularized kidney scaffold. The flow rate throughout the course was 15mL/min during the perfusion.
Preferably, during enzymolysis, the concentration of the extracellular matrix of the kidney obtained after crushing is 10mg/mL; the mass ratio of pepsin to extracellular matrix powder is 1:10. In addition, the pH was 2 during the enzymolysis, the temperature was room temperature, and the enzymolysis time was 72 hours.
Further, the concentration of the kidney extracellular matrix is 0.05-0.5 mg/mL;
preferably, the concentration of the kidney extracellular matrix is 0.1-0.5 mg/mL.
The kidney tumor organoid culture container is prepared by adopting a PDMS micro-pattern printing technology: firstly, a silicon wafer with a specific pattern is etched by using laser as a template, a micro-array PDMS stamp with a convex circular micro-pattern with a specific size is obtained by reverse molding, kidney extracellular matrix glue is coated on the surface of the PDMS stamp, the kidney extracellular matrix glue is stamped on the bottom surface of a culture container, an extracellular matrix micro-pattern array is obtained, and then the rest of the culture container is sealed by using a sealing agent, so that the kidney tumor organoid culture container is obtained.
The invention also provides a kidney tumor organoid culture method, which comprises the following steps:
inoculating the kidney cancer cells into the culture container, culturing for 4-6 h, washing off unattached cells, and culturing for 3-5 days to obtain the kidney tumor organoid.
Further, the inoculation density of the kidney cancer cells is 10 4 ～10 6 A personal/culture container;
and/or, the kidney cancer cells are derived from a human;
preferably, the inoculation density of the kidney cancer cells is 10 5 A culture vessel.
The invention also provides a kidney tumor organoid which is prepared by the method.
The invention also provides application of the kidney tumor organoid in constructing a kidney cancer model or screening medicines.
The invention provides a method for culturing kidney tumor organoids, which cultures kidney cancer cells by adopting kidney ECM pattern microarrays with specific lattice shapes, sizes and densities, prepares the kidney tumor organoids, shortens the in-vitro amplification culture time of the kidney tumor organoids, simplifies the culture mode of the kidney tumor organoids, and effectively overcomes the defects of complicated operation, higher cost, small scale, different organoid particle sizes, difficult analysis and observation and the like of the traditional tumor organoids. Effectively controls the particle size and arrangement of kidney tumor organoids and improves the repeatability and stability of drug screening. The kidney decellularized ECM provides a tumor microenvironment for kidney tumor organoid culture, is favorable for maintaining tumor heterogeneity, and simulates in vivo kidney cancer tissues. The kidney tumor organoid is used for drug screening, has higher efficiency and safety, and simultaneously PDO can be used for personalized treatment to guide clinical medication.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 shows the course of culture of kidney tumor organoids according to the invention.
Fig. 2 shows the results after decellularization of whole pig kidneys: a is the general appearance of the kidneys before and after decellularization; b is an electron microscope image before and after decellularization; c is DNA content detection before and after decellularization; d is H & E staining before and after decellularization; e is the dyeing of type I collagen fibers before and after decellularization; f is the staining of type IV collagen fibers before and after decellularization; g is fibronectin staining before and after decellularization; h is laminin staining before and after decellularization; scale = 20 μm; in the figure Native is non-decellularized and DLSs is decellularized.
Fig. 3 is a kidney decellularized ECM powder (left) kidney decellularized ECM gel (right).
FIG. 4 shows the results of the dead and alive staining of kidney tumor organoids after the action of each drug.
Fig. 5 shows a decellularized renal extracellular matrix micropattern array chip prepared by micropattern printing technology and a kidney tumor organoid cultured: a is PDMS seal; b is an extracellular matrix micropattern array; scale = 100 μm.
FIG. 6 shows the kidney tumor organoids formed after 3 days of culture.
Detailed Description
Unless otherwise indicated, the materials and equipment used in the embodiments of the present invention are all known products and are obtained by purchasing commercially available products.
The culture route of the kidney tumor organoid of the invention is shown in figure 1, and comprises the following steps:
(1) Preparation of kidney decellularized ECM: porcine whole kidney decellularized scaffold was prepared using Triton-SLES-Triton perfusion. The obtained decellularized kidney scaffold (kidney extracellular matrix) was further lyophilized, and after pulverization using a ball mill, the decellularized kidney extracellular matrix (ECM) gel was obtained by pepsin enzymolysis.
(2) Preparing a pattern micro-array master plate by adopting a PDMS micro-pattern printing technology: a silicon wafer with a specific pattern is etched by using laser as a template, the etching precision reaches 1 mu m, the etching area reaches 12 inches at maximum, and a Polydimethylsiloxane (PDMS) seal is obtained by reverse molding. The extracellular matrix gel (called extracellular matrix gel or matrigel) is coated on the surface of a seal, then a PDMS micro-pattern printing technology is utilized to lay an extracellular matrix gel pattern micro-array on the bottom of a cell culture dish which is subjected to non-attachment treatment, pluronic F-127 seals the space at the bottom of the rest culture dish, so that the added cells can only grow on the extracellular matrix gel pattern in an attached mode. The kidney decellularized ECM pattern microarray chip was obtained.
(3) Culture of kidney tumor organoids: the kidney decellularized ECM pattern microarray chip is used for culturing kidney tumor organoids, so that the in-vitro amplification culture time is shortened, and the particle size and arrangement of the kidney tumor organoids can be effectively controlled.
EXAMPLE 1 method of culturing kidney tumor organoids of the invention
The method for culturing the kidney tumor organoid comprises the following steps:
(1) Preparation of pig kidney decellularized extracellular matrix glue: after taking fresh pig kidneys, renal arteries were cannulated and flushed with PBS for 10 minutes to remove blood. Kidneys were then frozen and thawed, perfused with 1% triton X-100 for 3 hours, followed by 1% sodium laureth sulfate (SLES) for 6 hours, further perfused with 1% triton X-100 for 3 hours, and finally perfused with PBS for 2 hours to equilibrate the decellularized kidneys to give a decellularized renal stent (renal extracellular matrix). The flow rate throughout the course was 15mL/min during the perfusion. Decellularized kidney scaffolds (DLSs) were then cut into cubes of 1X 1cm and lyophilized. Further crushing the mixture by a ball mill to obtain kidney extracellular matrix powder, and carrying out enzymolysis on the kidney extracellular matrix powder by using pepsin to obtain the decellularized kidney extracellular matrix gel. During enzymolysis, extracellular matrix powder is dissolved in water, the concentration is 10mg/mL, the mass ratio of pepsin to extracellular matrix powder is 1:10, the pH is 2 during enzymolysis, the temperature is room temperature, and the enzymolysis time is 72 hours.
The pig kidney is white and transparent after decellularization, and after immunofluorescence, histochemical and enzyme-linked immunosorbent assay, extracellular matrix protein is well reserved (figure 2), and further, decellularized kidney extracellular matrix gel is obtained through crushing and enzymolysis (figure 3).
(2) A silicon wafer with a specific pattern is etched by using laser as a template, and a PDMS stamp is obtained by reverse molding, so that a pattern microarray with convex circular array points (each circular array point has a diameter of 100 μm, the distance between each circular array point is 50 μm, and the microarray size is 4 cm) 2 ) And a PDMS stamp.
(3) The matrigel concentration is regulated by PBS, 1mL of pig kidney decellularized extracellular matrigel with the concentration of 0.1mg/mL is coated on the surface of a PDMS seal, after incubation for 20 minutes at room temperature, the superfluous matrigel is washed off, and the mixture is dried in an incubator at 37 ℃.
(4) The PDMS stamp was pressed against the inner bottom surface of the cell non-attached culture dish, and a force of 0.2 newton was applied for 10 minutes, and the stamp was removed after the pattern was formed on the bottom surface of the culture dish.
(5) The mass fraction of 1% Pluronic F-127 (Prulinix F-127, a polypropylene glycol and ethylene oxide addition polymer (polyether)) was added and incubated at room temperature for 2 hours, and the space on the inner bottom surface of the remaining dish was closed to obtain an "extracellular matrix pattern microarray" having an adhesive effect on cells. Before the prepared microarray culture dish is used, PBS is used for cleaning for 3 times, so that the culture dish substrate cannot be completely dried, and then cells are inoculated.
(6) Human kidney cancer cells (os-rc-2) were inoculated in 1.5X10-dose in a petri dish 5 Dish) is cultured for 6 hours at 37 ℃, unattached cells are washed off, and the kidney tumor organoids which are visible with naked eyes can be formed after 3 days of culture. The kidney tumor organoids are all on the same plane, and have uniform size and controllable quantity.
Human kidney cancer cells are defined to attach to "extracellular matrix patterned microarrays" for ingrowth. The medium used in this step was 1640 medium containing 10% fetal bovine serum and 1% diabody.
The decellularized renal extracellular matrix pattern microarray chip is prepared by a micropattern printing technology, so that renal tumor cells are cultured, and the renal tumor organoid can be formed after 3 days of culture.
The beneficial effects of the present invention are demonstrated by specific test examples below.
Test example 1 screening of the kidney tumor organoids of the present invention for drugs
Experimental method
The kidney tumor organoids of 100 μm diameter cultured in example 1 were selected and screened with different concentrations (5. Mu.g/ml, 10. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml) of sunitinib, sirolimus and everolimus, respectively, and incubated for 24h and 48h. Dead and alive staining analysis was performed using calcein and propidium iodide fluorescent double staining and imaging analysis using confocal microscopy.
Experimental results
From the results of fig. 4, it can be seen that: the kidney tumor organoids cultured by the invention can effectively screen the influence of medicine types, medicine doses and medicine action time on kidney tumors. The results show that the activity of the kidney tumor organoids is related to the drug species, the drug dosage and the culture time. Sunitinib has the greatest inhibitory activity on kidney tumor organoids, and as sunitinib concentration and culture time increase, kidney tumor organoids stop growing. Sirolimus and everolimus have some inhibitory activity on kidney tumor organoids, but have weaker inhibitory activity than sunitinib. It was found that the organoids can be better analyzed and the results of drug screening observed after size control and alignment. The kidney tumor organoid pattern microarray chip is applied to personalized treatment and potential value of antineoplastic drug screening.
Test example 2 screening of the method for culturing kidney tumor organoids of the present invention
Early-stage researches show that if rectangular lattice points are used in the pattern microarray, the effect of preparing kidney tumor organoids is poor, and even kidney tumor organoids cannot be generated; while kidney tumor organoids prepared using circular lattice points are significantly better than rectangular lattice points. Thus, an extracellular matrix patterned microarray was prepared by the method described in example 1, and the size of the circular array spots (the diameters of the circular array spots were 50 μm, 100 μm, 150 μm, 200 μm, respectively) in the patterned microarray was changed, the interval between each circular array spot was 50 μm, and the microarray size was 4cm 2 . Each pattern microarray is shown in fig. 5.
According to realityExample 1A kidney tumor organoid was cultured separately using the above extracellular matrix patterned microarray, which was washed 1 time with PBS before use to ensure that the culture substrate was not completely dried, inoculated with human kidney cancer cells (os-rc-2, inoculum size 1.5X10 5 /dish). The patterned microarray was grown on spots defined in the extracellular matrix gel patterned microarray by restricting the growth and adhesion space of cells on the bottom of the cell culture dish, and kidney tumor organoids were formed by culturing for 3 days (fig. 6).
The optimal size of circular array spots in the microarray was selected based on the morphology of the kidney tumor organoids, the dead-alive function. The research finds that: when the diameter of the circular lattice point is too small (50 μm), kidney tumor organoids cannot be cultivated; with the increase of the diameter of the circular lattice points, dead cells in the kidney tumor organoids are increased, and the functional activity is poor, which is unfavorable for evaluation as a model. Considering comprehensively, the circular array point with the diameter of 100 μm is finally selected as the optimal array point for culturing the kidney tumor organoid pattern microarray, and the prepared kidney tumor organoid has optimal morphology and function.
In summary, the invention provides a method for culturing kidney tumor organoids, which cultures kidney cancer cells by adopting kidney ECM pattern microarrays with specific lattice shapes, sizes and densities, prepares the kidney tumor organoids, shortens the in-vitro amplification culture time of the kidney tumor organoids, simplifies the culture mode of the kidney tumor organoids, and effectively overcomes the defects of complicated operation, higher cost, small scale, different particle sizes of organoids, difficult analysis and observation and the like of the conventional tumor organoids. Effectively controls the particle size and arrangement of kidney tumor organoids and improves the repeatability and stability of drug screening. The kidney decellularized ECM provides a tumor microenvironment for kidney tumor organoid culture, is favorable for maintaining tumor heterogeneity, and simulates in vivo kidney cancer tissues. The kidney tumor organoid is used for drug screening, has higher efficiency and safety, and simultaneously PDO can be used for personalized treatment to guide clinical medication.
Claims (13)
1. A kidney tumor organoid culture container, characterized in that: the cell culture container is provided with a kidney extracellular matrix imprinting block array on the inner bottom surface;
the area of the bottom surface not covered by the extracellular matrix of the kidney is blocked by a blocking agent;
the kidney extracellular matrix imprinting block array consists of circular lattice points with the diameter of 100 mu m, and the interval between each circular lattice point is 50 mu m;
the concentration of the kidney extracellular matrix is 0.05-0.5 mg/mL.
2. A kidney tumor organoid culture vessel according to claim 1, wherein: the area of the kidney extracellular matrix imprinting block array is 1-10 cm 2 。
3. A kidney tumor organoid culture vessel according to claim 2, wherein: the area of the kidney extracellular matrix imprinting block array is 4cm 2 。
4. A kidney tumor organoid culture vessel according to any one of claims 1 to 3, wherein: the container is a culture dish, a culture box, a culture plate or a culture bottle;
and/or the blocking agent is Pluronic F-127.
5. A kidney tumor organoid culture vessel according to claim 4, wherein: the mass fraction of Pluronic F-127 is 1-5%.
6. A kidney tumor organoid culture vessel according to any one of claims 1 to 3, wherein: the kidney extracellular matrix is porcine kidney extracellular matrix.
7. A kidney tumor organoid culture vessel according to claim 6, wherein: the preparation method of the pig kidney extracellular matrix comprises the following steps:
(1) Preparing a pig whole kidney decellularized scaffold by adopting a Triton-SLES-Triton perfusion method;
(2) The obtained decellularized kidney stent is obtained by freeze-drying and crushing the decellularized kidney stent and then carrying out enzymolysis by pepsin.
8. A kidney tumor organoid culture vessel according to claim 1, wherein: the concentration of the kidney extracellular matrix is 0.1-0.5 mg/mL.
9. A kidney tumor organoid culture method, which is characterized in that: the method comprises the following steps:
inoculating the kidney cancer cells into the culture container according to any one of claims 1-8, culturing for 4-6 h, washing off unattached cells, and culturing for 3-5 days to obtain the kidney tumor organoid.
10. The culture method according to claim 9, wherein: the inoculation density of the kidney cancer cells is 10 4 ～10 6 A personal/culture container;
and/or, the kidney cancer cells are of human origin.
11. The culture method according to claim 10, wherein: the inoculation density of the kidney cancer cells is 10 5 A culture vessel.
12. A kidney tumor organoid, characterized in that: a kidney tumor organoid prepared by the method of any one of claims 9 to 11.
13. Use of the kidney tumor organoid of claim 12 in constructing a kidney cancer model or in drug screening.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111678865.XA CN114214284B (en) | 2021-12-31 | 2021-12-31 | Method for culturing kidney tumor organoids |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111678865.XA CN114214284B (en) | 2021-12-31 | 2021-12-31 | Method for culturing kidney tumor organoids |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114214284A CN114214284A (en) | 2022-03-22 |
| CN114214284B true CN114214284B (en) | 2024-01-26 |
Family
ID=80707553
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111678865.XA Active CN114214284B (en) | 2021-12-31 | 2021-12-31 | Method for culturing kidney tumor organoids |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114214284B (en) |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104208751A (en) * | 2014-08-19 | 2014-12-17 | 温州医科大学 | Preparation method for novel kidney acellularized biological scaffold |
| CN104841017A (en) * | 2015-05-28 | 2015-08-19 | 四川大学华西医院 | Cellularization biological liver stent with anticoagulation property and preparation method of cellularization biological liver stent |
| CN108102913A (en) * | 2016-11-25 | 2018-06-01 | 中国科学院苏州纳米技术与纳米仿生研究所 | Three-dimensional cell culture chip, its preparation method and application based on soft lithography |
| CN109182125A (en) * | 2018-09-25 | 2019-01-11 | 中国科学院苏州纳米技术与纳米仿生研究所 | Three-dimensional single cell source cell ball production chip, preparation method and application |
| CN110249044A (en) * | 2016-11-18 | 2019-09-17 | 洛桑联邦理工学院 | Organoid organizational project |
| CN111494718A (en) * | 2020-04-24 | 2020-08-07 | 四川大学华西医院 | Preparation method of animal decellularization lung biological scaffold material |
| CN111826338A (en) * | 2019-12-27 | 2020-10-27 | 四川大学华西医院 | Method for rapidly culturing liver bud organoid |
| CN112210536A (en) * | 2020-09-03 | 2021-01-12 | 电子科技大学 | 2D and 3D cell co-culture system capable of being continuously harvested without enzyme digestion and construction method and application thereof |
| CN113151177A (en) * | 2021-05-21 | 2021-07-23 | 四川大学华西医院 | Mammary gland or breast cancer tissue acellular matrix and preparation method and application thereof |
| CN113186165A (en) * | 2021-04-15 | 2021-07-30 | 北京大学 | Kidney cancer related organoid combination and application thereof |
| CN114214282A (en) * | 2021-12-31 | 2022-03-22 | 四川大学华西医院 | Method for culturing lung tumor organoid |
| CN114214283A (en) * | 2021-12-31 | 2022-03-22 | 四川大学华西医院 | Method for culturing liver tumor organoid |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2210589B1 (en) * | 2009-01-22 | 2015-05-06 | Ludwig-Maximilians-Universität München | Vesicular phospholipid gels comprising proteinaceous substances |
| WO2014169031A1 (en) * | 2013-04-10 | 2014-10-16 | Tufts University | Two and three dimensional decellularized ecm constructs and uses therefor |
| WO2017024206A1 (en) * | 2015-08-05 | 2017-02-09 | Colorado State University Research Foundation | Engineered platforms to stabilize both hepatocytes and endothelial cells in vitro |
-
2021
- 2021-12-31 CN CN202111678865.XA patent/CN114214284B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104208751A (en) * | 2014-08-19 | 2014-12-17 | 温州医科大学 | Preparation method for novel kidney acellularized biological scaffold |
| CN104841017A (en) * | 2015-05-28 | 2015-08-19 | 四川大学华西医院 | Cellularization biological liver stent with anticoagulation property and preparation method of cellularization biological liver stent |
| CN110249044A (en) * | 2016-11-18 | 2019-09-17 | 洛桑联邦理工学院 | Organoid organizational project |
| CN108102913A (en) * | 2016-11-25 | 2018-06-01 | 中国科学院苏州纳米技术与纳米仿生研究所 | Three-dimensional cell culture chip, its preparation method and application based on soft lithography |
| CN109182125A (en) * | 2018-09-25 | 2019-01-11 | 中国科学院苏州纳米技术与纳米仿生研究所 | Three-dimensional single cell source cell ball production chip, preparation method and application |
| CN111826338A (en) * | 2019-12-27 | 2020-10-27 | 四川大学华西医院 | Method for rapidly culturing liver bud organoid |
| CN111494718A (en) * | 2020-04-24 | 2020-08-07 | 四川大学华西医院 | Preparation method of animal decellularization lung biological scaffold material |
| CN112210536A (en) * | 2020-09-03 | 2021-01-12 | 电子科技大学 | 2D and 3D cell co-culture system capable of being continuously harvested without enzyme digestion and construction method and application thereof |
| CN113186165A (en) * | 2021-04-15 | 2021-07-30 | 北京大学 | Kidney cancer related organoid combination and application thereof |
| CN113151177A (en) * | 2021-05-21 | 2021-07-23 | 四川大学华西医院 | Mammary gland or breast cancer tissue acellular matrix and preparation method and application thereof |
| CN114214282A (en) * | 2021-12-31 | 2022-03-22 | 四川大学华西医院 | Method for culturing lung tumor organoid |
| CN114214283A (en) * | 2021-12-31 | 2022-03-22 | 四川大学华西医院 | Method for culturing liver tumor organoid |
Non-Patent Citations (4)
| Title |
|---|
| Adam D. McInnes等.Preparation and Use of Decellularized Extracellular Matrix for Tissue Engineering.Journal of Functional Biomaterials.2022,第13卷(第4期),1-54. * |
| Yi Li等.Decellularization of porcine whole lung to obtaina clinical-scale bioengineered scaffold.J Biomed Mater Res A.第109卷(第9期),第1624段左栏第3段. * |
| 刘春晓等.灌注法制备大鼠全肾脏脱细胞基质的研究.南方医科大学学报.2021,第29卷(第5期),979-982. * |
| 许洁.大鼠子宫去细胞支架及其细胞外基质凝胶的制备.生物医学工程学杂志.2018,第35卷(第2期),第238页左栏第1段. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114214284A (en) | 2022-03-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108624561A (en) | Primary tumor cell culture medium, cultural method and application | |
| CN106574242A (en) | Single cell-derived organoids | |
| CN110475860A (en) | Use the dimensional culture of the primary cancer cell of tumor tissues | |
| WO2020187081A1 (en) | Method for preparing three-dimensional biological construct, and three-dimensional biological construct and use thereof | |
| CN102250840B (en) | Human pancreatic cancer cell line and its application | |
| TWI666448B (en) | Method for predicting effect of drug | |
| CN104531620A (en) | Method for culturing lung cancer stem cells under 3D culture conditions | |
| CN112592897A (en) | Preparation method of tumor organoid | |
| CN102329775B (en) | A kind of human pancreatic cancer cell to gemcitabine resistance and application thereof | |
| CN110878285A (en) | Chip organ model for screening bladder tumor chemotherapy drugs and manufacturing method thereof | |
| Li et al. | On-chip modeling of tumor evolution: Advances, challenges and opportunities | |
| CN114214282B (en) | Method for culturing lung tumor organoids | |
| CN104673743A (en) | Tissue block culture method for obtaining primary cell from animal tissue | |
| US20200270557A1 (en) | Human in vitro orthotopic and metastatic models of cancer | |
| CN114214283B (en) | Method for culturing liver tumor organoids | |
| Nair et al. | Multi compartmental 3D breast cancer disease model–recapitulating tumor complexity in in-vitro | |
| CN106676074B (en) | Method for in-vitro amplification of liver cancer stem cells | |
| CN114214284B (en) | Method for culturing kidney tumor organoids | |
| CN114958756B (en) | Culture solution for culturing prostate cancer organoid and preparation method thereof | |
| EP3684915A1 (en) | Fabrication of a biomimetic platform system and methods of use | |
| EP4098740A1 (en) | Decellularized heart extracellular matrix-derived scaffold for culturing and transplanting heart organoid, and preparation method therefor | |
| Leighton et al. | The propagation of cancer, a process of tissue remodeling: studies in histophysiologic gradient culture | |
| Kolahi Azar et al. | The progressive trend of modeling and drug screening systems of breast cancer bone metastasis | |
| Liu et al. | Breast tumor-on-chip: from the tumor microenvironment to medical applications | |
| Chaji | Bioprinting of three-dimensional models to evaluate adipocyte breast cancer cell interactions and migration |
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 |