CN116751749A - Body fluid tumor organoid culture method and drug sensitivity detection method - Google Patents

Body fluid tumor organoid culture method and drug sensitivity detection method Download PDF

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CN116751749A
CN116751749A CN202311023031.4A CN202311023031A CN116751749A CN 116751749 A CN116751749 A CN 116751749A CN 202311023031 A CN202311023031 A CN 202311023031A CN 116751749 A CN116751749 A CN 116751749A
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tumor
organoid
growth factor
culture medium
fibroblast growth
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CN116751749B (en
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陈晔光
方芝
田军
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Guangzhou Huayi Regeneration Technology Co ltd
Nanchang University
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Guangzhou Huayi Regeneration Technology Co ltd
Nanchang University
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Abstract

The invention provides a method for culturing body fluid tumor organoids and a method for detecting drug sensitivity. The organoid culture medium can ensure the cell activity to a great extent and reduce the interference of other hybrid cells, can well maintain the characteristics of tissue cells, and experiments show that the organoid cultured by the organoid culture medium is highly similar to tumors in terms of tissue morphology and can reduce the tumor characteristics of original tumor cells to a great extent. The invention brings the tumor cells in the coagulated mass into the culture range, and can preserve the original tumor cells in the body fluid as much as possible, so that the finally cultured body fluid tumor organoids basically cover the characteristics of all the tumor cells of different types.

Description

Body fluid tumor organoid culture method and drug sensitivity detection method
Technical Field
The invention relates to the technical field of biological medicine, in particular to a body fluid tumor organoid culture method and a drug sensitivity detection method.
Background
Organoid technology is a recently discovered 3D culture technology that is widely used in its ability to mimic the properties of the original maternal tissue in structure and function. In recent years, the tumor organoids have shown great potential in-vitro basic research and clinical transformation application, and gradually become important and hot spot research fields, but so far, the culture system and culture conditions aiming at the tumor organoids are not completely mature, and the limitation condition greatly pulls the development and application of the tumor organoids.
Peritoneum, pleura, pericardium, etc. are common distant metastases after malignant tumor surgery and recurrent lesions after treatment, and some patients find chest and peritoneal metastases. Early metastatic patients have no obvious clinical features and are difficult to find by imaging examination, while late patients often combine a great deal of clinical manifestations such as hydrothorax, ascites or pericardial effusion, and the prognosis is often poor when found. The cytological diagnosis of body fluid is a gold standard for judging the malignancy degree of body fluid at present, but researches on 3D culture technology for tumor cells in malignant effusion of patients and a tumor microenvironment simulation and drug treatment reaction system by using the same are rarely reported.
Disclosure of Invention
In view of the above, the present invention provides a method for culturing a humoral tumor organoid and a drug sensitive detection method, so as to realize the culture of the humoral tumor organoid, and simulate a tumor microenvironment and a drug treatment reaction system by using the same.
In one aspect, the invention provides a method for culturing a humoral tumor organoid, comprising:
step 1, adding a sample preservation solution into malignant effusion to obtain tumor cells in the malignant effusion, wherein the tumor cells comprise a free tumor cell part a and a tumor cell part b in a coagulated mass, filtering and collecting the tumor cell part b in the coagulated mass by using a cell screen, centrifuging, and removing the supernatant to obtain the free tumor cell part a;
step 2, digesting and hydrolyzing the tumor cell part b in the obtained coagulated mass by using a digestive juice, stopping digestion by using a mild sample buffer solution after enzymolysis, collecting suspension, and centrifuging to obtain a tumor cell part c;
step 3, re-suspending the free tumor cell part a and the tumor cell part c in a sample buffer solution respectively, performing adherence treatment, repeating tumor cells subjected to the adherence treatment for the last time on the basis of the last time, collecting the adherence cell suspension for the last time, and centrifuging to obtain tumor cells d and e;
Step 4, washing and centrifuging the tumor cells d and the tumor cells e by using a sample washing liquid respectively, adding an extracellular matrix, uniformly mixing, then performing seed plates, reversely buckling and standing in a cell culture box, adding an organoid culture medium after gel fixation, continuously culturing in the cell culture box, and replacing the organoid culture medium every 3-4 days to complete the culture of the body fluid tumor organoids;
the organoid medium comprises the following components: basic culture medium DMEM/F12, streptomycin, N2, B27, HEPES, glutamax, N-acetylcysteine, epidermal cell growth factor, R-spinal protein 1, wnt3a, fibroblast growth factor 10 and basic fibroblast growth factor, wherein the contents of each component except the basic culture medium DMEM/F12 in the organoid culture medium are as follows: green streptomycin, 1-100X; n2, 50-100X; b27 50-100X; HEPES,1-100X; glutaMAX,1-10X; n-acetylcysteine, 0.5-5mM; epidermal cell growth factor, 30-50ng/mL; r-spinal protein 1, 200-500ng/mL; wnt3a,50-100ng/mL; fibroblast growth factor 10, 50-100ng/mL; basic fibroblast growth factor, 20-100ng/mL.
The digestive juice comprises the following components in percentage by weight: glutaMAX,1-10X; pancreatic proteolytic enzyme, 0.1-0.2%; HEPES,1-10X; type IV collagenase, 0.5-2mg/mL; insulin, 1-5 μg/mL, Y27632,1-20 μM; DNA lyase, 0.1-0.5mg/mL;
The adherence treatment conditions are as follows: the sticking times are 3-6 times, and the time is 20-50 min/time.
The humoral tumor organoid culture method, wherein the components of the sample preservation solution comprise a basic culture medium DMEM/F12 and one or more of the following components: green streptomycin, N2, B27, HEPES, N-acetylcysteine, epidermal growth factor, noggin, R-spinal protein 1, wnt3a, fibroblast growth factor 10, basic fibroblast growth factor, EDTA-K.
Specifically, according to the method for culturing the humoral tumor organoids, the contents of all components except the basic culture medium DMEM/F12 in the sample preservation solution are as follows: green streptomycin, 1-100X; n2, 90-100X; b27 40-60X; HEPES,50-100X; n-acetylcysteine, 0.5-5mM; epidermal cell growth factor, 30-50ng/mL; noggin,50-100ng/mL; r-spinal protein 1, 200-500ng/mL; wnt3a,50-100ng/mL; fibroblast growth factor 10, 10-50ng/mL; alkaline fibroblast growth factor, 10-30ng/mL; EDTA-K,1-10X.
The humoral tumor organoid culture method comprises the following steps of: glutaMAX,1-10X; pancreatic proteolytic enzyme, 0.1-0.2%; HEPES,1-10X; type IV collagenase, 0.5-2mg/mL; insulin, 1-5 μg/mL, Y27632,1-20 μM; DNA lyase, 0.1-0.5mg/mL.
The body fluid tumor organoid culture method comprises the following components in parts by weight: green streptomycin, 1-10X; primocin,10-1000 mug/mL; insulin, 1-5 μg/mL; y27632, 1-20. Mu.M; 0.1-0.3X N2 PBS buffer without calcium and magnesium ions is added.
The humoral tumor organoid culture method, wherein the gentle sample buffer solution comprises the following components in percentage by weight: n2,0.1-0.3X; BSA,2-10%; y27632, 1-20. Mu.M; dexamethasone, 1-5nM; blebtistein, 10-50. Mu.M; gentamicin, 10-100 mug/mL; 1-10 XHEPES added minimal medium DMEM/F12.
The body fluid tumor organoid culture method comprises the following steps: the sticking times are 3-6 times, and the time is 20-50 min/time.
The humoral tumor organoid culture method, wherein the centrifugation conditions in the step 1, the step 2 and the step 3 are as follows: the centrifugal speed is 500-1000rpm/min, the centrifugal time is 5-10min at 4 ℃;
the washing and centrifuging conditions in the step 4 are that the temperature is 4 ℃ and the centrifugation is 250-500g and 3-5min.
The method for culturing a humoral tumor organoid, wherein the malignant effusion is any one of malignant effusion caused by gastric cancer, malignant effusion caused by non-small cell lung cancer, malignant effusion caused by intestinal cancer, malignant effusion caused by malignant appendiceal tumor, and malignant effusion caused by pancreatic cancer.
The humoral tumor organoid culture method, wherein the malignant effusion is prepared from the following parts: any one of the thoracic, abdominal and pericardial cavities.
The method for culturing the humoral tumor organoid, wherein the malignant effusion is obtained from a patient or an animal suffering from non-small cell lung cancer, and the part source is pericardium.
In another aspect, the invention provides a method for detecting drug sensitivity based on a body fluid tumor organoid, comprising:
washing the body fluid tumor organoids successfully cultured by using PBS buffer solution, adding 200-500 mu L of organoid digestive juice, shaking and digesting, centrifuging to obtain precipitate, counting, adding a medicine sieve culture medium to be resuspended, wherein the medicine sieve culture medium is an organoid culture medium containing 1-3% Matrigel, plating the organoids in a pore plate, adding a medicine to be tested, and carrying out cell viability test after 5 days.
The drug sensitivity detection method based on the body fluid tumor organoid comprises the following components in percentage by weight: glutaMAX,1-10X; insulin, 1-5 μg/mL; y27632, 1-20. Mu.M; forskolin, 1-20 μm; nicotinamide, 1-10mM; contains 0.1-0.5mg/mL of DNA lyase; trypLE digest containing HEPES at 1-10X.
The beneficial effects are that:
The sample preservation solution and the body fluid tumor cell culture method can ensure the cell activity to a great extent, reduce the interference of other hybrid cells such as mesothelial cells and fibroblasts, and the organoid culture medium can well maintain the characteristics of tissue cells. The invention brings the tumor cells in the coagulated mass into the culture range, and can preserve the original tumor cells in the body fluid as much as possible, so that the finally cultured body fluid tumor organoids basically cover the characteristics of all the tumor cells of different types.
In addition, the body fluid tumor organoid obtained by the invention can be used for detecting the drug sensitivity of clinical tumor patients and testing and applying potential effective target drugs, and in the aspect of clinical guidance drug administration, the body fluid tumor organoid drug sensitivity result also shows consistent conformity with clinical treatment response, and data support and possibility are provided for personalized accurate treatment of tumor patients.
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FIG. 1 is a graph showing the results of the organoid culture of gastric ascites organoid by the humoral tumor organoid culture method of example 1;
FIG. 2 is a graph showing the results of the organoid culture of gastric carcinoma hydrothorax organoids using the humoral tumor organoid culture method of example 1;
FIG. 3 is a graph of organoid results obtained from the culture of ascites organoids from appendicular cancer using the humoral tumor organoid culture method of example 1;
FIG. 4 is a graph showing the organoid results obtained by culturing a hydrothorax organoid of colon cancer using the humoral tumor organoid culture method of example 1;
FIG. 5 is a graph showing the results of organoids obtained by culturing ascites organoids for colon cancer using the humoral tumor organoid culture method of example 1;
FIG. 6 is a graph of organoid results obtained from culturing pancreatic cancer hydrothorax organoids using the humoral tumor organoid culture method of example 1;
FIG. 7 is a graph of organoid results obtained from culturing a lung adenocarcinoma pericardial effusion organoid using the humoral tumor organoid culture method of example 1;
FIG. 8 is a graph of organoid results obtained from culturing small cell lung cancer ascites organoids using the humoral tumor organoid culture method of example 1;
FIG. 9 is a graph of organoid results obtained from culturing lung adenocarcinoma hydrothorax organoids using the humoral tumor organoid culture method of example 1;
FIG. 10 is a graph showing the results of the culture of gastric cancer ascites organoids for 3 days using the humoral tumor organoids culture method of example 1;
FIG. 11 is a graph showing the results of the culture of gastric ascites organoid for 3 days by the humoral tumor organoid culture method of example 2;
FIG. 12 is a graph showing the results of the culture of gastric cancer ascites organoids for 3 days using the humoral tumor organoid culture method of example 3;
FIG. 13 is a graph showing the results of the culture of gastric ascites organoid for 3 days by the humoral tumor organoid culture method of example 4;
FIG. 14 is a graph showing the results of the culture of gastric ascites organoid for 3 days by the humoral tumor organoid culture method of example 5;
FIG. 15 is a graph showing the effect of lung adenocarcinoma pericardial effusion organoids in culture during conventional direct plating;
FIG. 16 is a graph showing the effect of gastric ascites organoids in culture during conventional direct plating;
FIG. 17 is a graph showing the effect of lung adenocarcinoma pericardial effusion organoids in the attachment process;
FIG. 18 is a graph showing the effect of gastric ascites organoids during adherence;
FIG. 19 is a graph showing the effect of lung adenocarcinoma pericardial effusion organoids in culture during digestion;
FIG. 20 is a graph showing the effect of gastric ascites organoid culture during digestion;
FIG. 21 is a graph showing the effect of identifying the thoracic water organoids of gastric cancer;
fig. 22 is a diagram of imaging results.
Detailed Description
The invention will be described more fully hereinafter with reference to the accompanying examples in order to facilitate an understanding of the invention, however, the invention may be embodied in many different forms and is not limited to the examples described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, 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 terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The embodiment of the invention provides a body fluid tumor organoid culture method, which comprises the following steps:
step 1, adding a sample preservation solution into malignant effusion to obtain tumor cells in the malignant effusion, wherein the purpose of adding the sample preservation solution is to ensure the cell activity, the tumor cells comprise a free tumor cell part a and a tumor cell part b in a coagulated mass, filtering and collecting the tumor cell part b in the coagulated mass by using a cell screen, centrifuging, and removing the supernatant to obtain the free tumor cell part a;
Step 2, digesting and hydrolyzing the tumor cell part b in the obtained coagulated mass by using a digestive juice, stopping digestion by using a mild sample buffer solution after enzymolysis, collecting suspension, and centrifuging to obtain a tumor cell part c;
step 3, re-suspending the free tumor cell part a and the tumor cell part c in a sample buffer solution respectively, performing adherence treatment, repeating tumor cells subjected to the adherence treatment for the last time on the basis of the last time, collecting the adherence cell suspension for the last time, and centrifuging to obtain tumor cells d and e;
step 4, washing and centrifuging the tumor cells d and the tumor cells e by using a sample washing liquid respectively, adding an extracellular matrix, uniformly mixing, then performing seed plates, reversely buckling and standing in a cell culture box, adding an organoid culture medium after gel fixation, continuously culturing in the cell culture box, and replacing the organoid culture medium every 3-4 days to complete the culture of the body fluid tumor organoids;
the organoid medium comprises the following components: basic culture medium DMEM/F12, green streptomycin (P/S), N2, B27, HEPES, glutamax, N-acetylcysteine (N-acetylcysteine), epidermal Growth Factor (EGF), R-spinal protein 1 (R-spondin 1), wnt3a, fibroblast growth factor 10 (FGF 10) and basic fibroblast growth factor (FGF 2), wherein the organoid culture medium comprises the following components except the basic culture medium DMEM/F12: green streptomycin, 1-100X; n2, 50-100X; b27 50-100X; HEPES,1-100X; glutaMAX,1-10X; n-acetylcysteine, 0.5-5mM; epidermal cell growth factor, 30-50ng/mL; r-spinal protein 1, 200-500ng/mL; wnt3a,50-100ng/mL; fibroblast growth factor 10, 50-100ng/mL; basic fibroblast growth factor, 20-100ng/mL.
The humoral tumor organoid culture method, wherein the components of the sample preservation solution comprise a basic culture medium DMEM/F12 and one or more of the following components: green streptomycin, N2, B27, HEPES, N-acetylcysteine, epidermal growth factor, noggin, R-spinal protein 1, wnt3a, fibroblast growth factor 10, basic fibroblast growth factor, EDTA-K.
Specifically, according to the method for culturing the humoral tumor organoids, the contents of all components except the basic culture medium DMEM/F12 in the sample preservation solution are as follows: green streptomycin, 1-100X; n2, 90-100X; b27 40-60X; HEPES,50-100X; n-acetylcysteine, 0.5-5mM; epidermal cell growth factor, 30-50ng/mL; noggin,50-100ng/mL; r-spinal protein 1, 200-500ng/mL; wnt3a,50-100ng/mL; fibroblast growth factor 10, 10-50ng/mL; alkaline fibroblast growth factor, 10-30ng/mL; EDTA-K,1-10X.
The humoral tumor organoid culture method comprises the following steps of: glutaMAX,1-10X; pancreatic proteolytic enzyme, 0.1-0.2%; HEPES,1-10X; collagenase type IV (Collagenase IV), 0.5-2mg/mL; insulin (Insulin), 1-5 μg/mL, Y27632,1-20 μM; DNA lyase, 0.1-0.5mg/mL.
The body fluid tumor organoid culture method comprises the following components in parts by weight: green streptomycin, 1-10X; primocin,10-1000 mug/mL; insulin, 1-5 μg/mL; y27632, 1-20. Mu.M; 0.1-0.3X N2 PBS buffer without calcium and magnesium ions is added.
The humoral tumor organoid culture method, wherein the gentle sample buffer solution comprises the following components in percentage by weight: n2,0.1-0.3X; BSA,2-10%; y27632, 1-20. Mu.M; dexamethasone (Dexamethaname), 1-5nM; blebtistein, 10-50. Mu.M; gentamicin (Gentamicin), 10-100 μg/mL; 1-10 XHEPES added minimal medium DMEM/F12.
The body fluid tumor organoid culture method comprises the following steps: the sticking times are 3-6 times, and the time is 20-50 min/time.
The humoral tumor organoid culture method, wherein the centrifugation conditions in the step 1, the step 2 and the step 3 are as follows: the centrifugal speed is 500-1000rpm/min, the centrifugal time is 5-10min at 4 ℃;
the washing centrifugation condition in the step 4 is centrifugation at 4 ℃,250-500g,3-5min
The method for culturing a humoral tumor organoid, wherein the malignant effusion is any one of malignant effusion caused by gastric cancer, malignant effusion caused by non-small cell lung cancer, malignant effusion caused by intestinal cancer, malignant effusion caused by malignant appendiceal tumor, and malignant effusion caused by pancreatic cancer.
The humoral tumor organoid culture method, wherein the malignant effusion is prepared from the following parts: any one of the thoracic, abdominal and pericardial cavities.
The method for culturing the humoral tumor organoid, wherein the malignant effusion is obtained from a patient or an animal suffering from non-small cell lung cancer, and the part source is pericardium.
The embodiment of the invention also provides a body fluid tumor organoid culture method, which comprises the following steps:
washing the body fluid tumor organoids successfully cultured by using PBS buffer solution, adding 200-500 mu L of organoid digestive juice, shaking and digesting, centrifuging to obtain precipitate, counting, adding a medicine sieve culture medium to be resuspended, wherein the medicine sieve culture medium is an organoid culture medium containing 1-3% Matrigel, plating the organoids in a pore plate, adding a medicine to be tested, and carrying out cell viability test after 5 days.
The drug sensitivity detection method based on the body fluid tumor organoid comprises the following components in percentage by weight: glutaMAX,1-10X; insulin, 1-5 μg/mL; y27632, 1-20. Mu.M; forskolin (Forskolin), 1-20 μm; nicotinamide (Nicotinamide), 1-10mM; contains 0.1-0.5mg/mL of DNA lyase; trypLE digest containing HEPES at 1-10X.
The following examples are provided to further illustrate embodiments of the invention. The embodiments of the present invention are not limited to the following specific embodiments. The modification can be appropriately performed within the scope of the main claim.
Example 1:
a method of humoral tumor organoid culture comprising:
step 1, adding 10mL of sample preservation solution into malignant effusion to obtain tumor cells in the malignant effusion, wherein the sample preservation solution comprises the following components: basic culture medium DMEM/F12, green streptomycin, N2, B27, HEPES, N-acetylcysteine, epidermal cell growth factor, noggin, R-spinal protein 1, wnt3a, fibroblast growth factor 10, basic fibroblast growth factor and EDTA-K; the content of each component except the basic culture medium DMEM/F12 in the sample preservation solution is as follows: green streptomycin, 100X; n2, 100X; b27 50X; HEPES,100X; n-acetylcysteine, 2.5mM; epidermal growth factor, 50ng/mL; noggin,50ng/mL; r-spinal protein 1, 200ng/mL; wnt3a,50ng/mL; fibroblast growth factor 10, 10ng/mL; basic fibroblast growth factor, 20ng/mL; EDTA-K,10X; the tumor cells comprise a free tumor cell part a and a tumor cell part b in the coagulated mass, the tumor cell part b in the coagulated mass is collected by using a 100 mu m cell screen for filtration, and then the obtained mixture is centrifuged at 4 ℃, the centrifugal speed is 1000rpm/min, the centrifugal time is 5min, and the supernatant is removed to obtain the free tumor cell part a;
Step 2, performing digestion and enzymolysis on a tumor cell part b in the obtained coagulated mass at 37 ℃ by using a digestion liquid, wherein the digestion liquid comprises the following components in percentage by weight: glutaMAX,1X; pancreatic proteolytic enzyme, 0.2%; HEPES,1X; type IV collagenase, 0.5mg/mL; insulin, 1 μg/mL, Y27632, 10 μM; DNA lyase, 0.2mg/mL; after enzymolysis, digestion is stopped by using a mild sample buffer solution, wherein the mild sample buffer solution comprises the following components in percentage by weight: n2,0.1X; BSA,2%; y27632, 10. Mu.M; dexamethasone, 1nM; blebtistein, 20. Mu.M; gentamicin, 10 μg/mL; 1X HEPES-supplemented minimal medium DMEM/F12; collecting suspension, and obtaining a tumor cell part c after centrifugation, wherein the centrifugation conditions are as follows: the centrifugal speed is 1000rpm/min, the temperature is 4 ℃, and the centrifugal time is 5min;
step 3, the free tumor cell part a and the tumor cell part c are respectively resuspended in a sample buffer solution, and the adherence treatment is carried out under the following conditions: the sticking times are 3 times, and the time is 50 min/time; repeating the tumor cells subjected to the next adherence treatment on the basis of the last time, collecting the final adherence cell suspension, and centrifuging to obtain tumor cells d and e, wherein the centrifuging conditions are as follows: the centrifugal speed is 800rpm/min, the temperature is 4 ℃, and the centrifugal time is 6min;
Step 4, washing and centrifuging the tumor cells d and the tumor cells e by using a sample washing liquid, wherein the conditions of washing and centrifuging are that the centrifugation is carried out at 4 ℃,500g and 4min, and the sample washing liquid comprises the following components in percentage by weight: green streptomycin, 1X; primocin,100 μg/mL; insulin, 1 μg/mL; y27632, 10. Mu.M; 0.2X N2-containing no magnesium ion PBS buffer; then adding extracellular matrix, uniformly mixing, performing plating according to 2 ten thousand cells/30 mu L of glue, then standing in a cell culture box in a reverse buckling manner, adding 500 mu L of organoid culture medium after the gel is solidified, continuously culturing in the cell culture box, and replacing the organoid culture medium once every 3 days to complete the culture of the humoral tumor organoid, wherein the organoid culture medium comprises the following components: basic culture medium DMEM/F12, streptomycin, N2, B27, HEPES, glutamax, N-acetylcysteine, epidermal cell growth factor, R-spinal protein 1, wnt3a, fibroblast growth factor 10 and basic fibroblast growth factor, wherein the contents of each component except the basic culture medium DMEM/F12 in the organoid culture medium are as follows: green streptomycin, 10X; n2, 100X; b27 50X; HEPES,100X; glutaMAX,2X; n-acetylcysteine, 2.5mM; epidermal growth factor, 50ng/mL; r-spinal protein 1, 200ng/mL; wnt3a,50ng/mL; fibroblast growth factor 10, 50ng/mL; basic fibroblast growth factor, 20ng/mL.
Based on the cultured body fluid tumor organoids, a drug sensitivity detection method is provided, which comprises the following steps: taking the body fluid tumor organoid which is successfully cultured, washing the body fluid tumor organoid by using PBS buffer solution, and adding 200-500 mu L of organoid digestive juice, wherein the organoid digestive juice comprises the following components in percentage by weight: glutaMAX,5X; insulin, 2 μg/mL; y27632, 10. Mu.M; forskolin, 15 μm; nicotinamide, 9mM; contains 0.3mg/mL of DNA lyase; a TrypLE digest containing 5X HEPES; after shaking digestion, centrifuging to obtain precipitate, counting, adding a medicine sieve culture medium, re-suspending the medicine sieve culture medium into 384-well plates, adding the medicine to be tested, and performing cell viability test after 5 days, wherein the medicine sieve culture medium is an organoid culture medium containing 2% Matrigel.
Example 2:
a method of humoral tumor organoid culture comprising:
step 1, adding 10mL of sample preservation solution into malignant effusion to obtain tumor cells in the malignant effusion, wherein the sample preservation solution comprises the following components: basic culture medium DMEM/F12, green streptomycin, N2, B27, HEPES, N-acetylcysteine, epidermal cell growth factor, noggin, R-spinal protein 1, wnt3a, fibroblast growth factor 10, basic fibroblast growth factor and EDTA-K; the content of each component except the basic culture medium DMEM/F12 in the sample preservation solution is as follows: green streptomycin, 40X; n2, 90X; b27 40X; HEPES,60X; n-acetylcysteine, 1mM; epidermal growth factor, 45ng/mL; noggin,70ng/mL; r-spinal protein 1, 30ng/mL; wnt3a,95ng/mL; fibroblast growth factor 10, 20ng/mL; basic fibroblast growth factor, 10ng/mL; EDTA-K,4X; the tumor cells comprise a free tumor cell part a and a tumor cell part b in the coagulated mass, the tumor cell part b in the coagulated mass is collected by using a 100 mu m cell screen for filtration, and then the obtained mixture is centrifuged at 4 ℃, the centrifugal speed is 800rpm/min, the centrifugal time is 6min, and the supernatant is removed to obtain the free tumor cell part a;
Step 2, performing digestion and enzymolysis on a tumor cell part b in the obtained coagulated mass at 37 ℃ by using a digestion liquid, wherein the digestion liquid comprises the following components in percentage by weight: glutaMAX,50X; pancreatic proteolytic enzyme, 0.1%; HEPES,6X; type IV collagenase, 1mg/mL; insulin, 4 μg/mL, Y27632,5 μM; DNA lyase, 0.5mg/mL; after enzymolysis, digestion is stopped by using a mild sample buffer solution, wherein the mild sample buffer solution comprises the following components in percentage by weight: n2,0.3X; BSA,6%; y27632, 1. Mu.M; dexamethasone, 3nM; blebtistein, 40. Mu.M; gentamicin, 100 μg/mL; 10X HEPES-supplemented minimal medium DMEM/F12; collecting suspension, and obtaining a tumor cell part c after centrifugation, wherein the centrifugation conditions are as follows: the centrifugal speed is 500rpm/min, the temperature is 4 ℃, and the centrifugal time is 10min;
step 3, the free tumor cell part a and the tumor cell part c are respectively resuspended in a sample buffer solution, and the adherence treatment is carried out under the following conditions: the sticking times are 4 times, and the time is 30 min/time; repeating the tumor cells subjected to the next adherence treatment on the basis of the last time, collecting the final adherence cell suspension, and centrifuging to obtain tumor cells d and e, wherein the centrifuging conditions are as follows: the centrifugal speed is 1000rpm/min, the temperature is 4 ℃, and the centrifugal time is 5min;
Step 4, washing and centrifuging the tumor cells d and the tumor cells e by using a sample washing liquid, wherein the conditions of washing and centrifuging are that centrifugation is carried out at 4 ℃,250g and 5min, and the sample washing liquid comprises the following components in percentage by weight: green streptomycin, 5X; primocin,300 μg/mL; insulin, 4 μg/mL; y27632, 15. Mu.M; 0.1X of N2-containing no magnesium ion PBS buffer; then adding extracellular matrix, uniformly mixing, performing plating according to 2 ten thousand cells/30 mu L of glue, then standing in a cell culture box in a reverse buckling manner, adding 500 mu L of organoid culture medium after the gel is solidified, continuously culturing in the cell culture box, and replacing the organoid culture medium once every 4 days to complete the culture of the humoral tumor organoid, wherein the organoid culture medium comprises the following components: basic culture medium DMEM/F12, streptomycin, N2, B27, HEPES, glutamax, N-acetylcysteine, epidermal cell growth factor, R-spinal protein 1, wnt3a, fibroblast growth factor 10 and basic fibroblast growth factor, wherein the contents of each component except the basic culture medium DMEM/F12 in the organoid culture medium are as follows: green streptomycin, 60X; n2, 80X; b27 70X; HEPES,50X; glutaMAX,1X; n-acetylcysteine, 4.5mM; epidermal growth factor, 35ng/mL; r-spinal protein 1, 250ng/mL; wnt3a,80ng/mL; fibroblast growth factor 10, 90ng/mL; basic fibroblast growth factor, 30ng/mL.
Based on the cultured body fluid tumor organoids, a drug sensitivity detection method is provided, which comprises the following steps: taking the body fluid tumor organoid which is successfully cultured, washing the body fluid tumor organoid by using PBS buffer solution, and adding 200-500 mu L of organoid digestive juice, wherein the organoid digestive juice comprises the following components in percentage by weight: glutaMAX,7X; insulin, 3 μg/mL; y27632, 20. Mu.M; forskolin, 3 μm; nicotinamide, 5mM; contains 0.1mg/mL of DNA lyase; a TrypLE digest containing 10X HEPES; after shaking digestion, centrifuging to obtain precipitate, counting, adding a medicine sieve culture medium, re-suspending the medicine sieve culture medium into 384-well plates, adding the medicine to be tested, and performing cell viability test after 5 days, wherein the medicine sieve culture medium is an organoid culture medium containing 3% Matrigel.
Example 3:
a method of humoral tumor organoid culture comprising:
step 1, adding 10mL of sample preservation solution into malignant effusion to obtain tumor cells in the malignant effusion, wherein the sample preservation solution comprises the following components: basic culture medium DMEM/F12, green streptomycin, N2, B27, HEPES, N-acetylcysteine, epidermal cell growth factor, noggin, R-spinal protein 1, wnt3a, fibroblast growth factor 10, basic fibroblast growth factor and EDTA-K; the content of each component except the basic culture medium DMEM/F12 in the sample preservation solution is as follows: green streptomycin, 1X; n2, 100X; b27 45X; HEPES,95X; n-acetylcysteine, 0.5mM; epidermal cell growth factor, 30ng/mL; noggin,85ng/mL; r-spinal protein 1, 500ng/mL; wnt3a, 100ng/mL; fibroblast growth factor 10, 45ng/mL; basic fibroblast growth factor, 15ng/mL; EDTA-K,1X; the tumor cells comprise a free tumor cell part a and a tumor cell part b in the coagulated mass, the tumor cell part b in the coagulated mass is collected by using a 100 mu m cell screen for filtration, and then the obtained mixture is centrifuged at 4 ℃, the centrifugal speed is 600rpm/min, the centrifugal time is 8min, and the supernatant is removed to obtain the free tumor cell part a;
Step 2, performing digestion and enzymolysis on a tumor cell part b in the obtained coagulated mass at 37 ℃ by using a digestion liquid, wherein the digestion liquid comprises the following components in percentage by weight: glutaMAX,10X; pancreatic proteolytic enzyme, 0.2%; HEPES,3X; type IV collagenase, 2mg/mL; insulin, 5 μg/mL, Y27632, 20 μM; DNA lyase, 0.4mg/mL; after enzymolysis, digestion is stopped by using a mild sample buffer solution, wherein the mild sample buffer solution comprises the following components in percentage by weight: n2,0.2X; BSA,10%; y27632, 10. Mu.M; dexamethasone, 5nM; blebtistein, 25. Mu.M; gentamicin, 15 μg/mL; 5X HEPES-supplemented minimal medium DMEM/F12; collecting suspension, and obtaining a tumor cell part c after centrifugation, wherein the centrifugation conditions are as follows: the centrifugal speed is 900rpm/min, the temperature is 4 ℃, and the centrifugal time is 7min;
step 3, the free tumor cell part a and the tumor cell part c are respectively resuspended in a sample buffer solution, and the adherence treatment is carried out under the following conditions: the sticking times are 6 times, and the time is 20 min/time; repeating the tumor cells subjected to the next adherence treatment on the basis of the last time, collecting the final adherence cell suspension, and centrifuging to obtain tumor cells d and e, wherein the centrifuging conditions are as follows: the centrifugal speed is 600rpm/min, the centrifugal time is 8min at 4 ℃;
Step 4, washing and centrifuging the tumor cells d and the tumor cells e by using a sample washing liquid, wherein the conditions of washing and centrifuging are that the centrifugation is carried out at 4 ℃,300g and 3min, and the sample washing liquid comprises the following components in percentage by weight: green streptomycin, 10X; primocin,10 μg/mL; insulin, 5 μg/mL; y27632, 20. Mu.M; 0.3X of N2-containing no magnesium ion PBS buffer; then adding extracellular matrix, uniformly mixing, performing plating according to 2 ten thousand cells/30 mu L of glue, then standing in a cell culture box in a reverse buckling manner, adding 500 mu L of organoid culture medium after the gel is solidified, continuously culturing in the cell culture box, and replacing the organoid culture medium once every 3 days to complete the culture of the humoral tumor organoid, wherein the organoid culture medium comprises the following components: basic culture medium DMEM/F12, streptomycin, N2, B27, HEPES, glutamax, N-acetylcysteine, epidermal cell growth factor, R-spinal protein 1, wnt3a, fibroblast growth factor 10 and basic fibroblast growth factor, wherein the contents of each component except the basic culture medium DMEM/F12 in the organoid culture medium are as follows: green streptomycin, 1X; n2, 50X; b27 60X; HEPES,1X; glutaMAX,8X; n-acetylcysteine, 3mM; epidermal cell growth factor, 30ng/mL; r-vertebrate protein 1, 350ng/mL; wnt3a,75ng/mL; fibroblast growth factor 10, 75ng/mL; basic fibroblast growth factor, 100ng/mL.
Based on the cultured body fluid tumor organoids, a drug sensitivity detection method is provided, which comprises the following steps: taking the body fluid tumor organoid which is successfully cultured, washing the body fluid tumor organoid by using PBS buffer solution, and adding 200-500 mu L of organoid digestive juice, wherein the organoid digestive juice comprises the following components in percentage by weight: glutaMAX,10X; insulin, 5 μg/mL; y27632, 1. Mu.M; forskolin, 1 μm; nicotinamide, 10mM; contains 0.2mg/mL of DNA lyase; a TrypLE digest of HEPES containing 3X; after shaking digestion, centrifuging to obtain precipitate, counting, adding a medicine sieve culture medium, re-suspending the medicine sieve culture medium into 384-well plates, adding the medicine to be tested, and performing cell viability test after 5 days, wherein the medicine sieve culture medium is an organoid culture medium containing 1% Matrigel.
Example 4:
a method of humoral tumor organoid culture comprising:
step 1, adding 10mL of sample preservation solution into malignant effusion to obtain tumor cells in the malignant effusion, wherein the sample preservation solution comprises the following components: basic culture medium DMEM/F12, green streptomycin, N2, B27, HEPES, N-acetylcysteine, epidermal cell growth factor, noggin, R-spinal protein 1, wnt3a, fibroblast growth factor 10, basic fibroblast growth factor and EDTA-K; the content of each component except the basic culture medium DMEM/F12 in the sample preservation solution is as follows: green streptomycin, 10X; n2, 95X; b27 60X; HEPES,50X; n-acetylcysteine, 4.5mM; epidermal growth factor, 35ng/mL; noggin,100ng/mL; r-spinal protein 1, 200ng/mL; wnt3a,65ng/mL; fibroblast growth factor 10, 50ng/mL; basic fibroblast growth factor, 30ng/mL; EDTA-K,3X; the tumor cells comprise a free tumor cell part a and a tumor cell part b in the coagulated mass, the tumor cell part b in the coagulated mass is collected by using a 100 mu m cell screen for filtration, and then the obtained mixture is centrifuged at 4 ℃, the centrifugal speed is 500rpm/min, the centrifugal time is 10min, and the supernatant is removed to obtain the free tumor cell part a;
Step 2, performing digestion and enzymolysis on a tumor cell part b in the obtained coagulated mass at 37 ℃ by using a digestion liquid, wherein the digestion liquid comprises the following components in percentage by weight: glutaMAX,8X; pancreatic proteolytic enzyme, 0.1%; HEPES,2X; type IV collagenase, 1.5mg/mL; insulin, 2 μg/mL, Y27632,1 μM; DNA lyase, 0.1mg/mL; after enzymolysis, digestion is stopped by using a mild sample buffer solution, wherein the mild sample buffer solution comprises the following components in percentage by weight: n2,0.1X; BSA,8%; y27632, 20. Mu.M; dexamethasone, 1nM; blebtistein, 10. Mu.M; gentamicin, 35 μg/mL; 2X HEPES-supplemented minimal medium DMEM/F12; collecting suspension, and obtaining a tumor cell part c after centrifugation, wherein the centrifugation conditions are as follows: the centrifugal speed is 700rpm/min, the temperature is 4 ℃, and the centrifugal time is 8min;
step 3, the free tumor cell part a and the tumor cell part c are respectively resuspended in a sample buffer solution, and the adherence treatment is carried out under the following conditions: the sticking times are 5 times, and the time is 25 min/time; repeating the tumor cells subjected to the next adherence treatment on the basis of the last time, collecting the final adherence cell suspension, and centrifuging to obtain tumor cells d and e, wherein the centrifuging conditions are as follows: the centrifugal speed is 650rpm/min, the temperature is 4 ℃, and the centrifugal time is 8min;
Step 4, washing and centrifuging the tumor cells d and the tumor cells e by using a sample washing liquid, wherein the conditions of the washing and centrifuging are that the washing and centrifuging are at 4 ℃,450g and 3min, and the sample washing liquid comprises the following components in percentage by weight: green streptomycin, 3X; primocin,1000 μg/mL; insulin, 3 μg/mL; y27632, 1. Mu.M; 0.2X N2-containing no magnesium ion PBS buffer; then adding extracellular matrix, uniformly mixing, performing plating according to 2 ten thousand cells/30 mu L of glue, then standing in a cell culture box in a reverse buckling manner, adding 500 mu L of organoid culture medium after the gel is solidified, continuously culturing in the cell culture box, and replacing the organoid culture medium once every 3 days to complete the culture of the humoral tumor organoid, wherein the organoid culture medium comprises the following components: basic culture medium DMEM/F12, streptomycin, N2, B27, HEPES, glutamax, N-acetylcysteine, epidermal cell growth factor, R-spinal protein 1, wnt3a, fibroblast growth factor 10 and basic fibroblast growth factor, wherein the contents of each component except the basic culture medium DMEM/F12 in the organoid culture medium are as follows: green streptomycin, 50X; n2, 60X; b27 90X; HEPES,80X; glutaMAX,10X; n-acetylcysteine, 0.5mM; epidermal growth factor, 45ng/mL; r-spinal protein 1, 500ng/mL; wnt3a,100ng/mL; fibroblast growth factor 10, 80ng/mL; alkaline fibroblast growth factor, 80ng/mL.
Based on the cultured body fluid tumor organoids, a drug sensitivity detection method is provided, which comprises the following steps: taking the body fluid tumor organoid which is successfully cultured, washing the body fluid tumor organoid by using PBS buffer solution, and adding 200-500 mu L of organoid digestive juice, wherein the organoid digestive juice comprises the following components in percentage by weight: glutaMAX,1X; insulin, 1 μg/mL; y27632, 5. Mu.M; forskolin, 8 μm; nicotinamide, 2mM; contains 0.5mg/mL of DNA lyase; a TrypLE digest containing 1X HEPES; after shaking digestion, centrifuging to obtain precipitate, counting, adding a medicine sieve culture medium, re-suspending the medicine sieve culture medium into 384-well plates, adding the medicine to be tested, and performing cell viability test after 5 days, wherein the medicine sieve culture medium is an organoid culture medium containing 2% Matrigel.
Example 5:
a method of humoral tumor organoid culture comprising:
step 1, adding 10mL of sample preservation solution into malignant effusion to obtain tumor cells in the malignant effusion, wherein the sample preservation solution comprises the following components: basic culture medium DMEM/F12, green streptomycin, N2, B27, HEPES, N-acetylcysteine, epidermal cell growth factor, noggin, R-spinal protein 1, wnt3a, fibroblast growth factor 10, basic fibroblast growth factor and EDTA-K; the content of each component except the basic culture medium DMEM/F12 in the sample preservation solution is as follows: green streptomycin, 80X; n2, 100X; b27 55X; HEPES,75X; n-acetylcysteine, 5mM; epidermal growth factor, 50ng/mL; noggin,65ng/mL; r-spinal protein 1, 450ng/mL; wnt3a,80ng/mL; fibroblast growth factor 10, 15ng/mL; alkaline fibroblast growth factor, 25ng/mL; EDTA-K,8X; the tumor cells comprise a free tumor cell part a and a tumor cell part b in the coagulated mass, the tumor cell part b in the coagulated mass is collected by using a 100 mu m cell screen for filtration, and then the obtained mixture is centrifuged at 4 ℃ at 650rpm/min for 8min, and the supernatant is removed to obtain the free tumor cell part a;
Step 2, performing digestion and enzymolysis on a tumor cell part b in the obtained coagulated mass at 37 ℃ by using a digestion liquid, wherein the digestion liquid comprises the following components in percentage by weight: glutaMAX,2X; pancreatic proteolytic enzyme, 0.1%; HEPES,10X; type IV collagenase, 1mg/mL; insulin, 1 μg/mL, Y27632,8 μM; DNA lyase, 0.3mg/mL; after enzymolysis, digestion is stopped by using a mild sample buffer solution, wherein the mild sample buffer solution comprises the following components in percentage by weight: n2,0.2X; BSA,3%; y27632, 5. Mu.M; dexamethasone, 2nM; blebtistein, 15. Mu.M; gentamicin, 60 μg/mL; 9X HEPES-supplemented minimal medium DMEM/F12; collecting suspension, and obtaining a tumor cell part c after centrifugation, wherein the centrifugation conditions are as follows: the centrifugal speed is 600rpm/min, the temperature is 4 ℃, and the centrifugal time is 9min;
step 3, the free tumor cell part a and the tumor cell part c are respectively resuspended in a sample buffer solution, and the adherence treatment is carried out under the following conditions: the sticking times are 3 times, and the time is 45 min/time; repeating the tumor cells subjected to the next adherence treatment on the basis of the last time, collecting the final adherence cell suspension, and centrifuging to obtain tumor cells d and e, wherein the centrifuging conditions are as follows: the centrifugal speed is 500rpm/min, the temperature is 4 ℃, and the centrifugal time is 10min;
Step 4, washing and centrifuging the tumor cells d and the tumor cells e by using a sample washing liquid, wherein the conditions of the washing and centrifuging are 4 ℃ and 400g and 5min, and the components and the contents of the sample washing liquid are as follows: green streptomycin, 8X; primocin,800 μg/mL; insulin, 1 μg/mL; y27632, 10. Mu.M; 0.1X of N2-containing no magnesium ion PBS buffer; then adding extracellular matrix, uniformly mixing, performing plating according to 2 ten thousand cells/30 mu L of glue, then standing in a cell culture box in a reverse buckling manner, adding 500 mu L of organoid culture medium after the gel is solidified, continuously culturing in the cell culture box, and replacing the organoid culture medium once every 4 days to complete the culture of the humoral tumor organoid, wherein the organoid culture medium comprises the following components: basic culture medium DMEM/F12, streptomycin, N2, B27, HEPES, glutamax, N-acetylcysteine, epidermal cell growth factor, R-spinal protein 1, wnt3a, fibroblast growth factor 10 and basic fibroblast growth factor, wherein the contents of each component except the basic culture medium DMEM/F12 in the organoid culture medium are as follows: green streptomycin, 100X; n2, 90X; b27 80X; HEPES,20X; glutaMAX,5X; n-acetylcysteine, 5mM; epidermal cell growth factor, 40ng/mL; r-spinal protein 1, 450ng/mL; wnt3a,95ng/mL; fibroblast growth factor 10, 50ng/mL; alkaline fibroblast growth factor, 75ng/mL.
Based on the cultured body fluid tumor organoids, a drug sensitivity detection method is provided, which comprises the following steps: taking the body fluid tumor organoid which is successfully cultured, washing the body fluid tumor organoid by using PBS buffer solution, and adding 200-500 mu L of organoid digestive juice, wherein the organoid digestive juice comprises the following components in percentage by weight: glutaMAX,2X; insulin, 3 μg/mL; y27632, 15. Mu.M; forskolin, 20 μm; nicotinamide, 1mM; contains 0.4mg/mL of DNA lyase; a TrypLE digest of HEPES at 6X; after shaking digestion, centrifuging to obtain precipitate, counting, adding a medicine sieve culture medium, re-suspending the medicine sieve culture medium into 384-well plates, adding the medicine to be tested, and performing cell viability test after 5 days, wherein the medicine sieve culture medium is an organoid culture medium containing 1% Matrigel.
Culture experiments of different tumor organoids
The humoral tumor organoid culture method in example 1 was used to culture gastric cancer ascites, gastric cancer hydrothorax, appendiceal cancer ascites, colon cancer hydrothorax, colon cancer ascites, pancreatic cancer hydrothorax, lung adenocarcinoma pericardial effusion, small cell lung cancer ascites, lung adenocarcinoma hydrothorax organoids, respectively, and the culture effects are shown in fig. 1 to 9. Further, the results of culturing gastric cancer ascites organoids by the humoral tumor organoid culture methods of examples 1, 2, 3, 4 and 5 for 3 days are shown in fig. 10 to 14. The results of FIGS. 1 to 14 show that various organs were well cultured.
Analysis of gastric cancer ascites organoid and lung adenocarcinoma pericardial effusion organoid culture results are shown in fig. 15 to 20, and compared with the conventional direct seed culture method, the improved culture system (organoid culture medium) is more suitable for culturing malignant body fluid organoids, as the tumor cell organoids of the pericardial effusion of lung adenocarcinoma and gastric cancer ascites proliferate and differentiate faster under the conditions of adherence and digestion.
The results of the humoral tumor organoid viability of different sites derived from different tumor types were counted using the humoral tumor organoid culture method of example 1 and the methods in other literature are shown in table 1.
TABLE 1
As can be seen from Table 1, the body fluid tumor organoid culture method of example 1 can effectively increase the survival rate of organoids of different types of tumors.
Immunofluorescence assay
The invention also carries out immunofluorescence experiment identification on hydrothorax organoids of gastric cancer patients, and the identification result is shown in figure 21. The markers Ep-cam and CK7 of the epithelium are positive, and the tumor related markers CEA, CA125 and Ki67 are also high-expressed, which further suggests that the gastric carcinoma hydrothorax organoids cultured by the system of the invention are tumor organoids of epithelial origin.
Drug sensitivity result consistency analysis experiment
The results of the organoid culture and single blindness drug sensitivity test of one example of ascites from an advanced gastric cancer patient harvested at day 23 of 2023 using the body fluid tumor organoid culture method and drug sensitivity test method of example 1 are shown in Table 2 and FIG. 22.
TABLE 2
As can be seen from table 2 and fig. 22, the tumor organoids derived from the humoral tumor cells of the patient were insensitive to the chemotherapeutic drug 5-Fu, oxaliplatin, paclitaxel and the targeted drug lenvatinib. Imaging of the patient at 2023, 05, suggested disease progression consistent with organoid sensitization.
In summary, the humoral tumor organoid culture method and the drug sensitivity detection method provided by the invention have the following beneficial effects:
the sample preservation solution and the body fluid tumor cell culture method can ensure the cell activity to a great extent, reduce the interference of other hybrid cells such as mesothelial cells and fibroblasts, and the organoid culture medium can well maintain the characteristics of tissue cells. The invention brings the tumor cells in the coagulated mass into the culture range, and can preserve the original tumor cells in the body fluid as much as possible, so that the finally cultured body fluid tumor organoids basically cover the characteristics of all the tumor cells of different types.
In addition, the body fluid tumor organoid obtained by the invention can be used for detecting the drug sensitivity of clinical tumor patients and testing and applying potential effective target drugs, and in the aspect of clinical guidance drug administration, the body fluid tumor organoid drug sensitivity result also shows consistent conformity with clinical treatment response, and data support and possibility are provided for personalized accurate treatment of tumor patients.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (10)

1. A method of humoral neoplasm organoid culture, comprising:
step 1, adding a sample preservation solution into malignant effusion to obtain tumor cells in the malignant effusion, wherein the tumor cells comprise a free tumor cell part a and a tumor cell part b in a coagulated mass, filtering and collecting the tumor cell part b in the coagulated mass by using a cell screen, centrifuging, and removing the supernatant to obtain the free tumor cell part a;
Step 2, digesting and hydrolyzing the tumor cell part b in the obtained coagulated mass by using a digestive juice, stopping digestion by using a mild sample buffer solution after enzymolysis, collecting suspension, and centrifuging to obtain a tumor cell part c;
step 3, re-suspending the free tumor cell part a and the tumor cell part c in a sample buffer solution respectively, performing adherence treatment, repeating tumor cells subjected to the adherence treatment for the last time on the basis of the last time, collecting the adherence cell suspension for the last time, and centrifuging to obtain tumor cells d and e;
step 4, washing and centrifuging the tumor cells d and the tumor cells e by using a sample washing liquid respectively, adding an extracellular matrix, uniformly mixing, then performing seed plates, reversely buckling and standing in a cell culture box, adding an organoid culture medium after gel fixation, continuously culturing in the cell culture box, and replacing the organoid culture medium every 3-4 days to complete the culture of the body fluid tumor organoids;
wherein the organoid medium comprises the following components: basic culture medium DMEM/F12, streptomycin, N2, B27, HEPES, glutamax, N-acetylcysteine, epidermal cell growth factor, R-spinal protein 1, wnt3a, fibroblast growth factor 10 and basic fibroblast growth factor, wherein the contents of each component except the basic culture medium DMEM/F12 in the organoid culture medium are as follows: green streptomycin, 1-100X; n2, 50-100X; b27 50-100X; HEPES,1-100X; glutaMAX,1-10X; n-acetylcysteine, 0.5-5mM; epidermal cell growth factor, 30-50ng/mL; r-spinal protein 1, 200-500ng/mL; wnt3a,50-100ng/mL; fibroblast growth factor 10, 50-100ng/mL; alkaline fibroblast growth factor, 20-100ng/mL;
The digestive juice comprises the following components in percentage by weight: glutaMAX,1-10X; pancreatic proteolytic enzyme, 0.1-0.2%; HEPES,1-10X; type IV collagenase, 0.5-2mg/mL; insulin, 1-5 μg/mL, Y27632,1-20 μM; DNA lyase, 0.1-0.5mg/mL;
the adherence treatment conditions are as follows: the sticking times are 3-6 times, and the time is 20-50 min/time.
2. The method of claim 1, wherein the sample preservation fluid comprises a basic medium DMEM/F12, and a combination of one or more of the following components: green streptomycin, N2, B27, HEPES, N-acetylcysteine, epidermal growth factor, noggin, R-spinal protein 1, wnt3a, fibroblast growth factor 10, basic fibroblast growth factor, EDTA-K.
3. The method according to claim 2, wherein the sample preservation solution contains the following components except the basic medium DMEM/F12: green streptomycin, 1-100X; n2, 90-100X; b27 40-60X; HEPES,50-100X; n-acetylcysteine, 0.5-5mM; epidermal cell growth factor, 30-50ng/mL; noggin,50-100ng/mL; r-spinal protein 1, 200-500ng/mL; wnt3a,50-100ng/mL; fibroblast growth factor 10, 10-50ng/mL; alkaline fibroblast growth factor, 10-30ng/mL; EDTA-K,1-10X.
4. The method according to claim 1, wherein the components and contents of the sample washing solution are as follows: green streptomycin, 1-10X; primocin,10-1000 mug/mL; insulin, 1-5 μg/mL; y27632, 1-20. Mu.M; 0.1-0.3X N2 PBS buffer without calcium and magnesium ions is added.
5. The method of claim 1, wherein the gentle sample buffer has the following composition and content: n2,0.1-0.3X; BSA,2-10%; y27632, 1-20. Mu.M; dexamethasone, 1-5nM; blebtistein, 10-50. Mu.M; gentamicin, 10-100 mug/mL; 1-10 XHEPES added minimal medium DMEM/F12.
6. The method according to claim 1, wherein the malignant effusion is any one of malignant effusion caused by gastric cancer, malignant effusion caused by non-small cell lung cancer, malignant effusion caused by intestinal cancer, malignant effusion caused by malignant appendiceal tumor, and malignant effusion caused by pancreatic cancer.
7. The humoral tumor organoid culture method according to claim 1, wherein the site sources of the malignant effusion are: any one of the thoracic, abdominal and pericardial cavities.
8. The method of claim 1, wherein the centrifugation conditions in step 1, step 2, and step 3 are as follows: the centrifugal speed is 500-1000rpm/min, the centrifugal time is 5-10min at 4 ℃;
the washing and centrifuging conditions in the step 4 are that the temperature is 4 ℃ and the centrifugation is 250-500g and 3-5min.
9. A method for detecting drug sensitivity of a body fluid tumor organoid based on the method of claim 1, comprising: washing a body fluid tumor organoid which is successfully cultured by using PBS buffer solution, adding 200-500 mu L of organoid digestive juice, shaking and digesting, centrifuging to obtain precipitate, counting, adding a medicine sieve culture medium to be resuspended, wherein the medicine sieve culture medium is an organoid culture medium containing 1-3% Matrigel, plating the organoid culture medium in an orifice plate, adding a medicine to be tested, and carrying out cell viability test after 5 days.
10. The method for detecting drug sensitivity according to claim 9, wherein the organoid digestive juice comprises the following components in percentage by weight: insulin, 1-5 μg/mL; y27632, 1-20. Mu.M; forskolin, 1-20 μm; nicotinamide, 1-10mM; contains 0.1-0.5mg/mL of DNA lyase; trypLE digest containing HEPES at 1-10X.
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