CN116716236A - Construction method and application of cervical cancer organoids - Google Patents

Construction method and application of cervical cancer organoids Download PDF

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Publication number
CN116716236A
CN116716236A CN202310783193.1A CN202310783193A CN116716236A CN 116716236 A CN116716236 A CN 116716236A CN 202310783193 A CN202310783193 A CN 202310783193A CN 116716236 A CN116716236 A CN 116716236A
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cervical
organoid
cervical cancer
final concentration
mass
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邹冬玲
何密斯
王海霞
朱雪萍
郑倩
隆玲
毛泽佳
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Chongqing University Cancer Hospital
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Chongqing University Cancer Hospital
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Abstract

The application discloses a construction method and application of cervical cancer organoids, and provides a culture method, a culture medium and application of cervical cancer organoids. The cervical cancer organoid culture method provided by the application has higher culture success rate, provides a basis for research on pathogenesis of cervical cancer patients, especially cervical cancer patients, and selection of treatment schemes, and has wide application prospects.

Description

Construction method and application of cervical cancer organoids
Technical Field
The application belongs to the field of biological medicine, and in particular relates to a construction method and application of cervical cancer organoids.
Background
So far, the most commonly used in-vitro drug sensitivity test models for tumors mainly include a human tumor cell line and a human tumor xenograft model (PDX). Although both of these model systems have contributed significantly to the transformation study of cervical rare types of cancer, they also have drawbacks. Human tumor cell lines are a relatively inexpensive and rapid model system suitable for high throughput drug screening, but because of the extensive adaptation and selection of in vitro 2D culture conditions involved in their generation from raw patient material, no genomic features of the original tissue and only rare clones can expand and remain. The PDX model grown in immunodeficient mice better mimics the original tumor characteristics, but has low success rate of construction, long time and high cost, cannot be subjected to high-throughput drug screening, and PDX may undergo the special tumor evolution of mice, and cannot accurately reflect the characteristics of human tumor tissues.
In recent years, a human-derived organoid model is constructed by 3D culturing in vitro cells of normal tissue, tumor tissue or malignant body fluid obtained from a human body. Organoids belong to three-dimensional (3D) cell cultures, which contain some key properties that represent organs. Such in vitro culture systems contain a self-renewing stem cell population that can differentiate into a variety of organ-specific cell types, possess similar spatial organization arrangements as the corresponding organ and reproduce part of the function of the corresponding organ, thereby providing a highly pathophysiologically relevant system.
Therefore, compared with a humanized tumor cell line and a humanized tumor xenograft model, the humanized tumor organoid derived from tumor tissue or malignant body fluid of a patient can not only highly retain the histological and genetic characteristics of the original tumor and display the heterogeneity of the tumor among patients, but also has high efficiency and less time consumption, and is more suitable for high-throughput drug screening and new drug research and development.
Disclosure of Invention
In order to make up for the defects of the prior art, the application provides a cervical cancer organoid culture method and related application.
In order to achieve the above purpose, the application adopts the following technical scheme:
in a first aspect, the application provides a method of culturing cervical cancer organoids comprising culturing cervical cancer cells using a medium comprising a BMP inhibitor, an N-acetylcysteine, nicotinamide, p inhibitor, a TGF- β inhibitor, a FGF, forskolin, wnt agonist, a Rock inhibitor, estradiol, and/or a mitogenic proliferation factor.
Further, the BMP inhibitor is selected from Noggin.
Further, the p38 inhibitor is selected from SB-202190.
Further, the TGF-beta inhibitor is selected from A83-01.
Further, the FGF comprises FGF7 and/or FGF10.
Further, the Wnt agonist is selected from the group consisting of Rspo1.
Further, the Rock inhibitor is selected from Y27632.
Further, the Estradiol is selected from beta-Estradiol.
Further, the mitogenic proliferation factor is selected from EGF.
Further, the final concentration of Noggin is 97-103ng/mL, the final concentration of SB-202190 is 0.5-1.5 mu M, the final concentration of A83-01 is 450-550nM, the final concentration of FGF7 is 20-30ng/mL, the final concentration of FGF10 is 95-105ng/mL, the final concentration of Rspo1 is 240-260ng/mL, the final concentration of Y27632 is 9-11 mu M, the final concentration of beta-Estradiol is 95-105nM, the final concentration of EGF is 45-55ng/mL, the final concentration of Forskolin is 9-11 mu M, the final concentration of Nicotinamide is 2-3mM, and the final concentration of N-acetylcysteine is 1-1.5mM.
Further, the final concentration of Noggin is 100ng/mL, SB-202190 is 1. Mu.M, A83-01 is 500nM, FGF7 is 25ng/mL, FGF10 is 100ng/mL, rspo1 is 250ng/mL, Y27632 is 10. Mu.M, beta-Estradiol is 100nM, EGF is 50ng/mL, forskolin is 10. Mu.M, nicotinamide is 2.5mM, and N-acetylcysteine is 1.25mM.
Further, the SB-202190 is 0.000033-0.0000339% by mass volume, the A83-01 is 0.000015-0.000025% by mass volume, the FGF7 is 0.000002-0.000003% by mass volume, the Rspo1 is 0.00002-0.00003% by mass volume, the Y27632 is 0.0003-0.00038% by mass volume, the beta-Estradiol is 0.0000026-0.0000028% by mass volume, the EGF is 0.000004-0.000006% by mass volume, the Forskolin is 0.00035-0.00047% by mass volume, the Nicotinamide is 0.02-0.04% by mass volume, and the N-acetylcysteine is 0.01-0.03% by mass.
Further, the Noggin is 0.00001% by mass volume, the SB-202190 is 0.0000334% by mass volume, the A83-01 is 0.000021% by mass volume, the FGF7 is 0.0000025% by mass volume, the FGF10 is 0.00001% by mass volume, the Rspo1 is 0.000025% by mass volume, the Y27632 is 0.00032% by mass, the beta-Estradiol is 0.0000027238% by mass volume, the EGF is 0.000005% by mass, the Forskolin is 0.00041% by mass, the Nicotinamide is 0.03% by mass, and the N-acetylcysteine is 0.02% by mass.
Further, the culture medium also comprises nutritional additives, antibiotics and buffer solution.
Further, the nutritional additives include Glutamax and/or B27.
Further, the antibiotic is selected from Pen Strep.
Further, the buffer is selected from HEPES.
Further, the final concentration of Glutamax, pen Strep, B27 was 1×, and the final concentration of HEPES was 10mM.
Further, the volume percentage of the Glutamax, the Pen Strep and the HEPES is 1%, and the volume percentage of the B27 is 2%.
Further, the cervical cancer cells are obtained by digesting cervical cancer tissue.
Further, cervical cancer tissue was digested with collagenase and Tryple.
Further, the concentration of collagenase is 0.5-2mg/mL.
Further, the concentration of collagenase was 1mg/mL.
Further, the concentration of Tryple was 1 XTryple.
Further, the method also includes filtering the digested cervical cancer cells.
Further, filtration was performed using a 100 μm cell filter.
Further, the method further comprises washing the tissue prior to digestion.
Further, the tissue was washed with PBS.
Further, the method also includes adding ECM to co-culture with cervical cancer cells.
Further, the ECM includes BME and matrigel.
Further, the ECM is selected from matrigel.
Further, the cell and matrigel were 1×10 4 -1×10 6 50 (. Mu.l) of the seed was inoculated.
Further, the cell and matrigel are mixed in a ratio of 1.5-3×10 5 50 (. Mu.l) of the seed was inoculated.
Further, the method further comprises passaging the organoid.
Further, the passage of the organoids includes digestion of the organoids.
Further, the organoids were digested with Tryple.
Further, the concentration of the Tryple is 1×tryple.
Further, the method further comprises washing the organoid prior to digesting the organoid.
Further, HBSS was used to wash organoids.
Further, organoid passaging also included termination of digestion.
Further, the method comprises the steps of, adDF++ was used digestion is terminated.
Further, the passaging ratio of organoids was 1:2-4.
Further, the cervical cancer includes cervical squamous carcinoma, cervical adenocarcinoma, cervical endometrioid carcinoma, cervical small cell neuroendocrine carcinoma, cervical complex mucous carcinoma, cervical medium-and renal-tube carcinoma, cervical high-grade serous carcinoma, cervical gastric adenocarcinoma, cervical intestinal adenocarcinoma.
Further, the cervical cancer includes cervical adenocarcinoma, cervical endometrioid carcinoma, cervical small cell neuroendocrine carcinoma, cervical complex mucous carcinoma, cervical medium-tubular carcinoma, cervical high-grade serous carcinoma, cervical gastric adenocarcinoma, cervical intestinal adenocarcinoma.
In a second aspect the application provides a cervical cancer organoid obtainable using the method according to the first aspect of the application.
A third aspect of the application provides the use of any one of the following:
(1) The application of the organoid in the second aspect of the application in constructing cervical cancer radiotherapy efficacy/synchronous radiotherapy and chemotherapy efficacy prediction models;
(2) The application of the organoids of the second aspect of the application in constructing cervical cancer drug screening models;
(3) The application of the organoid of the second aspect of the application in screening medicines for preventing and/or treating cervical cancer or research and development of new medicines for cervical cancer;
(4) The use of the organoids of the second aspect of the application in toxicity testing or in cervical cancer drugs;
(5) The application of the organoid in constructing a cervical cancer drug toxicity test model is provided.
The fourth aspect of the application is a model as set forth in any one of the following:
(1) A cervical cancer radiotherapy efficacy/synchronous radiotherapy and chemotherapy efficacy prediction model, the model comprising the organoid of the second aspect of the application;
(2) An in vitro drug screening model for cervical cancer, said model comprising an organoid according to the second aspect of the application;
(3) A cervical cancer drug toxicity test model comprising an organoid according to the second aspect of the application;
(4) A new drug development model for cervical cancer, said model comprising an organoid according to the second aspect of the application.
In a fifth aspect the application provides a method of predicting sensitivity to radiotherapy/synchrotron radiation for cervical cancer, the method comprising contacting a candidate drug and/or therapeutic radiation with an organoid according to the second aspect of the application.
Further, the method further comprises contacting the organoid with radiation.
In a sixth aspect the application provides a method of testing the toxicity of a cervical cancer drug, the method comprising contacting a candidate drug with an organoid according to the second aspect of the application.
In a seventh aspect, the present application provides a method of screening for a drug for the prevention/treatment of cervical cancer, the method comprising contacting a candidate drug with an organoid according to the second aspect of the application.
The application has the advantages and beneficial effects that:
the cervical cancer organoid culture method provided by the application has higher culture success rate, provides a basis for research on pathogenesis of cervical cancer patients, especially cervical cancer patients, and selection of treatment schemes, and has wide application prospects.
Drawings
FIG. 1 is a cervical rare type carcinoma organoid growth state diagram, wherein 1A is a cervical adenocarcinoma organoid growth state diagram, 1B is a cervical endometrioid carcinoma organoid growth state diagram, 1C is a cervical small cell neuroendocrine carcinoma organoid growth state diagram, 1D is a cervical multiple layer mucous carcinoma organoid growth state diagram, 1E is a cervical medium renal tube carcinoma organoid growth state diagram, 1F is a cervical high grade serous carcinoma organoid growth state diagram, 1G is a cervical gastric adenocarcinoma organoid growth state diagram, 1H is a cervical intestinal adenocarcinoma organoid growth state diagram;
FIG. 2 is a chart of the state of growth of a passaged cervical adenocarcinoma organoid.
Detailed Description
The following provides definitions of some of the terms used in this specification. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
The present application provides a method of culturing cervical cancer organoids comprising culturing cervical cancer cells using a medium comprising a BMP inhibitor, an N-acetylcysteine, nicotinamide, p inhibitor, a TGF-beta inhibitor, a FGF, forskolin, wnt agonist, a Rock inhibitor, estradiol, and/or a mitogenic proliferation factor.
In the present application, organoids refer to cell clusters having a 3D three-dimensional structure, and refer to miniaturized and simplified versions of organs prepared by artificial culture. The source of the cells constituting the organoids is not limited. Organoids may be derived from tissues, body fluids, and stem cells. Organoids may have the ability to allow interaction with the surrounding environment during cell growth. Thus, the 3D organoids of the present application, which almost completely mimic in vivo interactions, can be an excellent model for studying disease. The human body model can similarly replicate the pathophysiological activity function of the human body, can carry out disease modeling according to the genetic information of a patient by constructing organ analogs from the tissues of the patient, and can carry out drug screening and predict the curative effect of radiotherapy/synchronous radiotherapy and chemotherapy by in vitro testing.
In the present application, BMP (bone morphogenic protein, bone morphogeneitc protein) inhibitors include, but are not limited to Noggin (Noggin), gremlin (lattice Lei Lin), chord (tenascin), tenascin domain and like tenascin-like proteins, follistatin (Follistatin), follistatin domain and like Follistatin-related proteins, DAN-like proteins including DAN cysteine domain and like proteins, sclerostin/SOST, decorin, alpha 2-macroglobulin.
In a specific embodiment of the application, the BMP inhibitor is selected from Noggin (Noggin).
In the present application, N-Acetylcysteine (N-Acetylcysteine) is also called Acetylcysteine (ACCESSIN) or N-acetyl-L-cysteine (N-ACCESSIN-L-cysteine) is often Jian Chen NAC.
In the present application, a P38 inhibitor (P38 inhibitor) refers to any inhibitor that directly or indirectly down-regulates P38 signaling. In general, p38 inhibitors bind to p38 and reduce its activity. Including but not limited to SB-202190 (4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole), SB-203580 (4- [4- (4-fluorophenyl) -2- [4- (methylsulfinyl) -phenyl ] -1H-imidazol-5-yl ] pyridine), VX-702 (6- (N-carbamoyl-2, 6-difluorophenylamino) -2- (2, 4-difluorophenyl) pyridine-3-carboxamide), VX-745 (5- (2, 6-dichlorophenyl) -2- [2, 4-difluorophenyl) thio ] -6H-pyrimido [1,6-b ] pyridazin-6-one), PD-169316 (4- (4-fluorophenyl) -2- (4-nitrophenyl) -5- (4-pyridyl) -1H-imidazole), RO-4402257 (6- (2, 4-difluorophenoxy) -2- { [ 3-hydroxy-1- (2-hydroxyethyl) propyl ] amino } -8-methylpyrido [2,3-D ] pyrimidine-8H), BIRB-796 (1- [ 5-tert-butyl-2- (4-methylphenyl) pyrazol-3-yl ] -3- [4- (2-morpholin-4-ylethoxy) naphthalen-1-yl ] urea).
In a specific embodiment of the application, the p38 inhibitor is selected from SB-202190 (4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole).
In the present application, TGF- β inhibitors include, but are not limited to, A83-01 (3- (6-methylpyridin-2-yl) -1-phenylthiocarbamoyl-4-quinolin-4-ylpyrazole), ALK5 inhibitor I (3- (pyridin-2-yl) -4- (4-quinolyl) -1H-pyrazole), LDN193189 (4- (6- (4- (piperazin-1-yl) phenyl) pyrazolo [1,5-a ] pyrimidin-3-yl) quinoline), SB431542 (4- [4- (1, 3-benzodioxazol-5-yl) -5-pyridin-2-yl-1H-imidazol-2-yl ] benzamide), SB-124 (2- (5-benzo [1,3] dioxazol-5-yl-2-tert-butyl-3H-imidazol-4-yl) -6-methylpyridin hydrochloride hydrate), SD-208 (2- (5-chloro-2-fluorophenyl) pteridin-4-yl) pyridin-4-yl-amine), SB431542 (4- [4- (1, 3-benzodioxazol-5-yl) -5-pyridin-2-yl ] benzamide), SB-124 (2- (5-benzo [1, 3-dioxazol-5-yl-2-imidazol-yl) imidazol-hydrochloride hydrate), SD-208 (1-methyl) pyridin-5-yl hydrochloride), LY-364947 (4- [3- (2-pyridinyl) -1H-pyrazol-4-yl ] -quinoline), LY2157299 (4- [2- (6-methyl-pyridin-2-yl) -5, 6-dihydro-4H-pyrrolo [1,2-b ] pyrazol-3-yl ] -quinoline-6-carboxamide), TGF-beta RI kinase inhibitor II 616452 (2- (3- (6-methylpyridin-2-yl) -1H-pyrazol-4-yl) -1, 5-naphthyridine), TGF-beta RI kinase inhibitor III 616453 (2- (5-benzo [1,3] dioxazol-4-yl-2-tert-butyl-1H-imidazol-4-yl) -6-methylpyridin, HCl), TGF-beta RI kinase inhibitor IX 616463 (4- ((4- ((2, 6-dimethylpyridin-3-yl) oxy) pyridin-2-yl) amino) benzenesulfonamide), TGF-beta RI kinase inhibitor VII 616458 (1- (2- ((6, 7-dimethoxy-4-quinolin-yl) oxy) - (4, 5-dimethylphenyl) -ethyl-1-ethyl-ketone, TGF-beta RI kinase inhibitor VIII 616459 (6- (2-tert-butyl-5- (6-methyl-pyridin-2-yl) -1H-imidazol-4-yl) -quinoxaline), AP12009 (TGF-beta 2 antisense compound "Trabedersen"), belagapumatecel-L (TGF-beta 2 antisense gene modified allogeneic tumor cell vaccine), CAT-152 (glaucoma-Lepiderman (anti-TGF-beta 2 monoclonal antibody)), CAT-192 (metimab (human IgG4 monoclonal antibody neutralizing TGF beta 1), GC-1008 (anti-TGF-beta monoclonal antibody).
In a specific embodiment of the application, the TGF- β inhibitor is selected from the group consisting of A83-01 (3- (6-methylpyridin-2-yl) -1-phenylthiocarbamoyl-4-quinolin-4-ylpyrazole).
In the present application, FGF (fibroblast growth factor ), also called heparin binding growth factor, mainly includes two major classes, acidic and basic, and refers to a class of active proteins or polypeptides capable of promoting cell growth. Including but not limited to FGF-basic, FGF1, FGF2, FGF4, FGF7, FGF10, FGF18, fibroblast growth factor-synthesized peptides.
In particular embodiments of the application, FGF comprises FGF7, FGF10.
In the present application Rock (Rho-kinase) inhibitors include, but are not limited to, Y-27632 ((R) - (+) -trans-4- (1-aminoethyl) -N- (4-pyridinyl) cyclohexanecarboxamide dihydrochloride monohydrate), HA1077 (fasudil) (5- (1, 4-diazepan-1-ylsulfonyl) isoquinoline), H-1152 ((S) - (+) -2-methyl-1- [ (4-methyl-5-isoquinolinyl) sulfonyl ] -hexahydro-1H-1, 4-diazepine dihydrochloride).
In a specific embodiment of the application, the Rock inhibitor is selected from the group consisting of Y-27632 ((R) - (+) -trans-4- (1-aminoethyl) -N- (4-pyridinyl) cyclohexanecarboxamide dihydrochloride monohydrate).
In the present application, wnt agonists refer to agents that activate T-cytokine (TCF)/lymphokine-mediated transcription (LEF) in cells. Wnt agonists are not limited to Wnt family proteins, but include Wnt agonists that bind to frizzled receptor family members for activation, intracellular β -catenin, and TCF/LEF activating substances. Wnt agonists include Wnt proteins, R-spondin (R-spongosine) and GSK-3 beta inhibitors.
Among them, wnt proteins derived from various organisms can be used. Preferred are Wnt proteins of mammalian origin. Examples of the mammal include humans, mice, rats, cows, pigs, and rabbits. The mammalian Wnt proteins include Wnt1, wnt2b, wnt3a, wnt4, wnt5a, wnt5b, wnt6, wnt7a, wnt7b, wnt8a, wnt8b, wnt9a, wnt9b, wnt10a, wnt10b, wnt11, and Wnt16.
GSK-3 beta inhibitors include CHIR-99021, CHIR-98014, lithium, kenparone, 6-bromoindirubin-30-acetoxime chem. Biol.10, 1255-1266), SB 216763 and SB 415286, and FRAT family members and FRAT derived peptides which prevent GSK-3 interaction with Axin.
R-spondin (R-spongehprin) includes R-spondin 1 (Rspo 1, R-spongehprin 1), R-spondin 2 (Rspo 2, R-spongehprin 2), R-spondin 3 (Rspo 3, R-spongehprin 3) and R-spondin 4 (Rspo 4, R-spongehprin 4). In the present application, R-spongin may be used in various combinations.
In an embodiment of the application, the Wnt agonist is selected from the group consisting of R-spondin (R-cavernosum).
In a specific embodiment of the present application, R-spondin (R-spongosine) is selected from the group consisting of R-spondin 1 (Rspo 1, R-spongosine 1).
In the present application, estradiol means (17 beta) -estra-1, 3,5 (10) -triene-3, 17-diol. Estradiol is also interchangeably referred to as 17β -estradiol (oestradiol) or E2. There are two types, α, β.
In a specific embodiment of the application, the Estradiol is selected from beta-Estradiol (beta-oestradiol).
In the present application, mitogenic proliferation factors include, but are not limited to, a family of growth factors such as EGF (epidermal growth factor ), BDNF (brain derived nerve growth factor, brain derived neurotrophic factor), KGF (keratinocyte growth factor ), and the like. These mitogenic proliferation factors may be used in various combinations.
In a specific embodiment of the application, the mitogenic proliferation factor is selected from EGF (epidermal growth factor ).
The culture medium also comprises nutritional additives, antibiotics and buffer solution.
In the application, the antibiotics comprise one or more of Pen Strep, primocin, penicillin streptomycin and metronidazole.
In a specific embodiment of the application, the antibiotic is selected from Pen Strep.
In the present application, the nutritional additives include Gluta Max, B27, and/or N2.
In a specific embodiment of the application, the nutritional additive is selected from Glutamax and/or B27.
The media of the present application also includes basal media including DMEM (Dulbecco's modified Igor's Medium, dulbecco's Modified Eagle Medium), DMEM/nutrient mixture F-12 (DMEM/F-12), higher DMEM/F-12, RPMI 1640, IMDM (Dulbecco's modified Igor's Medium), MEM (minimal basal medium), BME (basal medium Igor), knockOut DMEM/F12, advanced DMEM/F12, neurobasal, in one embodiment, basal media includes combinations of the above exemplary basal media in various proportions.
In a specific embodiment of the application, the basal medium is selected from Advanced DMEM/F12.
In the present application, the organoid medium may further comprise amino acids including L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and combinations thereof.
In the present application, the organoid medium may further comprise vitamins including, but not limited to, thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), calcium D-pantothenate (vitamin B5), pyridoxal/pyridoxamine/pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamine (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), DL-alpha-tocopherol (vitamin E), biotin (vitamin H), menaquinone (vitamin K).
In the present application, the organoid medium may further comprise inorganic salts including, but not limited to, salts of calcium, copper, iron, magnesium, potassium, sodium, zinc. Salts are generally used in the form of chlorides, phosphates, sulphates, nitrates and bicarbonates. More specifically, salts include, but are not limited to CaCl 2 、CuSO 4 ·5H 2 O、Fe(NO 3 )·9H 2 O、FeSO 4 ·7H 2 O、MgCl、MgSO 4 、KCl、NaHCO 3 、NaCl、Na 2 HPO 4 、Na 2 HPO 4 ·H 2 O、ZnSO 4 ·7H 2 O。
In the present application, the organoid medium may further comprise a sugar that can be a source of carbon energy. Sugars include, but are not limited to, glucose, galactose, mannose, fructose. Among them, glucose is preferable, and D-glucose (dextrose) is more preferable.
In the present application, the organoid medium may further comprise trace elements. The microelements comprise barium, bromine, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, aluminum or ions thereof.
The cervical cancer cells are obtained by digesting cervical cancer tissue.
Cervical cancer tissue was digested with collagenase and Tryple.
Wherein the collagenase comprises collagenase I, collagenase II and collagenase IV.
The method further comprises adding ECM to co-culture with cervical cancer cells.
In the present application, the ECM (extracellular matrix) is a three-dimensional matrix including BME (basement membrane extract) and Matrigel (Matrigel).
In a specific embodiment of the application, the ECM is selected from matrigel.
The present application provides a method for screening a drug for preventing/treating cervical cancer, which comprises contacting a candidate drug with the above-mentioned organoids.
In the present application, contacting a candidate drug with the above-described organoids involves exposing the organoids to the candidate drug. Comprising dissolving the candidate drug in solution to a (predicted) therapeutically effective concentration and administering it by injection or other suitable means into a container holding the organoid in contact with the organoid.
Further comprising a detection step comprising detecting any biochemical, genetic, phenotypic or phenomenological change of the organoid. Including decreased organoid viability, decreased organoid proliferation, increased organoid death, changes in organoid size, number of organoids, expression of marker proteins of cells comprising organoids. The change in the size of the organoid may be, for example, a measurement of the organoid size, and the organoid size may be measured by a known device (for example, a microscope or FACS). The organoid size may be, for example, the organoid circumference, diameter (e.g., major and minor diameters), or area. The expression of the marker protein of the cells constituting the organoid can be measured using a known device (e.g., a microscope or FACS) and a known reagent (e.g., an antibody labeled with a fluorescent substance). The expression of the marker protein of the cells constituting the organoid may be, for example, fluorescence intensity corresponding to the amount of the marker protein expressed per unit area of the organoid. The marker protein to be assayed may be one or a combination of two or more. The marker protein to be measured is preferably a combination of two or more.
In the present application, the organoid measurement value may be, for example, one measurement value measured at a time, an average value measured a plurality of times, or an average value measured a plurality of organoids. The organoid measurement may be, for example, a change (e.g., a difference or multiple) before and after exposure to the candidate drug. For example, when a value after the candidate drug is contacted is used as a measurement value of an organoid, a measurement value of a control organoid before or without the candidate drug is used as a control value.
For example, when the measurement is of the organoid size, the determination of whether the drug candidate is a substance capable of preventing and/or treating cervical cancer may include the steps of: when the measured value of the organoid after contacting the candidate drug is smaller than the control value (i.e., when the degree of increase in the organoid size is decreased or the size is reduced), the candidate drug is judged as a drug capable of preventing and/or treating cervical cancer.
In the present application, treatment refers to any method for partially or completely alleviating, ameliorating, reducing, inhibiting, preventing, delaying the onset of, reducing the severity and/or incidence of one or more symptoms or features of a particular disease, disorder and/or condition. The treatment may be administered to a subject that does not exhibit a sign of the disease and/or exhibits only an early sign of the disease in order to reduce the risk of developing a condition associated with the disease.
The application is further illustrated below in connection with specific embodiments. It should be understood that the particular embodiments described herein are presented by way of example and not limitation. The principal features of the application may be used in various embodiments without departing from the scope of the application.
Examples
1 Experimental materials
A special culture medium for cervical cancer organoids comprises the components and the final concentration or mass volume percent/volume percent of each component in the culture medium are shown in a table 1.
TABLE 1 organoid Medium composition
2 Experimental methods
2.1 method for constructing human cervical rare type cancer organoids
1) Cleaning: surgical or biopsy to obtain human cervical cancer tumor tissue, removing blood clot, connective tissue and necrosis part, and cleaning the tumor tissue with PBS until it is clean;
2) Grinding: the tissue is chopped into minced meat by scissors and a blade in sequence, and the minced meat is even, loose and not adhered (ice operation);
3) Digestion: transferring the tissue into a centrifuge tube, adding a proper amount of collagenase (1 mg/mL) digestive juice, blowing and mixing uniformly, placing in a constant temperature shaking table at 37 ℃ for 40-60min, adding 10 XTryple until the final concentration of the digestive juice is 1X, blowing and mixing uniformly, and placing in a constant temperature shaking table at 37 ℃ for 15-30min;
4) And (3) filtering: the digested cell suspension was filtered using a 100 μm filter, and the filtrate was centrifuged at 1200rmp,4℃for 5min, and the supernatant was discarded;
5) Split red: adding 1-3mL erythrocyte lysate, mixing, standing at 4deg.C for 5min, adding 4-5 times volume of PBS for neutralization, mixing, filtering with 100 μm filter, removing digestion residues, centrifuging at 1200rmp at 4deg.C for 5min, and discarding supernatant;
6) Counting: adding AdDF++ culture medium to re-suspend and counting;
7) Inoculating: re-suspending the cells with matrigel at a ratio of 150000-300000 cells/50 μl matrigel (ice operation), dripping the matrigel and cell mixture into the center of preheated 6/12/24 well plate, transferring into 37deg.C, and adding 5% CO 2 Solidifying for 20min in an incubator;
8) Slowly adding 2000/1000/500 μl of culture medium along the side wall of the well plate, standing at 37deg.C, and containing 5% CO 2 Culturing in an incubator;
9) Changing liquid every 3-5 days; passaging is performed every 14-21 days.
2.2 cervical rare type cancer organoid passaging step
1) Digestion: selecting stable organoids, removing medium from the culture plate, washing 1-2 times with HBSS, adding 1 XTryple digest (pre-warmed at 37deg.C for 10min before use) at 2 mL/well to 6 wellsBlowing matrigel into cell culture plate, mixing, adding 37deg.C and 5% CO 2 Digesting for 3-10min in an incubator, centrifuging for 5min at 1200rmp and 4 ℃ after stopping digestion by an equal volume of AdDF++, and discarding the supernatant;
2) And (3) paving: according to the following steps of 1:2-4, mixing appropriate amount of matrigel and cell precipitate, adding into 6-well cell culture plate at 50 μl/well, placing into 37 deg.C, 5% CO 2 An incubator, solidifying for 20min;
3) Adding a culture medium: after the gel is fixed, 2000 mu L of preheated self-made culture medium is added into each hole, and the mixture is put into 37 ℃ and 5% CO 2 And (5) incubating in an incubator.
3 results of experiments
Culturing for 14-21 days by the culture method to obtain the anthropogenic cervical rare type cancer organoids, wherein the growth conditions of the organoids are shown in figure 1; the culture method can stably passage human cervical rare type cancer organoids and enable the human cervical rare type cancer organoids to stably grow, the growth state is good, and the growth state of the passage organoids is shown as figure 2. Wherein, the success rate of the culture of the cervical adenocarcinoma is 95.90 percent (70/73); the success rate of the culture of cervical endometrial cancer is 100 percent (3/3); the success rate of culturing cervical small cell neuroendocrine cancer is 91.67% (11/12); the success rate of culturing the cervical stratified mucous-producing cancer is 83.33 percent (5/6); the success rate of culturing the cervical medium-renal tubular carcinoma is 100% (1/1); the success rate of Gong Genggao grade serous carcinoma culture is 100% (1/1); the success rate of cervical gastric adenocarcinoma culture is 100% (3/3); the success rate of cervical intestinal adenocarcinoma culture is 100% (1/1).
The above description of the embodiments is only for the understanding of the method of the present application and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present application without departing from the principle of the application, and these improvements and modifications will fall within the scope of the claims of the application.

Claims (10)

1. A method of culturing a cervical cancer organoid, said method comprising culturing cervical cancer cells using a medium comprising a BMP inhibitor, an N-acetylcysteine, nicotinamide, p inhibitor, a TGF- β inhibitor, a FGF, forskolin, wnt agonist, a Rock inhibitor, estradiol, and/or a mitogenic proliferation factor;
preferably, the BMP inhibitor is selected from Noggin;
preferably, the p38 inhibitor is selected from SB-202190;
preferably, the TGF-beta inhibitor is selected from A83-01;
preferably, the FGF comprises FGF7 and/or FGF10;
preferably, the Wnt agonist is selected from the group consisting of Rspo1;
preferably, the Rock inhibitor is selected from Y27632;
preferably, the Estradiol is selected from β -Estradiol;
preferably, the mitogenic proliferation factor is selected from EGF;
preferably, the final concentration of Noggin is 97-103ng/mL, the final concentration of SB-202190 is 0.5-1.5 mu M, the final concentration of A83-01 is 450-550nM, the final concentration of FGF7 is 20-30ng/mL, the final concentration of FGF10 is 95-105ng/mL, the final concentration of Rspo1 is 240-260ng/mL, the final concentration of Y27632 is 9-11 mu M, the final concentration of beta-Estradiol is 95-105nM, the final concentration of EGF is 45-55ng/mL, the final concentration of Forskolin is 9-11 mu M, the final concentration of Nicoamide is 2-3mM, and the final concentration of N-acetylcysteine is 1-1.5mM.
Preferably, the final concentration of Noggin is 100ng/mL, SB-202190 is 1 μM, A83-01 is 500nM, FGF7 is 25ng/mL, FGF10 is 100ng/mL, rspo1 is 250ng/mL, Y27632 is 10 μM, β -Estradiol is 100nM, EGF is 50ng/mL, forskolin is 10 μM, nicotinamide is 2.5mM, and N-acetylcysteine is 1.25mM;
preferably, the mass volume percentage of SB-202190 is 0.000033-0.0000339%, the mass volume percentage of A83-01 is 0.000015-0.000025%, the mass volume percentage of FGF7 is 0.000002-0.000003%, the mass volume percentage of Rspo1 is 0.00002-0.00003%, the mass volume percentage of Y27632 is 0.0003-0.00038%, the mass volume percentage of beta-Estradiol is 0.0000026-0.0000028%, the mass volume percentage of EGF is 0.000004-0.000006%, the mass volume percentage of Forskolin is 0.00035-0.00047%, the mass volume percentage of Nicotinamide is 0.02-0.04%, and the mass volume percentage of N-acetylcysteine is 0.01-0.03%;
preferably, the Noggin is 0.00001% by mass volume, the SB-202190 is 0.0000334% by mass volume, the a83-01 is 0.000021% by mass volume, the FGF7 is 0.0000025% by mass volume, the FGF10 is 0.00001% by mass, the Rspo1 is 0.000025% by mass, the Y27632 is 0.00032% by mass, the β -escadiol is 0.0000027238% by mass, the EGF is 0.000005% by mass, the Forskolin is 0.00041% by mass, the Nicotinamide is 0.03% by mass, and the N-acetylcysteine is 0.02% by mass.
2. The method of claim 1, wherein the medium further comprises nutritional additives, antibiotics, buffers;
preferably, the nutritional additives include Glutamax and/or B27;
preferably, the antibiotic is selected from Pen Strep;
preferably, the buffer is selected from HEPES;
preferably, the final concentration of Glutamax, pen Strep and B27 is 1×, and the final concentration of HEPES is 10mM;
preferably, the volume percentage of the Glutamax, the Pen Strep and the HEPES is 1%, and the volume percentage of the B27 is 2%.
3. The method of claim 1, wherein the cervical cancer cells are obtained by digesting cervical cancer tissue;
preferably, cervical cancer tissue is digested with collagenase and Tryple;
preferably, the concentration of collagenase is 0.5-2mg/mL;
preferably, the collagenase concentration is 1mg/mL;
preferably, the concentration of Tryple is 1 XTryple;
preferably, the method further comprises filtering the digested cervical cancer cells;
preferably, filtration is performed using a 100 μm cell filter;
preferably, the method further comprises washing the tissue prior to digestion;
preferably, the tissue is washed with PBS.
4. The method of claim 1, further comprising adding ECM to co-culture with cervical cancer cells;
preferably, the ECM includes BME and matrigel;
preferably, the ECM is selected from matrigel;
preferably, the cell is 1X 10 with matrigel 4 -1×10 6 Inoculating according to the proportion of 50;
preferably, the cell is mixed with matrigel in an amount of 1.5-3×10 5 Inoculating according to the proportion of 50;
preferably, the method further comprises passaging the organoid;
preferably, the passaging of the organoid comprises digesting the organoid;
preferably, the organoids are digested with Tryple;
preferably, the concentration of the Tryple is 1×tryple;
preferably, the method further comprises washing the organoid prior to digesting the organoid;
preferably, the organoids are washed using HBSS;
preferably, organoid passaging further comprises terminating digestion;
preferably, the method comprises the steps of, adDF++ was used terminating digestion;
preferably, the organoid is passaged in a ratio of 1:2-4;
preferably, the cervical cancer comprises cervical squamous carcinoma, cervical adenocarcinoma, cervical endometrioid carcinoma, cervical small cell neuroendocrine carcinoma, cervical multiple layer mucinous carcinoma, cervical medium-renal tubular carcinoma, cervical high-grade serous carcinoma, cervical gastric adenocarcinoma, cervical intestinal adenocarcinoma;
preferably, the cervical cancer includes cervical adenocarcinoma, cervical endometrioid carcinoma, cervical small cell neuroendocrine carcinoma, cervical complex mucous carcinoma, cervical medium-tubular carcinoma, cervical high-grade serous carcinoma, cervical gastric adenocarcinoma, cervical intestinal adenocarcinoma.
5. Cervical cancer organoid, characterized in that it is obtained using the method according to any one of claims 1-4.
6. The use of any one of the following:
(1) The use of the organoid of claim 5 in constructing a cervical cancer radiotherapy efficacy/synchronized radiotherapy and chemotherapy efficacy prediction model;
(2) The use of the organoid of claim 5 in constructing a drug screening model for cervical cancer;
(3) The use of the organoid of claim 5 in screening a medicament for the prophylaxis and/or treatment of cervical cancer, or in the development of a new medicament for cervical cancer;
(4) Use of the organoid of claim 5 in a toxicity test of a cervical cancer drug;
(5) The use of the organoid of claim 5 in constructing a model for testing toxicity of a drug against cervical cancer.
7. The model of any one of the following:
(1) A model for predicting the efficacy of radiotherapy and/or simultaneous radiotherapy and/or chemotherapy of cervical cancer, characterized in that the model comprises the organoid of claim 5;
(2) An in vitro drug screening model for cervical cancer, characterized in that said model comprises the organoid of claim 5;
(3) A cervical cancer drug toxicity test model, wherein said model comprises the organoid of claim 5;
(4) A model for developing a new drug for cervical cancer, characterized in that the model comprises the organoid of claim 5.
8. A method of predicting sensitivity to radiation/simultaneous radiation and chemotherapy of cervical cancer, the method comprising contacting a candidate agent and/or therapeutic radiation with the organoid of claim 5.
9. A method of testing the toxicity of a drug for cervical cancer, comprising contacting a candidate drug with the organoid of claim 5.
10. A method of screening for a drug for preventing/treating cervical cancer, comprising contacting a candidate drug with the organoid of claim 5.
CN202310783193.1A 2023-06-29 2023-06-29 Construction method and application of cervical cancer organoids Pending CN116716236A (en)

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