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CN118931839A - A culture medium and culture method for culturing a gastric cancer liver metastasis microtumor model - Google Patents

A culture medium and culture method for culturing a gastric cancer liver metastasis microtumor model Download PDF

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CN118931839A
CN118931839A CN202410996481.XA CN202410996481A CN118931839A CN 118931839 A CN118931839 A CN 118931839A CN 202410996481 A CN202410996481 A CN 202410996481A CN 118931839 A CN118931839 A CN 118931839A
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recombinant protein
gastric cancer
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席建忠
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Peking University
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Abstract

本发明公开了一种用于培养胃癌肝转移微肿瘤模型的培养基及培养方法。本发明提供了一种用于培养胃癌肝转移的微肿瘤模型的培养基，进一步地，本发明还提供了利用该培养基培养胃癌肝转移微肿瘤模型的方法。利用本发明方法得到的胃癌肝转移微肿瘤模型可以准确反应患者肝转移病灶的各种特征，是肿瘤精准诊疗领域良好的科研实验模型和临床前实验模型。可以预见，这种培养方法在胃癌的研究和临床诊断治疗领域具有广泛的应用前景。The present invention discloses a culture medium and a culture method for culturing a micro-tumor model of gastric cancer liver metastasis. The present invention provides a culture medium for culturing a micro-tumor model of gastric cancer liver metastasis. Furthermore, the present invention also provides a method for culturing a micro-tumor model of gastric cancer liver metastasis using the culture medium. The micro-tumor model of gastric cancer liver metastasis obtained by the method of the present invention can accurately reflect the various characteristics of the patient's liver metastasis lesions, and is a good scientific research experimental model and preclinical experimental model in the field of precise diagnosis and treatment of tumors. It can be foreseen that this culture method has broad application prospects in the field of gastric cancer research and clinical diagnosis and treatment.

Description

Culture medium and culture method for culturing gastric cancer liver metastasis micro-tumor model
Technical Field
The invention relates to the technical field of biology, in particular to a culture medium and a culture method for culturing a gastric cancer liver metastasis micro-tumor model.
Background
Gastric cancer is one of the malignant tumors that seriously threatens human health. Liver is the most important target organ for gastric cancer blood metastasis, 4-14% of patients have liver metastasis focus at the initial diagnosis, more than 13-30% of gastric cancer patients have recurrent metastasis with different degrees within months to years after radical treatment, and up to 45% of late gastric cancer patients have complicated liver metastasis. Even if patients with liver metastasis from gastric cancer undergo radical excision, the five-year survival time of the patients is only 20-40%.
Classical cytotoxic drug-based chemotherapy is one of the basic therapeutic approaches to gastric cancer, and is an effective treatment to increase patients' long-term survival rate and risk of recurrent metastasis. The standard treatment scheme for liver metastasis of gastric cancer is a multi-drug combined treatment scheme mainly comprising fluorouracil chemotherapeutics, sweater chemotherapeutics and platinum chemotherapeutics. Her2 positive patients may use targeted drugs or antibody European drugs. Gastric cancer has very strong tumor heterogeneity and very different individuals with chemotherapy effects, and most (80% -90%) liver metastases cannot be excised radically at first. This places new demands on the study of gastric liver metastases and on personalized treatments.
Gastric cancer is a complex system disease, and the occurrence, development, recurrence and metastasis of the disease and other biological processes are still very little known about the corresponding mechanisms of heterogeneity, drug resistance and tumor immunity. The cause and development process of liver metastasis of gastric cancer have strong individual variability, and the focus of liver metastasis is greatly different from the focus of primary stomach cancer in tumor microenvironment. Therefore, the personalized accurate research of taking the primary cell culture of the stomach cancer liver metastasis focus as a model is a trend in the stomach cancer research field and even the stomach cancer diagnosis and treatment field. Therefore, it is important to develop a tumor model capable of accurately reflecting the characteristics of liver metastasis of patients, and the existing primary tumor cell culture technology mainly comprises 2D culture, 3D culture, reprogramming culture and other types, and the methods have the problems of extremely long culture period, low culture success rate, difficult removal of mixed cells, incapability of reproducing tumor microenvironment and the like.
Disclosure of Invention
In order to effectively solve the technical problems, the invention provides a novel gastric cancer liver metastasis micro-tumor model culture technology and a matched reagent, and the core of the technology is as follows: (1) The stomach cancer liver metastasis focus tissue is treated by a mild cell dissociation reagent, the component proportion of the culture medium is adjusted according to the micro-environment characteristics of the stomach cancer liver metastasis focus, and the activity of various types of cells in the tissue is ensured to the greatest extent; (2) Preparing a special serum-free culture medium, and utilizing a suspension culture system to self-assemble various types of cells separated from stomach cancer liver metastasis focus tissues to form a cell mass structure with various cell components, which is called as a stomach cancer liver metastasis micro tumor model.
In a first aspect, the invention claims a medium for culturing a microtumor model of liver metastasis from gastric cancer.
The culture medium for culturing the micro-tumor model for liver metastasis of gastric cancer claimed by the invention consists of an antibacterial and antifungal agent, such as three antibodies, HEPES, glutaMax, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein Noggin, human recombinant protein R-spondin, human recombinant protein FGF-10, human recombinant protein Wnt-3a, SB202190 (4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole ）、CHIR99021、A83-01（3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H- pyrazole-1- carbothioamide）、Primocin、N- acetyl-L-cysteine (N-acetyl-L-cysteine), nicotine (Nicotinamide), N-2 additive (N2 supplement), cholera Toxin (Cholera Toxin), B27, ITS-X (Insulin, transfer, selenium, ethanolamine Solution), Y-27632, gastrin (Gastrin) and a basal culture medium.
The antibacterial and antifungal agent three antibodies are penicillin, streptomycin and amphotericin B.
In the medium, the final concentration of HEPES is 8-12mM (e.g.10 mM); the final concentration of the GlutaMax is 0.8-1.2% (such as 1%) by volume; the final concentration of the human recombinant protein EGF is 10-100ng/mL (such as 50 ng/mL); the final concentration of the human recombinant protein bFGF is 10-50ng/mL (such as 20 ng/mL); the final concentration of the human recombinant protein HGF is 5-25ng/mL (such as 20 ng/mL); the final concentration of the human recombinant protein Noggin is 100-200ng/mL (such as 100 ng/mL); the final concentration of the human recombinant protein R-spondin is 250-500ng/mL (such as 400 ng/mL); the final concentration of the human recombinant protein FGF-10 is 5-25ng/mL (such as 20 ng/mL); the final concentration of the human recombinant protein Wnt-3a is 200-300ng/mL (such as 200 ng/mL); the final concentration of SB202190 is 5-10. Mu.M (e.g., 10. Mu.M); the final concentration of CHIR99021 is 1.5-6. Mu.M (e.g. 3. Mu.M); the final concentration of A83-01 is 0.25-1.25. Mu.M (e.g., 1. Mu.M); the final concentration of Primocin is 1% by volume; the final concentration of N-acetyl-L-cysteine is 0.5-2mM (e.g., 1 mM); the final concentration of nicotine is 5-10mM (e.g., 10 mM); the final concentration of the N2 additive is 1% by volume; the final concentration of cholera toxin is 0.1-1nM (e.g., 0.5 nM); the final concentration of the B27 is 1.5-2.5% (such as 2%) by volume percent; the final concentration of ITS-X is 0.8-1.2% (e.g. 1%) by volume; the final concentration of Y-27632 is 5-20. Mu.M (e.g., 10. Mu.M); the final concentration of gastrin is 5-20nM (e.g., 10 nM).
In a specific embodiment of the invention, the basal medium is ADVANCED DMEM/F12 medium.
Further, the final concentration of penicillin in the antibacterial and antifungal agent triple antibody in the culture medium may be 100-200U/mL (e.g., 100U/mL); the final concentration of streptomycin in the antibacterial and antifungal agent three-antibody can be 100-200 mug/mL (such as 100 mug/mL); the final concentration of amphotericin B in the antimicrobial antifungal agent triple antibody may be 200-250ng/mL (e.g., 250 ng/mL).
The final concentrations of the above substances are all the final concentrations in the medium.
Further, the antibacterial and antifungal agent three antibodies consist of the following components: each milliliter contains 10000 units of penicillin (alkali), 10000 mug of streptomycin (alkali) and 25 mug of amphotericin B. The antibacterial and antifungal agent three antibodies are 'Antibiotic-Antimycotic, 100×' (such as Gibco #15240062, or other products with the same composition). The "Antibiotic-Antimycotic, 100×" contains 10000 units of penicillin (base), 10000 μg of streptomycin (base) and 25 μg of amphotericin B per ml, and penicillin G (sodium salt) in the form of a 0.85% salt solution, streptomycin sulfate and amphotericin B are used as Fungizone antifungals. The GlutaMax is an advanced cell culture additive and can directly replace L-glutamine in a cell culture medium. The GlutaMax is "GlutaMax ™ supply" (e.g., gibco #35050061, or other products of the same composition). The component of the 'GlutaMAX ™ supply' is L-alanyl-L-glutamine, which is an alternative to L-glutamine, the concentration is 200nM, and the solvent is 0.85% NaCl solution. The CHIR99021 is a high-specificity glycogen synthase kinase-3 (GSK-3) inhibitor for inhibiting GSK-3 alpha and GSK-3 beta, and the IC50 values are respectively 10nM and 6.7nM. Primocin is an antibacterial agent (such as Invivogene #ant-pm-1, or other products with the same composition) for primary cells, and is used for protecting primary cells from antibiotics contaminated by microorganisms, and has killing effect on gram-positive bacteria, gram-negative bacteria, mycoplasma and fungi. The N-2 Supplement is "N-2 Supplement (100X)" (such as Gibco # 17502001, or other products of the same composition). The "N-2 Supplement (100X)" contains 1mM final concentration of human whole iron transferrin (Human Transferrin (Holo)), 500mg/L recombinant insulin whole chain (Insulin Recombinant Full Chain), 0.63mg/L progesterone (Progesterone), 10mM putrescine (Putrescine), and 0.52mg/L selenite (Selenite). B27 is "B-27 ™ support (50×), minus vitamin A" (such as Gibco #12587010, or other products of the same composition). The "B-27 ™ Supplement (50×), minus vitamin A" contains Biotin (Biotin), DL-Alpha-tocopherol acetate (DL Alpha Tocopherol Acetate), DL-Alpha-tocopherol (DL Alpha-Tocopherol), BSA (FATTY ACID FREE Fraction V), catalase (Catalase), human recombinant insulin (Human Recombinant Insulin), Human transferrin (Human Transferrin), superoxide dismutase (Superoxide Dismutase), corticosterone (Corticosterone), D-galactose (D-Galactose), ethanolamine hydrochloride (Ethanolamine HCl), reduced glutathione (Glutathione (reduced)), L-carnitine hydrochloride (L-CARNITINE HCL), linoleic Acid (Linolenic Acid), linolenic Acid (Linolenic Acid), and pharmaceutical compositions, progesterone (Progesterone), putrescine (Putrescine HCl), sodium selenite (Sodium Selenite), triiodothyronine (T3 (triodo-I-thyronine)). the solvent of ITS-X is EBSS solution (Earle's balanced salt solution), and the solute and concentration are as follows: insulin 1g/L; transferrin 0.55g/L; 0.00067g/L of sodium selenite; ethanolamine 0.2g/L. The Y-27632 is "Y-27632 dihydrochloride (an ATP-competitive ROCK-I and ROCK-II inhibitor, ki 220nM and 300nM, respectively)" (e.g. MCE#129830-38-2, or other products of the same composition).
In a specific embodiment of the invention, the anti-bacterial and anti-fungal agent is given a brand number of Gibco#15240062; the brand number of the HEPES is Gibco#15630080; the brand goods number of the GlutaMax is Gibco#35050061; the brand goods number of the human recombinant protein EGF is Peprotech AF-100-15-100; the brand goods number of the human recombinant protein bFGF is Peprotech AF-100-18B-50; the brand goods number of the human recombinant protein HGF is Peprotech AF-100-39-100; the brand number of the human recombinant protein FGF-10 is Peprotech AF-100-26-100; the brand goods number of the human recombinant protein Wnt-3a is R & D5036-WN-500; the brand goods number of the human recombinant protein Noggin is Shanghai offshore # CB89; the brand goods number of the human recombinant protein R-spondin is Shanghai offshore # CX83; the brand goods number of the CHIR99021 is Selleck # S2924; the brand number of SB202190 is Sigma#S7067; the brand number of the A83-01 is Tocris #2939; the brand goods number of Primocin is Invivogene #ant-pm-1; the brand number of the N-acetyl-L-cysteine is Sigma#A9165; the brand number of Nicotinamide is Sigma#N0636; the brand number of the N-2 Supplement is Gibco #17502001; the brand number of the Cholera axin is Listlab #100B; the brand number of the B27 is Gibco#12587010; the brand number of the ITS-X is Gibco#51500056; the brand goods number of the Y-27632 is MCE#129830-38-2; the brand goods number of Gastrin is NJPeptide # Pep12307; the brand number of ADVANCED DMEM/F12 medium is Gibco #12634010.
Further, the culture medium may exist in two forms:
The culture medium is a solution formed by mixing the antibacterial and antifungal agent three antibodies, HEPES, glutaMax, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein FGF-10, human recombinant protein Wnt-3a, human recombinant protein Noggin, human recombinant protein R-spondin, CHIR99021, SB202190, A83-01, primocin, N-acetyl-L-cysteine, nicotine, N2 additive, cholera toxin, B27, ITS-X, Y-27632, gastrin and ADVANCED DMEM/F12 culture medium.
The medium was prepared and sterilized by filtration using a 0.22. Mu.M needle filter (Millipore SLGP RS) and stored at 4℃for two weeks.
And secondly, each component in the culture medium exists independently, and the culture medium is prepared according to a formula when in use.
Furthermore, the human recombinant protein EGF, the human recombinant protein bFGF, the human recombinant protein HGF, the human recombinant protein FGF-10, the human recombinant protein Wnt-3a, the human recombinant protein Noggin and the human recombinant protein R-spondin can exist in a liquid storage (mother solution) form (-80 ℃ can be stored for a long time), and can be specifically 1000 times of the liquid storage (mother solution). SB202190, N-acetyl-L-cysteine, nicotinamide, Y-27632 and Gastrin may be present in stock solution (mother liquor) form (-20 ℃ C. For long term storage), in particular 1000 times stock solution (mother liquor). CHIR99021 and Cholera axin may exist in the form of stock solution (mother liquor) which can be stored for a long period of time at (-20 ℃), and specifically may be 10000 times stock solution (mother liquor). A83-01 can exist in the form of stock solution (mother liquor), which can be stored at (-20deg.C for a long period of time), specifically 100000 times stock solution (mother liquor).
1000 Xstock solution of human recombinant protein EGF consists of human recombinant protein EGF, BSA and PBS, wherein the final concentration of the human recombinant protein EGF is 20 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.
1000 Xstock solution of human recombinant protein bFGF consists of human recombinant protein bFGF, BSA and PBS, wherein the final concentration of the human recombinant protein bFGF is 20 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.
1000 Xstock solution of human recombinant protein HGF consists of human recombinant protein HGF, BSA and PBS, wherein the final concentration of the human recombinant protein HGF is 20 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.
1000 Xstock solution of human recombinant protein FGF-10 consists of human recombinant protein FGF-10, BSA and PBS, wherein the final concentration of the human recombinant protein FGF-10 is 20 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.
1000 Xstock solution of human recombinant protein Wnt-3a consists of human recombinant protein Wnt-3a, BSA and PBS, wherein the final concentration of the human recombinant protein Wnt-3a is 200 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.
The 1000 Xhuman recombinant protein Noggin stock solution consists of human recombinant protein Noggin, BSA and PBS, wherein the final concentration of the human recombinant protein Noggin is 100 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.
1000 Xstock solution of human recombinant protein R-spondin consists of human recombinant protein R-spondin, BSA and PBS, wherein the final concentration of the human recombinant protein R-spondin is 250 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.
Of the seven 1000-fold stock solutions, BSA was present in 100-fold stock (stock solution) form (as-prepared), and consisted of BSA and PBS, with final concentration of BSA (Sigma#A1933) of 0.1g/mL and the balance PBS.
In addition, 10000 XCHIR 99021 stock solution consists of CHIR99021 and DMSO, wherein the final concentration of the CHIR99021 is 30mM, and the rest is DMSO.
1000 XSB 202190 stock solution consisted of SB202190 and DMSO, where the final concentration of SB202190 was 10mM, with the remainder being DMSO.
100000 XA 83-01 stock consists of A83-01 and DMSO, wherein the concentration of A83-01 is 25mM and the balance is DMSO.
The 1000 XN-acetyl-L-cysteine stock solution consists of N-acetyl-L-cysteine and ultrapure water, wherein the concentration of the N-acetyl-L-cysteine is 0.5M, and the balance is the ultrapure water.
The 1000 XNicotinamide stock solution consists of Nicotinamide and ultrapure water, wherein the concentration of the Nicotinamide is 5M, and the balance is the ultrapure water.
10000 XCholera Toxin stock solution consists of Cholera Toxin and Cholera Toxin lysate, wherein the final concentration of Cholera Toxin is 10 μm, and the rest is Cholera Toxin lysate. The Cholera Toxin solution consists of the following components: each 10mL of the Cholera axin solution contains Tris (1M) pH 7.0 0.05M,NaCl 0.2M, sodium azide 3mM, EDTA (0.5M) pH 8.0 1mM, and the balance of ultrapure water.
1000 XY-27632 consists of Y-27632 and ultrapure water, wherein the final concentration of Y-27632 is 10mM, and the balance is ultrapure water.
The 1000 Xgastrin (Gastrin) stock solution consisted of gastrin (Gastrin) and ultrapure water, wherein Gastrin had a final concentration of 10. Mu.M, the balance being ultrapure water.
In a second aspect, the invention claims a kit for culturing a micro-tumor model of liver metastasis from gastric cancer.
The kit of parts for culturing a micro-tumor model of liver metastasis from gastric cancer claimed in the present invention consists of all or part of the culture medium described in the first aspect above and: sample dissociation liquid, sample preservation liquid, sample washing liquid and digestion termination liquid.
The sample preservation solution can be used for temporary preservation after the sample is isolated, and can maintain the activity of cells in the sample in a short time after the sample is isolated. The sample preservation solution can be preserved for 1 month at 4 ℃ after being prepared.
The sample cleaning solution can be used for cleaning and disinfecting the sample. The sample cleaning liquid needs to be prepared at present.
The sample dissociation liquid can be used for dissociation of a sample. The sample dissociation solution needs to be prepared at present, wherein collagenase I, collagenase II and collagenase IV can be stored for a long time at the temperature of 20 ℃ below zero in a liquid storage (mother solution) form, and can be particularly 10 times of the liquid storage (mother solution). A 10 x collagenase I stock solution consisting of said collagenase I and PBS; wherein the final concentration of collagenase I is 2000U/mL; the balance was PBS.10 x collagenase II stock solution consisting of the collagenase II and PBS; wherein the final concentration of collagenase II is 2000U/mL; the balance was PBS. A 10 x collagenase IV stock solution consisting of the collagenase IV and PBS; wherein the final concentration of collagenase IV is 2000U/mL; the balance was PBS. The enzyme activities of collagenase I, collagenase II and collagenase IV are defined below.
The digestion stop solution may be used to terminate the sample dissociation process. The digestion stop solution can be stored at 4 ℃ for one month after being prepared.
The sample dissociation solution consists of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of collagenase I is 150-250U/mL (such as 200U/mL); the final concentration of collagenase II is 150-250U/mL (such as 200U/mL); the final concentration of collagenase IV is 150-250U/mL (such as 200U/mL); the balance was PBS.
Wherein the unit U of collagenase (said collagenase I or said collagenase II or said collagenase IV) is defined by the enzymatic activity of a protease: l-leucine can be released by treating collagenase (either said collagenase I or said collagenase II or said collagenase IV) with 1U protease at 37℃and pH7.5 for 5 hours.
In a specific embodiment of the invention, the brand number of the collagenase I is Gibco#17100-017; the brand goods number of the collagenase II is Gibco#17101-015; the brand goods number of the collagenase IV is Gibco#17104-019; the PBS was designated by the brand name Gibco #21-040-CVR.
The sample preservation solution consists of fetal bovine serum, an antibacterial antifungal agent, HEPES and HBSS; the antibacterial and antifungal agent three antibodies are penicillin, streptomycin and amphotericin B; the final concentration of said fetal bovine serum in said sample-holding fluid is 1-5% (e.g., 2%) by volume; the final concentration of penicillin in the antibacterial antifungal agent three antibody is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the antibacterial and antifungal agent is 100-200 mug/mL (such as 100 mug/mL); the final concentration of amphotericin B in the antibacterial antifungal agent is 200-250ng/mL (such as 250 ng/mL); the final concentration of HEPES is 8-12mM (e.g.10 mM); the balance is HBSS.
Further, the antibacterial and antifungal agent three antibodies consist of the following components: each milliliter contains 10000 units of penicillin (alkali), 10000 mug of streptomycin (alkali) and 25 mug of amphotericin B. The antibacterial and antifungal agent three antibodies are 'Antibiotic-Antimycotic, 100×' (such as Gibco #15240062, or other products with the same composition). The "Antibiotic-Antimycotic,100×" contains 10000 units of penicillin (base), 10000 μg of streptomycin (base) and 25 μg of amphotericin B per ml, and penicillin G (sodium salt) in the form of 0.85% salt solution, streptomycin sulfate and amphotericin B are used as Fungizone antifungals.
In a specific embodiment of the invention, the fetal bovine serum is given a brand number of Gibco #16000-044; the brand goods number of the double antibody P/S is Gibco#15140122; the brand number of the HEPES is Gibco# 15630080; the brand number of the HBSS is Gibco# 14170161.
The sample cleaning liquid consists of an antibacterial antifungal agent three antibody and PBS; the antibacterial and antifungal agent three antibodies are penicillin, streptomycin and amphotericin B; in the sample cleaning solution, the final concentration of penicillin in the antibacterial and antifungal agent three antibodies is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the antibacterial and antifungal agent is 100-200 mug/mL (such as 100 mug/mL); the final concentration of amphotericin B in the antibacterial antifungal agent is 200-250ng/mL (such as 250 ng/mL); the balance was PBS.
Further, the antibacterial and antifungal agent three antibodies consist of the following components: comprises 10000 units of penicillin (base), 10000 μg of streptomycin (base) and 25 μg of amphotericin B per ml. The antibacterial and antifungal agent three antibodies are 'Antibiotic-Antimycotic, 100×' (such as Gibco #15240062, or other products with the same composition). The "Antibiotic-Antimycotic,100×" contains 10000 units of penicillin (base), 10000 μg of streptomycin (base) and 25 μg of amphotericin B per ml, and penicillin G (sodium salt) in the form of 0.85% salt solution, streptomycin sulfate and amphotericin B are used as Fungizone antifungals.
In a specific embodiment of the invention, the anti-bacterial and anti-fungal agent is given a brand number of Gibco#15240062; the PBS was designated by the brand name Gibco #21-040-CVR.
The digestion stopping solution consists of fetal bovine serum, an antibacterial antifungal agent, a third antibody and a DMEM culture medium; the antibacterial and antifungal agent three antibodies are penicillin, streptomycin and amphotericin B; in the digestion stop solution, the final concentration of the fetal bovine serum is 8-12% (e.g., 10%) by volume; the final concentration of penicillin in the antibacterial antifungal agent three antibody is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the antibacterial and antifungal agent is 100-200 mug/mL (such as 100 mug/mL); the final concentration of amphotericin B in the antibacterial antifungal agent is 200-250ng/mL (such as 250 ng/mL); the balance being DMEM medium.
Further, the antibacterial and antifungal agent three antibodies consist of the following components: comprises 10000 units of penicillin (base), 10000 μg of streptomycin (base) and 25 μg of amphotericin B per ml. The antibacterial and antifungal agent three antibodies are 'Antibiotic-Antimycotic, 100×' (such as Gibco #15240062, or other products with the same composition). The "Antibiotic-Antimycotic,100×" contained 10000 units of penicillin (base), 10000 μg of streptomycin (base), and 25 μg of amphotericin B per ml, using penicillin G (sodium salt) in the form of 0.85% salt solution, streptomycin sulfate, and amphotericin B as Fungizone antimycotics.
In a specific embodiment of the invention, the fetal bovine serum is given a brand number of Gibco #16000-044; the brand number of the antibacterial and antifungal agent three antibodies is Gibco#15240062; the DMEM medium is given a brand number of Gibco #11965-092.
In a third aspect, the invention claims the use of a culture medium as described in the first aspect hereinbefore or a kit of reagents as described in the second aspect hereinbefore for culturing a micro-tumor model of liver metastasis from gastric cancer.
In a fourth aspect, the invention claims a method of culturing a microtumor model of liver metastasis from gastric cancer.
The method for culturing the micro-tumor model for liver metastasis of gastric cancer, which is claimed by the invention, can comprise the following steps:
(a1) Dissociating gastric cancer liver metastasis focus tissue with the sample dissociation solution described in the second aspect;
(a2) And (3) performing suspension culture on the dissociated cells in the step (a 1) by using the culture medium in the first aspect to form cell clusters, thus obtaining the micro-tumor model for liver metastasis of gastric cancer.
Further, in the step (a 1), the gastric cancer liver metastasis lesion tissue may be dissociated with the sample dissociation liquid according to a method comprising the steps of: and (3) according to the dosage of not more than 0.5mg of tissue in 1mL of the sample dissociation solution, performing sample dissociation on the cut gastric cancer liver metastasis focus tissue by using the sample dissociation solution at 37 ℃ for 30 minutes to 2 hours (such as 1 hour).
Further, in step (a 2), the dissociated cells of (a 1) may be suspension-cultured with the medium according to a method comprising the steps of: the dissociated cells of (a 1) were cultured in suspension using the medium in a cell culture vessel having a low adsorption surface (low-adsorption-surface), and the culture was performed under conditions of 37℃and 5% CO 2.
Wherein the initial seeding density may be 10 5 cells/cm 2 container bottom area, for example, a six well plate, plated at a density of 10 6 cells per well.
Further, the time of the cultivation in the step (a 2) is 3 to 5 days.
Further, before step (a 1), the method may further comprise the step of subjecting the gastric cancer liver metastasis tissue to a dissociation pretreatment as follows: ethanol with the volume percentage of 70-75% is used for cleaning the surface of a tissue sample of a stomach cancer liver metastasis focus for 10-30 seconds; washing the gastric cancer liver metastasis tissue sample with the sample washing solution of the second aspect 5-10 times (e.g., 5 times), and washing the gastric cancer liver metastasis tissue sample with sterile PBS solution 5-10 times (e.g., 5 times); and then removing impurities, connective tissues, adipose tissues, necrotic tissues and other components affecting the cultivation of the micro-tumor model in the gastric cancer liver metastasis focus tissue sample.
The step of dissociating the stomach cancer liver metastasis tissue is performed on ice, and the whole operation step is completed within 10 minutes.
Further, in the step (a 1), the dissociation treatment of the gastric cancer liver metastasis focus tissue by the sample dissociation liquid may further include the following steps: terminating the dissociation reaction with 8-15 (e.g. 10) volumes of the digestion stop solution as described in the second aspect above, and collecting the cell suspension; filtering the cell suspension with a 100 μm sterile cell strainer to remove tissue fragments and adherent cells; centrifuging at room temperature for 10-15 min (such as 10 min) at 800-1000g (such as 800 g), and discarding supernatant; cells were then resuspended with 3-5mL (e.g., 5 mL) of sterile PBS; centrifuging at room temperature for 10-15 min (such as 10 min) at 800-1000g (such as 800 g), and discarding supernatant; the cell pellet is then resuspended with the medium described in the first aspect above.
Further, the gastric cancer liver metastasis tissue sample subjected to the dissociation pretreatment is stored in the sample storage solution according to the second aspect before being subjected to the dissociation pretreatment for an in vitro time of 12 hours or less.
In a fifth aspect, the invention claims a micro-tumor model of liver metastasis from gastric cancer cultured by the method described in the fourth aspect.
In the present invention, all of the above-mentioned PBS's may be 1 XPBS, pH7.3-7.5. The concrete composition is as follows: the solvent is water, and the solute and the concentration are as follows: KH 2PO4144mg/L,NaCl 9000 mg/L,Na2HPO4·7H2 O795 mg/L.
The invention provides a method for extracting and culturing a gastric cancer liver metastasis microtumor model from fresh gastric cancer liver metastasis focus tissue and a matched reagent, and the method has the following advantages:
1. the dosage of the tissue sample is small, and only about 20 mg tissue samples are needed for the gastric cancer liver metastasis;
2. The culture period is short, and a gastric cancer micro-tumor model of 10 5-106 orders of magnitude can be obtained only by 3-5 days;
3. The culture stability is high, and the success rate of in vitro culture of the qualified gastric cancer liver metastasis focus operation specimen by the method is up to 70%.
The gastric cancer liver metastasis micro-tumor model obtained by the method can accurately reflect various characteristics of liver metastasis focus of a patient, and is a scientific research experimental model and a preclinical experimental model in the field of tumor accurate diagnosis and treatment. It is expected that the culture method has wide application prospect in the fields of research and clinical diagnosis and treatment of gastric cancer.
Drawings
FIG. 1 is a bright field image of a microtumor obtained by culturing samples of primary gastric cancer and hepatic metastases of the same patient.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 preparation of reagents for culturing gastric cancer liver metastasis microtumor model
1. Sample preservation solution (100 mL)
The specific formulation of the sample storage solution (100 mL) is shown in Table 1.
After the sample preservation solution is prepared, split charging is carried out by using a 15mL centrifuge tube, and each tube is 5mL. Can be stored at 4deg.C for 1 month after packaging.
2. Sample cleaning solution (100 mL)
The specific formulation of the sample rinse (100 mL) is shown in Table 2.
The sample cleaning liquid needs to be prepared at present.
3. Sample dissociation liquid (10 mL)
The specific formulation of the sample dissociation solution (10 mL) is shown in Table 3.
TABLE 3 sample dissociation liquid (10 mL)
Note that: the sample dissociation solution is prepared at present.
In Table 3, the collagenase stock solutions were prepared as shown in tables 4 to 6.
Table 4, 10 Xcollagenase I stock solution (100 mL)
After preparation of the 10 Xcollagenase I stock solution, 1.5mL sterile centrifuge tubes were used for split charging, 1mL per tube. The stock solution can be stored at-20deg.C for a long period.
Tables 5, 10 Xcollagenase II stock solution (100 mL)
After preparation of the 10 Xcollagenase II stock solution, 1.5mL sterile centrifuge tubes were used for split charging, 1mL per tube. The stock solution can be stored at-20deg.C for a long period.
Table 6, 10 Xcollagenase IV stock solution (100 mL)
After preparation of the 10 Xcollagenase IV stock solution, 1.5mL sterile centrifuge tubes were used for split charging, 1mL per tube. The stock solution can be stored at-20deg.C for a long period.
In tables 4, 5 and 6, the unit U of collagenase (either said collagenase I or said collagenase II or said collagenase IV) is defined by the enzymatic activity of the protease: l-leucine can be released by treating collagenase (either said collagenase I or said collagenase II or said collagenase IV) with 1U protease at 37℃and pH7.5 for 5 hours.
4. Digestion stop liquid (100 mL)
The specific formulation of the digestion terminator (100 mL) is shown in Table 7.
TABLE 7 digestion stop liquid (100 mL)
After the digestion stop solution is prepared, the mixture can be stored at 4 ℃ for one month.
5. Gastric cancer liver metastasis micro tumor model culture medium (100 mL)
The specific formulation of the gastric cancer liver metastasis microtumor model medium (100 mL) is shown in Table 8.
Table 8, gastric cancer microtumor model medium (100 mL)
After the preparation of the gastric cancer microtumor model medium, the medium was sterilized by filtration through a 0.22. Mu.M needle filter (MilliporeSLGP 033 RS) and stored at 4℃for two weeks.
In Table 8, the formulations of the human recombinant protein stock solutions are shown in tables 10 to 16 (the formulation of the BSA stock solution is shown in Table 9), the formulation of the SB202190 stock solution is shown in Table 17, the formulation of the CHIR99021 stock solution is shown in Table 18, the formulation of the A83-01 stock solution is shown in Table 19, the formulation of the N-acetyl-L-cysteine stock solution is shown in Table 20, the formulation of the Nicotinamide stock solution is shown in Table 21, the formulation of the Cholera Toxin stock solution is shown in tables 22 and 23, and the formulation of the Y-27632 stock solution is shown in Table 24. The formulations of Gastrin stock solutions are shown in table 25.
Table 9, 100 XBSA solution (1 mL)
The 100 XBSA solution was ready for use.
TABLE 10 1000 Xstock solution of human recombinant protein EGF (5 mL)
After 1000 Xstock solution of human recombinant protein EGF was prepared, it was sub-packaged with 1.5mL sterile centrifuge tubes, and the stock solution could be stored at-80℃for a long period of time.
Table 11, 1000 Xstock solution of human recombinant protein bFGF (2.5 mL)
After preparation of 1000 Xstock solution of human recombinant protein bEGF, the stock solution was dispensed using a 1.5mL sterile centrifuge tube and stored at-80℃for a long period of time.
Table 12, 1000 Xstock solution of human recombinant protein HGF (5 mL)
After 1000 Xhuman recombinant protein HGF stock solution was prepared, split-packed with 1.5mL sterile centrifuge tubes, the stock solution could be stored at-80℃for a long period of time.
TABLE 13 1000 Xstock solution of human recombinant protein FGF-10 (5 mL)
After 1000 Xhuman recombinant protein FGF-10 stock solution is prepared, the stock solution can be stored for a long time at-80 ℃ by sub-packaging with a 1.5mL sterile centrifuge tube.
Table 14, 1000 Xstock solution of human recombinant protein Wnt-3a (2.5 mL)
After 1000 Xhuman recombinant protein Wnt-3a stock solution is prepared, the stock solution is split-packed by a 1.5mL sterile centrifuge tube, and the stock solution can be stored at-80 ℃ for a long time.
Table 15, 1000 Xstock solution of human recombinant protein Noggin (5 mL)
After the 1000 Xhuman recombinant protein Noggin stock solution is prepared, the stock solution is subpackaged by a 1.5mL sterile centrifuge tube, and the stock solution can be stored at-80 ℃ for a long time.
Table 16, 1000 Xstock solution of human recombinant protein R-spondin (4 mL)
After 1000 Xhuman recombinant protein R-spondin stock solution is prepared, the stock solution is split-packed with 1.5mL sterile centrifuge tubes, and the stock solution can be stored at-80 ℃ for a long time.
Table 17, 1000 XSB 202190 stock solution (1.51 mL)
After the 1000 XSB 202190 stock solution is prepared, the stock solution is split-packed with 0.5mL sterile centrifuge tubes, and the stock solution can be stored at-20 ℃ for a long time.
Table 18, 10000 XCHIR 99021 stock solution (1.16 mL)
10000 XCHIR 99021 stock solution is prepared, and then subpackaged with 0.5mL sterile centrifuge tube, and the stock solution can be stored at-20deg.C for a long period.
TABLE 19 100000 XA 83-01 stock solution (1.05 mL)
After 1000 XA 83-01 stock solution is prepared, the stock solution is split-packed by a 0.5mL sterile centrifuge tube, and the stock solution can be stored for a long time at-20 ℃.
TABLE 20 1000 XN-acetyl-L-cysteine stock solution (5 mL)
After 1000 XN-acetyl-L-cysteine stock solution was prepared, it was sub-packaged with 0.5mL sterile centrifuge tubes and the stock solution could be stored at-20℃for a long period of time.
Table 21, 1000 XNicotinamide stock solution (4 mL)
After the 1000 XNicotinamide stock solution is prepared, the stock solution is split-packed by a 0.5mL sterile centrifuge tube, and the stock solution can be stored for a long time at-20 ℃.
Table 22, 10000 XCholera Toxin stock solution (1.17 mL)
After the 1000 XCholera Toxin stock solution is prepared, the stock solution is split into 0.5mL sterile centrifuge tubes, and the stock solution can be stored at-20 ℃ for a long time.
TABLE 23 Cholera Toxin solution (10 mL)
After the Cholera Toxin solution is prepared, the solution can be packaged in a 0.5mL sterile centrifuge tube, and the solution can be stored at-20 ℃ for a long time.
Table 24, 1000 XY-27632 stock solution (3.125 mL)
After the 1000 XY-27632 stock solution is prepared, the stock solution is split-packed by a 0.5mL sterile centrifuge tube, and the stock solution can be stored at-80 ℃ for a long time.
Table 25, 1000 XGastrin (Gastrin) stock solution (48 mL)
After the 1000 Xgastrin stock solution is prepared, the stock solution is split-packed by a 0.5mL sterile centrifuge tube, and the stock solution can be stored for a long time at-20 ℃.
Example 2 acquisition of surgical specimens of primary carcinoma of gastric cancer and liver metastasis
1. In cooperation with a three-dimensional hospital, samples are obtained in the form of a clinical study initiated by a researcher, and the cooperation is developed through regular medical ethical examination.
2. The attending physician selects the group of patients according to clinical indications prescribed by the medical guidelines, and selects the appropriate sample for in vitro culture according to the intraoperative clinical indications, the criteria for selection of the sample are: primary gastric cancer accompanies liver metastasis, and patients with primary tumors and liver metastasis are excised simultaneously in surgery, and the weight of both specimens exceeds 20 mg specimens.
3. All cases in the group are uniformly coded by adopting a mode of sample collection date and four positions after patient hospitalization number, for example, samples provided by 1 month and 1 day in 2020, and the patient hospitalization number is T001537474, and then the sample experiment number is 202001017474. And concealing information related to privacy of the patient, such as the name, the identification card number and the like of the patient. The hospital provides the sample with basic clinical information such as sex, age, medical history, family history, smoking history, pathological staging, clinical diagnosis, etc.
4. After tumor tissue is isolated in operation, a sample collection technician collects a fresh sample in an operating room sterile environment, and the collected sample needs to select a part with abundant fresh blood vessels, so that the part with poor cell activity such as necrotic tissue, adipose tissue, fibrotic tissue and the like is avoided. The collected sample was placed in a sample preservation solution (see example 1) pre-cooled at 4 ℃. The sample holding tube containing the sample is temporarily stored on ice and transported to a laboratory for the next operation within 12 hours, and the temperature is controlled to be 2-8 ℃ during the transportation.
Example 3 pretreatment for dissociation of tissue samples
The following operations are performed on ice, and the whole operation steps are completed within 10 minutes.
The surgical instruments used in the following operations all need to be sterilized by high-temperature steam (120 ℃ for 20 minutes) in advance and can be used after being dried.
1. After weighing the sample, the surface of the sample was rinsed with medical alcohol (75% by volume) for 10 to 30 seconds.
2. The sample was washed 5 times with the sample wash and 5 times with sterile PBS solution.
3. The adipose tissue, connective tissue and necrotic tissue in the sample are carefully peeled off by using an ophthalmic scissors, an ophthalmic forceps, a surgical knife and other devices.
Example 4 tissue sample dissociation
The surgical instruments used in the following examples were sterilized by high-temperature steam (120 ℃ C., 20 minutes) and dried before use.
1. The tissue was cut into small pieces of about 0.5mm 3 with an ophthalmic scissors.
2. Tissue was treated with a sample dissociation solution (see example 1), 1 mL sample dissociation solution was used for tissue with a sample size of no more than 0.5mg, and 0.1 mL sample dissociation solution was added for each 0.1mg increase in tissue weight for a sample size of more than 0.5 mg. Sample dissociation solution treatment condition is 37 ℃, and dissociation time is 1 hour. Dissociation of the sample was observed under a microscope every 15 minutes during dissociation until most cells were observed to be shed from the tissue.
3. The dissociation reaction was stopped with 10 volumes of digestion stop solution (see example 1), and after the cell suspension was filtered through a 100 μm sterile cell screen to remove tissue debris and adherent cells, the supernatant was discarded by centrifugation at 800 g for 10 minutes at room temperature.
4. Cells were resuspended in 5 mL sterile PBS, centrifuged at 800 g for 10 min at room temperature and the supernatant discarded.
5. Cell pellet was resuspended in gastric cancer liver metastasis microtumor model medium (see table 8 in example 1), cell counted, trypan blue staining was used to determine cell viability, and cell inoculation culture was performed with isolated cell viability greater than 70%.
Example 5 cultivation of a micro tumor model
1. The low adsorption surface (low-adsorption-surface) was used for suspension culture of gastric cancer primary focus and liver metastasis micro tumor model, wherein the culture medium used was the gastric cancer liver metastasis micro tumor model culture medium in Table 8 of example 1 (wherein the final concentration of human recombinant protein EGF was 50ng/mL, the final concentration of human recombinant protein bFGF was 20ng/mL, the final concentration of human recombinant protein HGF was 20ng/mL, the final concentration of human recombinant protein Noggin was 100ng/mL, the final concentration of human recombinant protein R-spondin was 400ng/mL, the final concentration of human recombinant protein FGF-10 was 20ng/mL, the final concentration of human recombinant protein Wnt-3a was 200ng/mL, the final concentration of SB202190 was 10. Mu.M, the final concentration of CHIR99021 was 3. Mu.M, the final concentration of A83-01 was 1. Mu.M, the final concentration of N-acetyl-L was 100ng/mL, the final concentration of human recombinant protein R-spondin was 400ng/mL, the final concentration of human recombinant protein R-spondin was 20ng/mL, the final concentration of human recombinant protein FGF-10 nM was 200ng/mL, the final concentration of human recombinant protein FGF-10 nM was 10mM, the final concentration of human recombinant protein F-3 a was 10mM, the final concentration of 35 mM, and the culture plate was used for culturing was 10mM, the final concentration of human recombinant protein was 10nM, 30 mM, and the final concentration was 10mM, per well plate was used. The inoculated cells were cultured at 37℃under 5% CO 2 in a cell incubator.
2. The state of the cells was observed every day until the cells formed a mass with a diameter of about 100 μm, after which the medium was changed every 2-3 days to maintain the growth state of the gastric cancer micro-tumor.
As shown in FIG. 1, during the first 48 hours of culture, many different types of cells from cancer tissue spontaneously aggregate, self-assembling to form a 100 μm-sized cell mass structure, which we call a micro-tumor model. The total number of micro tumor cell clusters can reach 10 5-106. Through a large number of sample tests, the success rate of the micro tumor model culture of the gastric cancer primary focus and liver metastasis focus operation sample can reach 70%.
Example 6 comparison of the different Medium's ability to form micro-tumor Structure on samples of gastric cancer primary foci and liver metastasis lesions
The procedure for primary culture of all samples in this example was identical (see above), with only the medium formulation being different. There are two media for comparison test, see chinese patent application CN113736738B (a method for culturing gastric cancer microtumor cell model) and chinese patent application CN111808816a (a medium for culturing gastric cancer solid tumor primary cells), specifically see table 26 and table 27 below.
Wherein the final concentration of the human recombinant protein EGF is 50ng/mL; the final concentration of the human recombinant protein bFGF is 20ng/mL; the final concentration of the human recombinant protein HGF is 20ng/mL; the final concentration of the human recombinant protein FGF-10 is 20ng/mL; the final concentration of the human recombinant protein Wnt-3a is 200ng/mL; the final concentration of the human recombinant protein Noggin is 100g/mL; the final concentration of the human recombinant protein R-spondin is 400ng/mL; the final concentration of the human recombinant protein IL-2 is 20ng/mL; the final concentration of the human recombinant protein IL-15 is 20ng/mL; the final concentration of CHIR99021 is 3 μm; the final concentration of SB202190 was 10. Mu.M; the final concentration of A83-01 is 1 mu M; the final concentration of the N-acetyl-L-cysteine is 1mM; the final concentration of Nicotinamide is 10mM; the final concentration of Cholera Toxin was 1nM; the final concentration of Y-27632 is 10 mu M; the final concentration of Gastrin was 10nM.
Wherein the final concentration of the human recombinant protein EGF is 50ng/mL; the final concentration of the human recombinant protein bFGF is 20ng/mL; the final concentration of the human recombinant protein HGF is 20ng/mL; the final concentration of the human recombinant protein FGF-10 is 20ng/mL; the final concentration of the human recombinant protein R-spondin is 400ng/mL; the final concentration of the human recombinant protein Wnt-3a is 200ng/mL; the final concentration of the human recombinant protein Noggin is 100g/mL; the final concentration of SB202190 was 10. Mu.M; the final concentration of A83-01 is 1 mu M; the final concentration of the N-acetyl-L-cysteine is 1mM; the final concentration of Nicotinamide is 10mM; the final concentration of Cholera Toxin was 1nM; the final concentration of Y-27632 is 10 mu M; the final concentration of Gastrin was 10nM.
The primary lesion of gastric cancer and the metastatic lesion of liver of the same patient were cultured with three media of Table 8 (inventive medium), table 26 (control medium 1) and Table 27 (control medium 2), respectively, and the number and size of the micro-tumor were observed, and the results obtained are shown in Table 28 and Table 29.
Table 28, culture conditions of gastric carcinoma primary focus
It can be seen that the three media had the same success rate of culture when the gastric cancer primary samples were cultured, but the number and size of cell clusters (i.e., the microtumor model) obtained by the culture of table 26 (control 1) were more advantageous.
Table 29, liver metastasis of gastric cancer culture conditions
It can be seen that the culture medium (invention) in Table 8 has a higher success rate of cultivation in the micro-tumor culture of liver metastasis compared to that of primary foci. Meanwhile, the medium (the present invention) in Table 8 has lower cost due to simpler composition.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims (10)

1.一种用于培养胃癌肝转移的微肿瘤模型的培养基，其特征在于：所述培养基由抗菌抗真菌剂三抗、HEPES、GlutaMax、人重组蛋白EGF、人重组蛋白bFGF、人重组蛋白HGF、人重组蛋白Noggin、人重组蛋白R-spondin、人重组蛋白FGF-10、人重组蛋白Wnt-3a、SB202190、CHIR99021、A83-01、Primocin、N-乙酰-L-半胱氨酸、烟碱、N-2添加剂、霍乱毒素、B27、ITS-X、Y-27632、胃泌素和基础培养基组成；1. A culture medium for culturing a microtumor model of gastric cancer liver metastasis, characterized in that: the culture medium consists of antibacterial and antifungal agents, three antibodies, HEPES, GlutaMax, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein Noggin, human recombinant protein R-spondin, human recombinant protein FGF-10, human recombinant protein Wnt-3a, SB202190, CHIR99021, A83-01, Primocin, N-acetyl-L-cysteine, nicotine, N-2 additive, cholera toxin, B27, ITS-X, Y-27632, gastrin and basal culture medium; 所述抗菌抗真菌剂三抗为青霉素、链霉素和两性霉素B；The three antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B; 在所述培养基中，所述HEPES的终浓度为8-12mM；所述GlutaMax的终浓度为0.8-1.2%体积百分含量；所述人重组蛋白EGF的终浓度为10-100ng/mL；所述人重组蛋白bFGF的终浓度为10-50ng/mL；所述人重组蛋白HGF的终浓度为5-25ng/mL；所述人重组蛋白Noggin的终浓度为100-200ng/mL；所述人重组蛋白R-spondin的终浓度为250-500ng/mL；所述人重组蛋白FGF-10的终浓度为5-25ng/mL；所述人重组蛋白Wnt-3a的终浓度为200-300ng/mL；所述SB202190的终浓度为5-10μM；所述CHIR99021的终浓度为1.5-6μM；所述A83-01的终浓度为0.25-1.25μM；所述Primocin的终浓度为1%体积百分含量；所述N-乙酰-L-半胱氨酸的终浓度为0.5-2mM；所述烟碱的终浓度为5-10mM；所述N2添加剂的终浓度为1%体积百分含量；所述霍乱毒素的终浓度为0.1-1nM；所述B27的终浓度为1.5-2.5%体积百分含量；所述ITS-X的终浓度为0.8-1.2%体积百分含量；所述Y-27632的终浓度为5-20μM；所述胃泌素的终浓度为5-20nM。In the culture medium, the final concentration of HEPES is 8-12 mM; the final concentration of GlutaMax is 0.8-1.2% volume percentage; the final concentration of the human recombinant protein EGF is 10-100 ng/mL; the final concentration of the human recombinant protein bFGF is 10-50 ng/mL; the final concentration of the human recombinant protein HGF is 5-25 ng/mL; the final concentration of the human recombinant protein Noggin is 100-200 ng/mL; the final concentration of the human recombinant protein R-spondin is 250-500 ng/mL; the final concentration of the human recombinant protein FGF-10 is 5-25 ng/mL; the final concentration of the human recombinant protein Wnt-3a is 200-300 ng/mL; the final concentration of S The final concentration of B202190 is 5-10 μM; the final concentration of CHIR99021 is 1.5-6 μM; the final concentration of A83-01 is 0.25-1.25 μM; the final concentration of Primocin is 1% by volume; the final concentration of N-acetyl-L-cysteine is 0.5-2 mM; the final concentration of nicotine is 5-10 mM; the final concentration of N2 additive is 1% by volume; the final concentration of cholera toxin is 0.1-1 nM; the final concentration of B27 is 1.5-2.5% by volume; the final concentration of ITS-X is 0.8-1.2% by volume; the final concentration of Y-27632 is 5-20 μM; and the final concentration of gastrin is 5-20 nM. 2. 根据权利要求1所述的培养基，其特征在于：所述基础培养基为Advanced DMEM/F12培养基。2. The culture medium according to claim 1, characterized in that the basal culture medium is Advanced DMEM/F12 culture medium. 3.根据权利要求1或2所述的培养基，其特征在于：在所述培养基中，所述抗菌抗真菌剂三抗中的青霉素的终浓度为100-200U/mL；所述抗菌抗真菌剂三抗中的链霉素的终浓度为100-200μg/mL；所述抗菌抗真菌剂三抗中的两性霉素B的终浓度为200-250ng/mL。3. The culture medium according to claim 1 or 2 is characterized in that: in the culture medium, the final concentration of penicillin in the triple antibiotics of antibacterial and antifungal agents is 100-200U/mL; the final concentration of streptomycin in the triple antibiotics of antibacterial and antifungal agents is 100-200μg/mL; the final concentration of amphotericin B in the triple antibiotics of antibacterial and antifungal agents is 200-250ng/mL. 4.一种用于培养胃癌肝转移的微肿瘤模型的成套试剂，由权利要求1所述培养基和如下中的全部或部分组成：样本解离液、样本保存液、样本清洗液和消化终止液；4. A set of reagents for culturing a microtumor model of gastric cancer liver metastasis, comprising the culture medium of claim 1 and all or part of the following: a sample dissociation solution, a sample preservation solution, a sample washing solution, and a digestion termination solution; 所述样本解离液由胶原酶I、胶原酶II、胶原酶IV和PBS组成；其中，所述胶原酶I的终浓度为150-250U/mL；所述胶原酶II的终浓度为150-250U/mL；所述胶原酶IV的终浓度为150-250U/mL；余量均为PBS；The sample dissociation solution is composed of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of collagenase I is 150-250U/mL; the final concentration of collagenase II is 150-250U/mL; the final concentration of collagenase IV is 150-250U/mL; and the remainder is PBS; 所述样本保存液由胎牛血清、抗菌抗真菌剂三抗、HEPES和HBSS组成；所述抗菌抗真菌剂三抗为青霉素、链霉素和两性霉素B；在所述样本保存液中，所述胎牛血清的终浓度为1-5%体积百分含量；所述抗菌抗真菌剂三抗中的青霉素的终浓度为100-200U/mL；所述抗菌抗真菌剂三抗中的链霉素的终浓度为100-200µg/mL；所述抗菌抗真菌剂三抗中的两性霉素B的终浓度为200-250ng/mL；所述HEPES的终浓度为8-12mM；余量均为HBSS；The sample preservation solution is composed of fetal bovine serum, three antibiotics of antibacterial and antifungal agents, HEPES and HBSS; the three antibiotics of antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B; in the sample preservation solution, the final concentration of the fetal bovine serum is 1-5% by volume; the final concentration of penicillin in the three antibiotics of antibacterial and antifungal agents is 100-200U/mL; the final concentration of streptomycin in the three antibiotics of antibacterial and antifungal agents is 100-200µg/mL; the final concentration of amphotericin B in the three antibiotics of antibacterial and antifungal agents is 200-250ng/mL; the final concentration of HEPES is 8-12mM; the remainder is HBSS; 所述样本清洗液由抗菌抗真菌剂三抗和PBS组成；所述抗菌抗真菌剂三抗为青霉素、链霉素和两性霉素B；在所述样本清洗液中，所述抗菌抗真菌剂三抗中的青霉素的终浓度为100-200U/mL；所述抗菌抗真菌剂三抗中的链霉素的终浓度为100-200µg/mL；所述抗菌抗真菌剂三抗中的两性霉素B的终浓度为200-250ng/mL；余量均为PBS；The sample cleaning solution is composed of three antibiotics of antibacterial and antifungal agents and PBS; the three antibiotics of antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B; in the sample cleaning solution, the final concentration of penicillin in the three antibiotics of antibacterial and antifungal agents is 100-200U/mL; the final concentration of streptomycin in the three antibiotics of antibacterial and antifungal agents is 100-200µg/mL; the final concentration of amphotericin B in the three antibiotics of antibacterial and antifungal agents is 200-250ng/mL; the rest is PBS; 所述消化终止液由胎牛血清、抗菌抗真菌剂三抗和DMEM培养基组成；所述抗菌抗真菌剂三抗为青霉素、链霉素和两性霉素B；在所述消化终止液中，所述胎牛血清的终浓度为8-12%体积百分含量；所述抗菌抗真菌剂三抗中的青霉素的终浓度为100-200U/mL；所述抗菌抗真菌剂三抗中的链霉素的终浓度为100-200µg/mL；所述抗菌抗真菌剂三抗中的两性霉素B的终浓度为200-250ng/mL；余量均为DMEM培养基。The digestion stop solution is composed of fetal bovine serum, three antibacterial and antifungal antibodies and DMEM culture medium; the three antibacterial and antifungal antibodies are penicillin, streptomycin and amphotericin B; in the digestion stop solution, the final concentration of the fetal bovine serum is 8-12% volume percentage; the final concentration of penicillin in the three antibacterial and antifungal antibodies is 100-200U/mL; the final concentration of streptomycin in the three antibacterial and antifungal antibodies is 100-200µg/mL; the final concentration of amphotericin B in the three antibacterial and antifungal antibodies is 200-250ng/mL; the rest is DMEM culture medium. 5.权利要求1-3中任一所述的培养基或权利要求4所述成套试剂在培养胃癌肝转移的微肿瘤模型中的应用。5. Use of the culture medium according to any one of claims 1 to 3 or the reagent set according to claim 4 in culturing a microtumor model of gastric cancer liver metastasis. 6.一种培养胃癌肝转移的微肿瘤模型的方法，包括如下步骤：6. A method for culturing a microtumor model of gastric cancer liver metastasis, comprising the following steps: （a1）用权利要求4中所述的样本解离液对胃癌肝转移病灶组织进行解离处理；(a1) dissociating gastric cancer liver metastasis tissue using the sample dissociation solution described in claim 4; （a2）利用权利要求1所述培养基悬浮培养步骤（a1）解离出来的细胞，形成细胞团，即得胃癌肝转移的微肿瘤模型。(a2) Using the culture medium of claim 1 to suspend and culture the cells dissociated in step (a1) to form cell clusters, thereby obtaining a microtumor model of gastric cancer liver metastasis. 7.根据权利要求6所述的方法，其特征在于：步骤（a1）中，是按照包括如下步骤的方法用所述样本解离液对所述胃癌肝转移病灶组织进行解离的：按1mL所述样本解离液不超过0.5mg组织的用量，将剪碎后的所述胃癌肝转移病灶组织用所述样本解离液在37℃条件下进行样本解离，解离时间30分钟至2小时；和/或7. The method according to claim 6, characterized in that: in step (a1), the gastric cancer liver metastasis lesion tissue is dissociated with the sample dissociation solution according to a method comprising the following steps: the minced gastric cancer liver metastasis lesion tissue is dissociated with the sample dissociation solution at 37°C in an amount of no more than 0.5 mg of tissue per 1 mL of the sample dissociation solution, and the dissociation time is 30 minutes to 2 hours; and/or 步骤（a2）中，是按照包括如下步骤的方法用所述培养基悬浮培养（a1）解离出来的细胞的：使用低吸附表面的细胞培养容器，利用所述培养基悬浮培养（a1）解离出来的细胞，37℃，5% CO2条件下进行培养。In step (a2), the cells dissociated from (a1) are suspended and cultured using the culture medium according to a method comprising the following steps: using a cell culture container with a low adsorption surface, suspending and culture the cells dissociated from (a1 ) using the culture medium, and culturing at 37°C and 5% CO2. 8.根据权利要求6或7所述的方法，其特征在于：在步骤（a1）之前，还包括如下对所述胃癌肝转移病灶组织进行解离前处理的步骤：用体积百分含量为70-75%的乙醇清洗胃癌肝转移病灶组织样本表面；用权利要求4中所述样本清洗液和无菌的PBS溶液先后清洗所述胃癌肝转移病灶组织样本；和/或8. The method according to claim 6 or 7, characterized in that: before step (a1), it also includes the following steps of pre-dissociation treatment of the gastric cancer liver metastasis lesion tissue: washing the surface of the gastric cancer liver metastasis lesion tissue sample with 70-75% by volume ethanol; washing the gastric cancer liver metastasis lesion tissue sample with the sample washing solution according to claim 4 and a sterile PBS solution in sequence; and/or 在步骤（a1）中，用所述样本解离液对所述胃癌肝转移病灶组织进行解离处理后还包括如下步骤：用权利要求4中所述消化终止液终止解离反应，收集细胞悬液；过滤所述细胞悬液，去除组织残片和粘连细胞；离心后用无菌PBS重悬细胞；再离心，然后用权利要求1-3中任一所述培养基重悬细胞沉淀。In step (a1), after the gastric cancer liver metastasis lesion tissue is dissociated with the sample dissociation solution, the following steps are also included: terminating the dissociation reaction with the digestion stop solution described in claim 4 and collecting the cell suspension; filtering the cell suspension to remove tissue debris and adherent cells; resuspending the cells with sterile PBS after centrifugation; centrifuging again, and then resuspending the cell precipitate with the culture medium described in any one of claims 1-3. 9.根据权利要求8所述的方法，其特征在于：进行所述解离前处理的所述胃癌肝转移病灶组织样本的离体时间为12小时以内，且在进行所述解离前处理之前一直保存于权利要求4中所述样本保存液中。9. The method according to claim 8, characterized in that the gastric cancer liver metastasis lesion tissue sample subjected to the pre-dissociation treatment is isolated from the body within 12 hours and is kept in the sample preservation solution of claim 4 before the pre-dissociation treatment. 10.利用权利要求6-9中任一所述方法培养得到的胃癌肝转移的微肿瘤模型。10. A microtumor model of gastric cancer liver metastasis cultured by the method according to any one of claims 6 to 9.
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