CN116355850A - Construction method and application of colorectal cancer organoids - Google Patents

Construction method and application of colorectal cancer organoids Download PDF

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CN116355850A
CN116355850A CN202310175815.2A CN202310175815A CN116355850A CN 116355850 A CN116355850 A CN 116355850A CN 202310175815 A CN202310175815 A CN 202310175815A CN 116355850 A CN116355850 A CN 116355850A
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organoid
colorectal cancer
organoids
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余佳
王芳
陈益莹
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Institute of Basic Medical Sciences of CAMS
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Abstract

The invention discloses a colorectal cancer organoid construction method and application thereof, and provides a colorectal cancer organoid culture medium, a colorectal cancer organoid construction method and application of the constructed organoid in predicting drug sensitivity and researching pathogenesis or pathogenicity factors of colorectal cancer. The organoid culture medium and the construction method provided by the invention have the advantages of high success rate, good organoid growth state, capability of well predicting the sensitivity of a patient to medicines, construction of a technical platform for the realization of accurate medical treatment of colorectal cancer, and wide application prospect.

Description

Construction method and application of colorectal cancer organoids
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a construction method and application of colorectal cancer organoids.
Background
The high incidence rate and the high mortality rate of colorectal cancer (Colorectal cancer, CRC) seriously threaten human health, and the statistical analysis of cancer data from 2015 to 2020 in China shows that the incidence rate of CRC in 2015 is 38.8%, the incidence rate of CRC in 2020 is increased to 55.5%, and the death rate of CRC is also increased to 28.6% from 18.7% in 2015. CRC occurs in countries throughout the world, but is most common in developed countries, and with increasing westernization of eating habits and rapid economic level development in densely populated countries such as China, the incidence of CRC is expected to rise year by year. Therefore, the prevention and treatment situation of CRC is very severe, the occurrence and development of CRC are deeply researched, and the exploration of an effective treatment scheme has important significance.
As technology advances, organoids are increasingly known as an emerging model for in vitro disease research. Organoids are 3D cell culture models that more closely approximate physiological or pathological conditions in the body. Organoids not only can be used as preclinical models for broader cancer studies, but also can provide personalized treatment advice for patients with advanced disease.
Therefore, constructing CRC organoids is of great significance in achieving accurate and effective treatment of CRC.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention provides a colorectal cancer organoid culture medium and a method for constructing colorectal cancer organoids.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect the invention provides a colorectal cancer organoid medium comprising BMP inhibitors, prostigndin E2, wnt agonists, TGF- β inhibitors, gastrin I, p38 inhibitors.
Further, the BMP inhibitors include Noggin, gremlin, chordin, follistatin, sclerostin, decorin.
Further, the BMP inhibitor is selected from Noggin.
Further, the Wnt agonist comprises Wnt protein, R-spondin.
Further, the Wnt agonist is selected from the group consisting of R-spondin.
Further, the R-spondin includes R-spondin1, R-spondin2, R-spondin3, R-spondin4.
Further, the R-spondin is selected from R-spondin1.
Further, the TGF- β inhibitors include a8301, SB431542, SB505124, SB525334, SD208, LY36494, and SJN2511.
Further, the TGF-beta inhibitor is selected from A8301.
Further, the p38 inhibitors include SB202190, SB203580, VX702, VX745, PD169316, RO4402257, BIRB796.
Further, the p38 inhibitor is selected from SB-202190.
Further, the medium also includes an antibiotic.
Further, the antibiotics include Primocin, penicillin streptomycin, and metronidazole.
Further, the antibiotic is selected from Primocin.
Further, the medium also includes a nutritional additive.
Further, the nutritional additives include Gluta Max, B27, and N2.
Further, the nutritional additive is selected from Gluta Max.
Further, the medium also includes mitogenic proliferation factor, FGF-basic, N-acetyl-L-cysteine, rock inhibitors.
Further, the mitogenic proliferation factor includes EGF, BDNF, KGF.
Further, the mitogenic proliferation factor is selected from EGF.
Further, the Rock inhibitors include Y-27632, HA1077, H-1152.
Further, the Rock inhibitor is selected from the group consisting of Y-27632.
In a second aspect the invention provides a method of culturing colorectal cancer organoids, the method comprising culturing colorectal cancer cells using the medium of the first aspect of the invention.
Further, the method further comprises adding ECM to co-culture with colorectal cancer cells. Further, the ECM includes BME and matrigel.
Further, the ECM is selected from matrigel.
Further, the colorectal cancer cells are obtained by digesting colorectal cancer tissue.
Further, colorectal cancer tissue is digested using the tissue digestion solution.
Further, the stock solution of the tissue digestion solution comprises Tryple and/or collagenase.
Further, the mother liquor of the tissue digestion solution is selected from the group consisting of Tryple.
Further, the concentration of Tryple is 2 XTryple-10 XTryple.
Further, the concentration of the Tryple is 5×tryple.
Further, the digestion time of the tissue is 30min-12h.
Further, the digestion time of the tissue was 30min.
Further, digestion was terminated using PBS solution or medium.
Further, digestion was terminated using PBS solution.
Further, the method further comprises washing the tissue prior to digesting the tissue.
Further, the tissue was washed with PBS.
Further, the method also includes amplifying the organoid.
Further, organoid augmentation includes digestion of organoids.
Further, the organoids were digested with Tryple.
Further, the concentration of Tryple was 2×Tryple.
Further, the digestion time is 5min-30min.
Further, the digestion time was 20min.
Further, the method further comprises passaging the organoid.
Further, the passaging organoids include digestive organoids.
Further, the organoids were digested with passaged digest.
Further, the mother liquor of the passaged digestive juice comprises Tryple and/or collagenase.
Further, the mother liquor of the passaged digest is selected from the group consisting of Tryple.
Further, HBSS was used to terminate digestion.
Further, the digestive juice: hbss=1: 2.
in a third aspect the present invention provides a colorectal cancer organoid cultivated using a medium according to the first aspect of the invention or obtainable by a method according to the second aspect of the invention.
A fourth aspect of the invention provides the use of any one of the following:
(1) The use of a medium according to the first aspect of the invention for culturing colorectal cancer organoids;
(2) The use of an organoid according to the third aspect of the invention for screening for a medicament for the prevention and/or treatment of colorectal cancer;
(3) The use of an organoid according to the third aspect of the invention for the establishment of a drug susceptibility model for colorectal cancer;
(4) The use of an organoid according to the third aspect of the invention in the study of colorectal cancer pathogenesis or causative factor.
In a fifth aspect the invention provides an in vitro drug screening model for colorectal cancer, said model comprising the organoids according to the third aspect of the invention.
In a sixth aspect the invention provides a method of predicting colorectal cancer drug sensitivity, the method comprising contacting an organoid according to the third aspect of the invention or a model according to the fifth aspect of the invention with a test drug.
In a seventh aspect the present invention provides a method of screening for a drug for the prevention and/or treatment of colorectal cancer, the method comprising contacting a candidate drug with an organoid according to the third aspect of the invention or a model according to the fifth aspect of the invention.
The invention has the advantages and beneficial effects that:
the organoid culture medium and the construction method provided by the invention have the advantages of high success rate, good organoid growth state, capability of well predicting the sensitivity of a patient to medicines, construction of a technical platform for the realization of accurate medical treatment of colorectal cancer, and wide application prospect.
Drawings
FIG. 1 is a diagram of tissue digestion condition screening;
FIG. 2 is an enrichment map of colorectal cancer cells, wherein 2A is a tissue state map after digestion treatment, and 2B is a microscopic view of the enriched cell map;
FIG. 3 is a diagram of CRC organoid growth process;
FIG. 4 is a plot of stable amplification of CRC organoids, wherein 4A is a microscopic plot of organoids after 5min, 10min, 15min digestion; 4B is a morphological map of organoids amplified from P1 to P10 generation;
FIG. 5 is a graph of organoid (P1-O) morphology change after 6 days of irinotecan administration;
FIG. 6 is a graph comparing the clinical medication results of a patient with the corresponding organoid drug sensitivity test results, wherein 6A is a graph of the clinical medication results of a P3 patient, 6B is a graph of the clinical medication results of a P4 patient, 6C is a graph of the organoid drug sensitivity test of a P3 patient, and 6D is a graph of the organoid drug sensitivity test of a P4 patient;
FIG. 7 is a graph of the inhibition of TNFR2 protein expression in CRC organoids;
FIG. 8 is a morphology of TNFR 2-inhibiting organoids;
FIG. 9 is a graph of the proliferation potency of TNFR2 organoids;
FIG. 10 is a graph of the number of TNFR 2-inhibiting organoids;
FIG. 11 is a graph of the size of a TNFR 2-inhibiting organoid;
FIG. 12 is a reduced medium component organoid growth state diagram, wherein 12A is a total component additive organoid growth state diagram, 12B is a no Noggin organoid growth state diagram, 12C is a no SB202190 organoid growth state diagram, 12D is a no gaslin organoid growth state diagram, 12E is a no A8301 organoid growth state diagram, 12F is a no R-Spondin1 organoid growth state diagram, and 12G is a no Prostaglandin E2 organoid growth state diagram;
FIG. 13 is a graph of reduced medium composition organ viability statistics;
FIG. 14 is a graph of the growth state of the organs in the addition medium, wherein 14A is the growth state of the organs in the addition of HGF, 14B is the growth state of the organs in the addition of IL-6, and 14C is the growth state of the organs in the addition of SDF 1.
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 invention belongs.
The invention provides a colorectal cancer organoid medium, which comprises BMP inhibitor, prostaglandin E2, wnt agonist, TGF-beta inhibitor, gastin I and p38 inhibitor.
In the present invention, organoids refer to cell clusters having a 3D three-dimensional structure, and refer to miniaturized and simplified versions of organs prepared by artificial culture processes. The source of the cells constituting the organoids is not limited. Organoids may be derived from tissues, stem cells. Organoids may have an environment that allows for interaction with the surrounding environment during cell growth. Thus, the 3D organoids of the present invention, which almost completely mimic in vivo interactions, can be an excellent model for observing the development of disease therapeutics. It can similarly replicate the physiological activity function of the human body, and by constructing organ analogs from the patient's tissues, disease modeling can be performed based on the patient's genetic information, and drug screening can be performed by repeated tests.
In a specific embodiment of the invention, the organoids are derived from colorectal cancer tissue.
Among these BMP (bone morphogenic protein, bone morphogeneitc protein) inhibitors include, but are not limited to Noggin (Noggin), gremlin (lattice Lei Lin), chord (tenascin), tenascin-like proteins such as tenascin domain, follistatin (Follistatin), follistatin-like proteins such as Follistatin domain, DAN-like proteins such as DAN-containing cysteine knot domain, sclerostin/SOST, decorin, alpha 2-macroglobulin.
In a specific embodiment of the invention, the BMP inhibitor is selected from Noggin (Noggin).
Wnt agonists refer to agents that activate T-cytokine (TCF)/lymphokine-mediated transcription in cells (LEF). 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. The Wnt agonist is preferably at least 1 of Wnt protein, R-spondin (R-cavernosum) and GSK-3 beta inhibitor.
Among them, wnt proteins derived from various organisms can be used. Among them, preferred is a Wnt protein derived from mammals. 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 (Sigma Aldrich), lithium (Sigma), kenparone (Biomol International, leost, M.et al (2000) Eur J biochem267, 5983-5994), 6-bromoindirubin-30-acetoxime (Meyer, L et al (2003) chem. Biol.10, 1255-1266), SB 216763 and SB 415286 (Sigma Aldrich), and FRAT family members and FRAT derived peptides that prevent GSK-3 interaction with Axin.
R-spondin (R-spongehprin) includes R-spondin 1 (R-spongehprin 1), R-spondin2 (R-spongehprin 2), R-spondin 3 (R-spongehprin 3) and R-spondin 4 (R-spongehprin 4). In the present invention, R-spongin may be used in various combinations.
In an embodiment of the invention, the Wnt agonist is selected from the group consisting of R-spondin (R-cavernosum).
In a specific embodiment of the present invention, R-spondin (R-spongosine) is selected from the group consisting of R-spondin 1 (R-spongosine 1).
TGF-beta (transforming growth factor-beta ) inhibitors include, but are not limited to, A8301, SB431542, SB505124, SB525334, SD208, LY36494, SJN2511.
In a specific embodiment of the invention, the TGF-beta inhibitor is selected from A8301.
p38 inhibitors include, but are 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) -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) -8-propyl ] amino } -8-methylpyrido-2, 3-pyri-ne [1,6-b ] pyridazin-6-one, PD-16931-6-one, 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 invention, the p38 inhibitor is selected from SB-202190 (4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole).
The culture medium of the invention also includes antibiotics.
The antibiotics comprise one or more of Primocin, penicillin streptomycin and metronidazole.
The media of the present invention 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 invention, the basal medium is selected from Advanced DMEM/F12.
The culture medium of the invention also comprises inhibitors of mitogenic proliferation factors, FGF-basic, N-acetyl-L-cysteine, rock.
Among these, mitogenic proliferation factors include, but are not limited to, the 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 invention, the mitogenic proliferation factor is selected from EGF (epidermal growth factor ).
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-diaza-pine dihydrochloride).
In a specific embodiment of the invention, the Rock inhibitor is selected from the group consisting of Y-27632 ((R) - (+) -trans-4- (1-aminoethyl) -N- (4-pyridinyl) cyclohexanecarboxamide dihydrochloride monohydrate).
The organoid medium of the present invention may further comprise at least 1 amino acid. The amino acids include 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.
The organoid medium of the present invention may further comprise at least 1 vitamin. The vitamins include, but are 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), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), DL-alpha-tocopherol (vitamin E), biotin (vitamin H), menaquinone (vitamin K).
The organoid medium of the present invention may further comprise at least 1 inorganic salt. The inorganic salts include, but are 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, the salt may be exemplified by CaCl 2 、CuSO 4 ·5H 2 O、Fe(NO 3 )·9H 2 O、FeSO4·7H 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。
The organoid medium of the present invention may further comprise at least 1 sugar that can be a source of carbon energy. Sugar includes glucose, galactose, mannose, fructose, etc. Among them, glucose is preferable, and D-glucose (dextrose) is more preferable.
The organoid medium of the present invention may further comprise at least 1 trace element. 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 invention provides a colorectal cancer organoid culture method, which comprises the step of culturing colorectal cancer cells by using the culture medium.
The method further comprises adding ECM to co-culture with colorectal cancer cells.
In the present invention, the ECM (extracellular matrix) is a three-dimensional matrix including BME (basement membrane extract) and Matrigel (Matrigel).
In a specific embodiment of the invention, the ECM is selected from matrigel.
In the present invention, the matrigel-cell pellet is added to the culture plate, and the culture medium is added at the edge of the culture plate, or directly added dropwise, preferably at the edge of the culture plate. The amount of culture required per well is 400. Mu.L to 600. Mu.L; preferably 500 μl.
The colorectal cancer cells are obtained by digesting colorectal cancer tissue.
In the present invention, colorectal cancer tissue is digested with a tissue digestion solution, the mother liquor of which comprises Tryple and/or collagenase, wherein the collagenase comprises collagenase I, collagenase II, collagenase IV.
As an embodiment of the present invention, colorectal cancer tissue may be digested using Tryple and collagenase as mother liquor; as an embodiment of the invention, colorectal cancer tissue may be digested using Tryple as a mother liquor; as an embodiment of the present invention, colorectal cancer tissue may be digested using collagenase as a mother liquor.
In a specific embodiment of the invention, the colorectal cancer tissue is digested using Tryple as a mother liquor.
In the present invention, the concentration of Tryple in the tissue digestion solution ranges from 2 XTryple to 10 XTryple, and colorectal cancer tissue can be digested with any of the above concentrations, preferably 5 XTryple; the time for colorectal cancer tissue digestion is 30min-12h. The preferred time is 30 minutes.
In a specific embodiment of the invention, 5 XTryple digestion for 30min is used as colorectal cancer tissue digestion condition.
The present invention provides a method of predicting colorectal cancer drug susceptibility comprising contacting the organoids or the model described above with a test drug.
The concentration of the test drug is determined based on the morphological changes of the organoids. Morphological changes of the organoids include a decrease in the number of organoids or a change in the organoid size. If the number of organoids is reduced or organoid growth is inhibited, then organoids are indicated to be sensitive at the test drug concentration.
10 organoids were selected for sensitivity testing of 11 clinical drugs. These 11 drugs contain 5 conventional chemotherapeutic drugs and 6 targeted drugs. Part of patient prognosis conditions are consistent with the detection result of the drug sensitivity of the organoids, and the feasibility and the reliability of predicting the drug response in the body by using the organoids model are realized.
The present invention provides a method of screening for a drug for the prevention and/or treatment of colorectal cancer, the method comprising contacting a candidate drug with the above organoids or the above model.
Further comprising determining the organoids or models after contact with the candidate substance; and comparing the measured value of the organoid or model after contact with the candidate agent with a control value. In one embodiment, the method comprises determining whether the candidate substance is a drug capable of preventing and/or treating colorectal cancer based on the comparison result.
In the present invention, contacting the candidate drug with the above-mentioned organoids or the above-mentioned model means that the above-mentioned organoids or model and the candidate drug are placed in contact. The contacting of the organoids or models with the candidate drug may be, for example, adding the candidate drug to a solution containing the organoids or models.
In the present invention, the candidate drug may be, for example, a low molecular compound, a protein (e.g., an antibody), DNA, RNA, low molecular interfering RNA, or an antisense oligonucleotide. The candidate drug may be, for example, an agent for treating a disease or cancer other than colorectal cancer. The drug candidate may be, for example, one or a mixture of two or more. The drug candidate is preferably a substance.
In the present invention, the measurement of the organoids or models after contact with the candidate substances described above includes reduction of organoid viability, reduction of organoid proliferation, increase of organoid death, change of organoid size, number of organoids, expression of marker proteins of cells constituting 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 invention, the measurement value of the organoid or model may be, for example, one measurement value measured at a time, may be an average value measured a plurality of times, or may be 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 test substance. 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 organoid size, the "determination" of whether the drug candidate is a substance capable of treating colorectal 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 colorectal cancer.
In the present invention, treatment refers to any method for partially or completely alleviating, ameliorating, alleviating, 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 invention 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 invention may be used in various embodiments without departing from the scope of the invention.
Experimental materials and experimental methods
1. Experimental materials
Agents required for organoid culture
1) Tissue preservation solution (500 mL for example): HEPES (1M) 5mL, gluta MAX (100X) 5mL, penicillin streptomycin solution (100X) 5mL,Advanced DMEM/F12 485mL;
2) Tissue digestate (10 mL for example): 20. Mu.L of 0.5M EDTA solution, 5mL of 10 XTryple solution, 4.98mL of DPBS;
3) Organoid passaging digest (10 mL for example): 20. Mu.L of 0.5M EDTA solution, 1mL of Tryple solution, 8.98mL of DPBS;
4) Organoid activity assay kit: cell Titer-Glo 3D Cell Viability Assay (cat# G9682), available from Promega corporation;
5) Organoid medium (table 1).
TABLE 1 organoid culture medium
Figure BDA0004100799950000121
Figure BDA0004100799950000131
6) Patient information
Patient details are shown in table 2.
Table 2 patient information
Figure BDA0004100799950000132
2. Experimental method
2.1 tumor cell purification
2.1.1 preparation before experiment
(1) The tissue transfer process needs to maintain a low temperature environment, and the tissue is put into a tissue protection culture medium for preservation at the first time after the tissue is isolated.
(2) Surgical forceps, surgical scissors, surgical blades and the like are sterilized for standby.
(3) Opening the water bath kettle and pre-cooling the water bath kettle at 4 ℃ by a centrifugal machine.
(4) The matrigel is placed at-20deg.C overnight into ice box for melting, and the ice box is placed in a refrigerator at 4deg.C, and can be used after shaking and observing before use without caking and with clear and transparent color.
(5) The incubation hole plate is placed into an incubator, 1mL of PBS is added to be paved uniformly on a culture dish (60 mm, low adsorption force), 50 mu L of CD16/32 antibody is added to be beaten and uniformly mixed by a pipetting gun, the bottom is paved fully, and the incubator is incubated for at least 2 hours.
2.2 organoid culture
2.2.1 tumor tissue pretreatment
After the tissue arrives, the medium attached to the surface of the sample is sucked off with a piece of absorbent paper, and the total weight of the sample is recorded by weighing.
2.2.2 tissue digestion
(1) Cleaning tissue: pouring PBS into 2 common culture dishes (60 mm), taking out tissues from a 50mL centrifuge tube by using long tweezers, and placing the tissues into the dishes for cleaning for 2-3 times; after the tissue is cleaned, the tissue is clamped into another dish by forceps, the tissue is sheared by scissors, and the tissue is repeatedly cut by a blade for 3-5min until no obvious large tissue block exists.
(2) Digesting the tissue: the minced tissue is clamped into a 5mL EP tube by forceps, 3mL of tissue digestion solution is added, and residual tissue in a dish is washed by 1mL of tissue digestion solution and then added into the EP tube, and the mixture is blown and evenly mixed by a gun head. Sealing the periphery of the cover with a sealing film, putting the cover in a water bath kettle at 37 ℃ for 30min after the film glove is knotted. After the water bath, pouring into a 15mL centrifuge tube, adding 6mL of PBS solution to stop digestion, blowing and mixing uniformly, taking 10 mu L of the solution, dripping the solution into a small culture dish, observing the solution under a microscope, photographing and preserving the solution. The remaining liquid in the centrifuge tube was sealed with a sealing film, and centrifuged at 1200rpm and 4℃for 4min.
2.2.3 tumor cell purification
(1) Sieving: after centrifugation, the supernatant was aspirated into another 15mL centrifuge tube, sealed, and centrifuged again. About 3-4mL of the basal medium is sucked and added into a 15mL tube containing the sediment, and the mixture is blown and evenly mixed.
(2) And (3) filtering: two 50mL centrifuge tubes, two 1mL syringes, 100 μm,40 μm filters were taken one each.
(3) Grinding: taking a 50mL centrifuge tube, and placing a 100 μm filter screen at a 50mL centrifuge tube orifice; pouring the precipitate suspension into a 100 μm sieve, and grinding with a small syringe needle handle; adding the sediment after supernatant centrifugation into a basic culture medium, pouring the sediment into a 100-mu m filter screen, cleaning a centrifuge tube by using the basic culture medium, pouring the sediment into the 100-mu m filter screen together, and repeatedly grinding the sediment.
(4) And (3) collecting: another 50mL centrifuge tube was taken, a 40 μm sieve was placed on the 50mL centrifuge tube, the filtrate passing through the 100 μm sieve was poured into the 40 μm sieve, the centrifuge tube was washed with the basal medium, and the mixture was poured into the 40 μm sieve together with repeated grinding, and 10. Mu.L of the 40 μm filtrate was dropped on a small dish for observation.
(5) Incubation: taking out the incubated Petri Dish plate at 37 ℃, sucking off the liquid, pouring the filtrate which passes through a 40 mu m filter screen into a small culture Dish, cleaning a centrifuge tube by cleaning liquid, adding the centrifuge tube into the small culture Dish, marking and placing an incubator for incubation for 1h. Sealing PBS and basal medium, placing in refrigerator, placing a box of ice in biosafety cabinet, irradiating with ultraviolet for 30min, and then placing in PBS. After 1h, the liquid in the small culture dish is sucked into a 15mL centrifuge tube, marking is done, 1mL of basic culture medium is taken to clean the culture dish, the culture dish is added into the 15mL centrifuge tube, and after sealing, the centrifuge is carried out at 1200rpm,4 ℃ for 4min.
If the pellet is red after centrifugation, a split red is required. The supernatant was pipetted into another 15mL centrifuge tube and centrifuged and the red sediment centrifuge tube placed on ice. Centrifuging the supernatant, removing the supernatant, taking 500 mu L of erythrocyte lysate to the supernatant sediment, blowing and mixing uniformly, sucking the supernatant into a red sediment centrifuge tube, and putting the supernatant on a frame for cracking for 5min. 1mL (2 times) of PBS was added and the mixture was centrifuged at 1200rpm at 4℃for 4min after blowing and mixing. After centrifugation, the supernatant was removed, 1ml of LPBS was added, the mixture was blown and mixed well, 20. Mu.L of the suspension was spotted on a counting plate, counted and recorded, and centrifuged (as above), and the supernatant was removed.
2.2.4 organoid culture
(1) Paving matrix glue: matrigel was placed on ice, and the required matrigel amount (total cell count/(20000-40000) =number of wells; matrigel=45 to 50 μl) was calculated and added to the cell pellet, and was blown and mixed with an ice gun head. The mixture of matrigel and cells was dropped in the center of the 24/48 well plate, and no bubbles were generated as much as possible. Then, the lid is marked with the sample application area, time, operator name, organoid type. Placing into incubator for 20min to gel and fix.
(2) Preheating a culture medium: a certain amount of organoid medium (500. Mu.L of medium was required for each well, Y-27632 was added in 1mL of medium plus 1. Mu. L Y-27632) was selected according to the number of wells of the adhesive, and the mixture was blown into a clean 15mL tube and mixed well. Sealing a 15mL pipe, putting the pipe into a plastic film glove, and then putting the glove into a 60 ℃ constant-temperature water bath kettle for preheating.
(3) Adding a culture medium: taking out the culture plate from the incubator, adding culture medium from the edge of the culture plate, shaking the culture plate slightly after the culture medium is added, placing the culture plate into the incubator at 37 ℃ for incubation, replacing the culture medium every three days, observing regularly, photographing and preserving.
After white particles are arranged at the bottom of the culture medium matrigel, the culture medium is passaged, and the frequency of changing the culture medium mainly depends on the growth speed of cells.
2.3 organoid liquid exchange
In order to ensure the nutrition required by organoid growth, the culture medium should be replaced in time. Essentially every 3-5 days, the medium should be changed from red to orange or yellow. When the liquid is changed, the culture medium is preheated at 37 ℃, then the culture plate is taken out from the inner box of the incubator, the old culture medium is discarded, the culture plate is slightly shaken after the new culture medium is added from the edge, no bubbles are generated, and the culture plate is placed into the incubator at 37 ℃ for incubation.
2.4 organoid passage
Standard of passaging: the frequency of medium replacement is shortened, white spots are visible to the naked eye at the bottom of the matrigel, and cells with full field are observed under a microscope.
(1) Early preparation: checking whether the articles in the safety cabinet are complete, and putting the ice box into the cabinet to irradiate ultraviolet.
(2) Subculture: HBSS and passage digestive juice are placed on ice, a culture plate needing passage is taken out, the original culture medium in each hole is sucked out by a 1mL single-channel pipetting gun, and the culture plate is washed 1-2 times by 1-2mL HBSS.
(3) Organoid digestion: digesting matrigel with 500 μl of passage digestive juice, taking care of blowing off matrigel, and placing into a 37 ℃ incubator for 15min after blowing off uniformly to make it fully digested.
(4) Terminating digestion: after 15min 1mL HBSS was added to terminate digestion (digests: hbss=1:2), transferred to a clean 15mL enzyme-free tube and the culture wells were washed once with 1-2mL HBSS and added to the 15mL tube.
(5) And (3) centrifuging: the supernatant was discarded at an equivalent amount of water trim, 1200rpm, leaving behind a cell pellet. Note that: after centrifugation, if the upper layer has a small amount of glue, the supernatant is discarded, and 1-2mL of HBSS is added for cleaning and removal. After a moderate amount of gum, the supernatant was discarded, the gum was transferred to a separate 15mL enzyme-free centrifuge tube and resuspended in HBSS and centrifuged. When the matrigel amount is large, the operation of 1.2 is repeated, and the cell recovery liquid can be used after washing for several times without improvement.
(6) Spreading glue: and (3) placing the matrix glue which is melted in advance on ice, calculating the required amount of the matrix glue, adding the calculated amount of the matrix glue into the cell sediment, and blowing and uniformly mixing. After being mixed with the cells, the matrigel is dripped in the center of a 6 or 24-pore plate preheated in advance, so that bubbles are avoided as much as possible. After matrigel is dripped, the sampling area, time, organoid type and passage batch are marked on the cover. Placing into a 37 ℃ incubator for 25min, and solidifying the matrigel.
(7) Preheating a culture medium: a certain amount of organoid culture was selected based on the number of wells passaged in 15mL tubes, Y-27632 and primary antibiotic (medium: Y-27632: primary antibiotic = 1mL: 1. Mu.L: 2. Mu.L) were added, and mixed by pipetting. The 15mL tube is placed into a plastic film glove and then placed into a constant temperature water bath kettle at 37 ℃ for preheating.
(8) Adding a culture medium: the culture plate is taken out from the incubator, a 1mL liquid-transferring gun is used for adding culture medium, and the culture medium is slowly dripped from the edge, so that the matrigel is prevented from being broken due to direct dripping on the gel. After the culture medium is added, the culture plate is slightly shaken, and then placed into a 37 ℃ incubator for incubation, the culture medium is replaced every three days, and the culture plate is observed regularly, photographed and stored.
2.5 organoids cryopreserved
Frozen organoid standard: organoids were grown substantially stable and partially frozen, usually after three generations of expansion.
(1) Early preparation: checking whether the articles in the safety cabinet are complete, putting the ice box into the cabinet, irradiating ultraviolet, and placing the freezing box at room temperature.
(2) HBSS, cell recovery and 15mL centrifuge tubes were placed on ice.
(3) The plate was removed, the medium that had been yellow in the culture well was discarded, and the remaining medium was washed with 2mL of HBSS by shaking.
(4) After cleaning, 2mL of cell recovery liquid is added into each hole, and the whole organoid is ensured as much as possible by taking gentle care when blowing, so that the organoid is not blown away.
(5) The mixed solution in the last step is collected into a 15mL tube which is placed on ice in advance, then 2-3mL of cell recovery solution is used for washing the culture holes once, and the culture holes are collected into a 15mL centrifuge tube.
(6) A15 mL centrifuge tube was inserted into crushed ice, and after 30min of standing, centrifuged.
(7) After centrifugation, the supernatant was discarded, washed 1-2 times with HBSS (gentle blowing) and centrifuged again. Discarding supernatant, adding frozen stock solution, and stirring.
(8) Transferring to a freezing tube, placing into a freezing box which is restored to room temperature, and placing into a refrigerator at-80 ℃ for overnight.
(9) The next day, the frozen stock is transferred from the refrigerator at the temperature of minus 80 ℃ to a liquid nitrogen tank.
2.6 organoid resuscitation
(1) The 15mL centrifuge tube was added with 3mL of culture medium and heated in a 37℃water bath. The frozen tube was placed in a plastic glove and thawed by shaking in a 37 ℃ water bath.
(2) The frozen stock solution was sucked into a 15mL centrifuge tube, and after mixing, it was centrifuged at 1200rpm at 25℃for 4min.
(3) The supernatant was removed, washed with 1mL of HBSS, and centrifuged at 1200rpm,4℃for 4min after mixing. After centrifugation, the supernatant was removed and placed on ice.
(4) Adding matrigel into the sedimentation centrifuge tube, and blowing and mixing uniformly.
(5) The glue is dripped into the center of the preheated orifice plate by using the head of a cold pipetting gun, 45-50 mu L of each drip is placed in a 37 ℃ incubator for 20min.
(6) Proper amount of Y-27632 and primary antibiotic are added and the culture medium is preheated.
(7) The culture medium was added in an amount and incubated in an incubator at 37 ℃.
2.7 organoid drug screening
2.7.1 Pre-preparation
(1) Preparing an organoid culture sample to be tested.
(2) 384-well plates are prepared, preheated at 37 ℃ in advance, corresponding marks (organoid types, batches, drug region division of each group and the like) are made on a cover plate, a water bath kettle is preheated at 37 ℃ in advance, a centrifugal machine is precooled at 4 ℃, and a sufficient amount of culture medium is prepared.
(3) The medicine screen scheme of this time is designed in advance and comprises medicines and concentration gradients thereof which need to be done so as to facilitate the follow-up plate paving and the medicine dispensing experiment.
2.7.2 organoid plating
(1) The organoid sample culture plate to be detected is taken out, the culture medium in the culture well is sucked and removed by a 1mL single-channel pipetting gun, and the residual culture medium is washed out by 1-2mL HBSS for 1-2 times.
(2) 1.5mL of passage digestive juice is added into a culture plate, matrigel is lightly blown, and the culture plate is placed into a incubator at 37 ℃ for 15min after being blown uniformly.
(3) After 15min, the digestion was stopped by adding HBSS (digests: medium=2:1), and the digested organoids were transferred to a clean 15mL enzyme-free centrifuge tube, washed with 200 μl HBSS, centrifuged at 1200rpm at 4 ℃ for 4min, and the supernatant was discarded, leaving a cell pellet.
(4) And (3) placing the matrix glue which is melted in advance on ice, calculating the required amount of the matrix glue, adding the calculated amount of the matrix glue into the cell sediment, and blowing and uniformly mixing. After mixing, the mixture was added to 384 well plates pre-heated and labeled in advance and placed into incubator for 20min.
(5) Adding culture medium (preheated in water bath at 37deg.C in advance), and culturing in incubator.
2.7.3 medicine and additive
(1) According to the design drawing of the experimental scheme, the amount of the medicine and the amount of the culture medium required for preparing each concentration of each medicine are calculated in advance.
(2) And preparing a drug-containing culture medium with the required concentration according to the calculation result. The negative control wells were medium without drug.
(3) The clean absorbent paper is laid on an ultra-clean workbench in advance for ultraviolet irradiation for 30min. The next day of plating out 384-well plates from the incubator, and the names of the drugs added to each region are indicated on the 384-well plate cover. The medium was gently tapped on absorbent paper. And adding the prepared corresponding medicine-containing culture medium from top to bottom according to the marked medicine area, and putting the culture medium into a incubator for culturing after the addition is finished.
2.7.4 replacement of culture Medium
The clean absorbent paper is laid on an ultra-clean workbench in advance for ultraviolet irradiation for 30min. Gently shooting out the medicine-containing culture medium, adding the corresponding medicine-containing culture medium again, and placing into an incubator for culture.
2.7.5 on-machine detection
(1) 3D activity detection reagent is prepared in advance and put into a refrigerator at 4 ℃ in advance to be melted overnight. The detection reagent is balanced for 30min in a water bath at 22 ℃ before use, the detection reagent is lightly stirred uniformly and then is packaged for use, the culture plate is taken out of the incubator before the detection reagent is added, and the detection reagent is balanced for 30min at room temperature (22-25 ℃).
(2) After two days of dressing change, adding a detection reagent (sample adding under a dark condition) which is equal to the culture reagent into a 384-well plate balanced at room temperature, vibrating for 5min on a micropore vibration instrument, fully and uniformly mixing to crack cells, standing for 25min at room temperature, balancing luminous signals, and reading by an enzyme-labeling instrument after removing bubbles.
2.7.6 data analysis
(1) Analysis of results: and screening out the hole with the largest deviation data among each group of complex holes according to the data measured by the enzyme label instrument, and calculating the relative survival rate of each remaining hole.
(2) And (3) drawing: and drawing a survival trend graph of the organoids under various medicaments according to the survival rate of the medicaments under different concentrations.
2.8 lentiviral infection of organoids
Organoids were recovered with recovery solution and counted, 1X 10 wells (24 well plate for example) were added 5 Organoids were added simultaneously with 50 μl of lentivirus and an equal amount of matrigel, and medium was supplemented to 500 μl. After 24 hours, the recovered liquid is used for recovering organoids and culturing normally.
EXAMPLE 1 organoid culture
Pre-experiments are performed on the concentration selection and digestion time of the tissue digestion solution to determine the optimal digestion conditions of colorectal cancer tissues, so that more isolated cells are obtained. As shown in fig. 1, 10×tryple was selected as the mother liquor of the digestion solution, and the digestion was performed by first setting a concentration of 2×tryple, and by setting a digestion time gradient, it was found that the tissue did not become loose after overnight digestion at 37 ℃ for 12 hours, a large number of tissue aggregates remained and free cells were less. The concentration of the digestive juice is increased to 2.5 times of the original concentration, and the tissue mass disappears after digestion for 30min at 37 ℃ at the concentration, so that more free cells exist and part of the free cells exist as cell mass. Whereas in this condition the digestion time was prolonged to 60min, the tissue mass was completely disappeared, shrinkage of the cells was observed under the mirror, and the cell state was markedly worse.
Then, it was determined that 5×tryple was used as a tissue digestion condition for colorectal cancer, and then enriched into a mixed cell population containing colorectal cancer cells and other cells such as normal cells by low-temperature centrifugation (fig. 2A, arrow indicates free cells). After purification steps such as immune cell removal and erythrocyte removal, colorectal cancer cells with relatively high purity are obtained (figure 2B).
The addition of antibiotics to organoid media prevents bacterial or fungal infection and CRC organoids were observed to grow from single cells, as shown in FIG. 3.
For long-term expansion of the organoids, the organoids were digested under conditions of low digestibility (2×tryple) and time gradients of 5min, 15min, 30min were set (fig. 4A). The results showed that the organoids remained globular at 37 ℃ for 5min of digestion, and the organoids became loose at 15min from globular, whereas at 30min of digestion time the organoids were completely dissociated, and finally we selected 20min of digestion at 37 ℃ as the conditions for organoid amplification. To confirm that this condition is universal, organoids were observed for morphological features after multiple subcultures under this condition, and it was found that there was no significant difference in organoid morphology after long-term subcultures (from P1 to P10) and good growth status (fig. 4B).
Example 2 use of organoids in drug sensitivity detection
The drug sensitivity test was performed by selecting 10 organoids, and 11 drugs including Oxaliplatin (Oxaliplatin), fluorouracil (Fluorouracil), irinotecan (Irinotecan), cetuximab (Cetuximab), regorafenib (Regorafenib), trifluralin (trifluradine), lapatinib (Lapatinib), trametinib (Trametinib), kang Naifei ni (Encorafenib), dabrafenib (Dabrafenib), and larrotinib (larrotinib) were used as the drugs to be tested.
Morphological changes after organoid administration were observed under a microscope, and normal growth was found in the control group 6 days after administration of irinotecan at gradient concentrations. As the drug concentration increased, the amount of P1-O gradually decreased, and organoid growth was completely inhibited at 100. Mu.M (FIG. 5). Follow-up is then performed on a portion of the patient's prognosis, and from the information obtained, it is found that both patients have undergone a chemotherapeutic agent, oxaliplatin, in a prior treatment, until the current post-operative recovery is not shown to be abnormal. In combination with their corresponding organoid drug response, both of their corresponding organoids, oxaliplatin, were found to be sensitive (fig. 6). The prediction of drug response by the organoids is suggested to have a certain reliability.
EXAMPLE 3 use of organoids in research for the treatment of colorectal cancer
The effect of TNFR2 on CRC organoid growth was examined. In three cases (# 1, #2, # 3) CRC organoids, TNFR2 was knocked down, the knockdown sequences were shTNFR2-1, shTNFR2-2 (FIG. 7), and organoid growth was indeed affected as compared to the control group (shControl), and it was observed under a microscope that the morphology had been significantly changed, that part had been changed to cell debris (FIG. 8), that the proliferation potency had been significantly reduced (FIG. 9), that the organoid number had been significantly reduced (FIG. 10), and that the size had been affected (FIG. 11).
Comparative example 1 reduction of the Effect of additives on organoid culture
Based on the medium of example 1, noggin, SB202190, gastin I, A8301, R-Spondin1, prostaglandin E2 were subtracted, respectively, and organoids were cultured according to the same experimental procedure as in example 1, and after 7 days of culture, it was found that removal of either additive affected normal organoid growth. The morphology of organoids no longer changed, had no growing trend, could not meet the requirements of passage expansion, had vacuoles in the middle, and lost the morphological features of colorectal cancer tissues (fig. 12).
Organoid survival was counted and decreased significantly in the absence of either component (0.0329,0.0072,0.0003,0.0002,0.0002,0.0021 from left to right between figure 13 and P values for the full addition group, respectively).
Comparative example 2 Effect of increasing additives on organoid culture
The organoids were cultured in the same manner as in example 1, and HGF, IL-6, and SDF1 were added to the medium of example 1, respectively, and it was found that the organoids were grown after addition of HGF, but the time for maintenance was short, death occurred in the latter organoids, and further growth was not continued (fig. 14A), and organoids could not be produced by addition of IL-6 (fig. 14B), and organoids were grown with no improvement by addition of SDF1, and the growth rate was slow (fig. 14C).
The above comparative examples 1 and 2 show that the organoids were cultured with less effectiveness than the organoids cultured in the medium of example 1, regardless of whether Noggin, SB202190, gastin I, A8301, R-Spondin1, prostaglandin E2 or HGF, IL-6, SDF1 were reduced or increased based on the original medium, indicating that the technical effect of the present invention could be achieved without reducing or increasing any factor.
The above description of the embodiments is only for the understanding of the method of the present invention 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 invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims (10)

1. A colorectal cancer organoid medium, wherein the medium comprises BMP inhibitors, prostigndin E2, wnt agonists, TGF- β inhibitors, gastrin I, p38 inhibitors;
preferably, the BMP inhibitor comprises Noggin, gremlin, chordin, follistatin, sclerostin, decorin;
preferably, the BMP inhibitor is selected from Noggin;
preferably, the Wnt agonist comprises Wnt protein, R-spondin;
preferably, the Wnt agonist is selected from the group consisting of R-spondin;
preferably, the R-spondin comprises R-spondin1, R-spondin2, R-spondin3, R-spondin4;
preferably, the R-spondin is selected from R-spondin1;
preferably, the TGF- β inhibitor comprises a8301, SB431542, SB505124, SB525334, SD208, LY36494, and SJN2511;
preferably, the TGF- β inhibitor is selected from a8301;
preferably, the p38 inhibitor comprises SB202190, SB203580, VX702, VX745, PD169316, RO4402257, BIRB796;
preferably, the p38 inhibitor is selected from SB-202190.
2. The medium of claim 1, wherein the medium further comprises an antibiotic;
preferably, the antibiotics comprise Primocin, penicillin streptomycin and metronidazole;
Preferably, the antibiotic is selected from Primocin;
preferably, the medium further comprises a nutritional additive;
preferably, the nutritional additives include Gluta Max, B27, and N2;
preferably, the nutritional additive is selected from Gluta Max;
preferably, the medium further comprises mitogenic proliferation factor, FGF-basic, N-acetyl-L-cysteine, rock inhibitor;
preferably, the mitogenic proliferation factor comprises EGF, BDNF, KGF;
preferably, the mitogenic proliferation factor is selected from EGF;
preferably, the Rock inhibitor comprises Y-27632, HA1077, H-1152;
preferably, the Rock inhibitor is selected from Y-27632.
3. A method of culturing colorectal cancer organoids, said method comprising culturing colorectal cancer cells using the medium of claim 1 or 2;
preferably, the method further comprises adding ECM to co-culture with colorectal cancer cells;
preferably, the ECM includes BME and matrigel;
preferably, the ECM is selected from matrigel.
4. The method of claim 3, wherein the colorectal cancer cells are obtained by digesting colorectal cancer tissue;
preferably, the colorectal cancer tissue is digested with a tissue digestion solution;
Preferably, the stock solution of the tissue digestion solution comprises Tryple and/or collagenase;
preferably, the mother liquor of the tissue digestion solution is selected from the group consisting of Tryple;
preferably, the concentration of Tryple is 2 XTryple-10 XTryple;
preferably, the concentration of the Tryple is 5×tryple;
preferably, the digestion time of the tissue is 30min-12h;
preferably, the digestion time of the tissue is 30min;
preferably, the digestion is stopped using a PBS solution or medium;
preferably, the digestion is terminated using a PBS solution;
preferably, the method further comprises washing the tissue prior to digesting the tissue;
preferably, the tissue is washed with PBS.
5. The method of claim 3, further comprising amplifying the organoid;
preferably, the organoid amplification comprises digestive organoids;
preferably, the organoids are digested with Tryple;
preferably, the concentration of Tryple is 2 XTryple;
preferably, the digestion time is 5min-30min;
preferably, the digestion time is 20min;
preferably, the method further comprises passaging the organoid;
preferably, the passaging organoids comprise digestive organoids;
preferably, the organoids are digested with passaged digestive fluids;
preferably, the mother liquor of the passaged digestive juice comprises Tryple and/or collagenase;
Preferably, the mother liquor of the passaged digest is selected from Tryple;
preferably, HBSS is used to terminate digestion;
preferably, the digestive juice: hbss=1: 2.
6. colorectal cancer organoids characterized in that they are cultivated using the medium according to claim 1 or 2 or obtained by the method according to any one of claims 3 to 5.
7. The use of any one of the following:
(1) Use of the medium of claim 1 or 2 for culturing colorectal cancer organoids;
(2) Use of the organoid of claim 6 for screening for a medicament for the prevention and/or treatment of colorectal cancer;
(3) Use of the organoid of claim 6 for modeling colorectal cancer drug susceptibility;
(4) Use of the organoid of claim 6 in the study of colorectal cancer pathogenesis or causative factor.
8. An in vitro drug screening model for colorectal cancer, characterized in that said model comprises the organoid of claim 6.
9. A method of predicting colorectal cancer drug sensitivity, comprising contacting the organoid of claim 6 or the model of claim 8 with a test drug.
10. A method of screening for a drug for preventing and/or treating colorectal cancer, comprising contacting a candidate drug with the organoid of claim 6 or the model of claim 8.
CN202310175815.2A 2023-02-28 2023-02-28 Construction method and application of colorectal cancer organoids Pending CN116355850A (en)

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