A culture medium for culturing liver cancer microtumor model and a culture method thereof

Abstract

The present invention discloses a culture medium for culturing a liver cancer microtumor model and a culture method thereof. The present invention relates to the field of biotechnology, and provides a culture medium for culturing a liver cancer microtumor model, which is composed of three antibacterial and antifungal agents, HEPES, GlutaMax, human recombinant protein EGF, FGF‑10, HGF, IL‑2, IL‑15, R‑spondin 1, forskolin, gastrin, A83‑01, N‑acetyl‑L‑cysteine, nicotinamide, N2 additive, B27 and basal culture medium. The liver cancer microtumor model obtained by the method of the present invention can accurately reflect the various characteristics of the original lesion of the patient, 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 research and clinical diagnosis and treatment of liver cancer.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a culture medium for culturing a liver cancer microtumor model and a culture method thereof. Background Art

[0002] The liver is the largest solid organ in the human body, mainly composed of hepatic parenchymal cells (hepatocytes) and non-parenchymal cells (hepatic stellate cells, sinusoidal endothelial cells, bile duct cells, immune cells), etc. It plays a variety of roles in detoxification, biosynthesis, metabolism and host defense. Specifically, the liver can produce bile, participate in the metabolism of various substances such as sugars, proteins, lipids and vitamins, and regulate the body's physiological processes such as detoxification, phagocytosis, defense and hematopoiesis. In recent years, with the increase in people's living pressure, the incidence and mortality of liver cancer have been increasing year by year.

[0003] Primary liver cancer mainly includes three subtypes: hepatocellular carcinoma, intrahepatic cholangiocarcinoma, and hepatocellular carcinoma-intrahepatic cholangiocarcinoma mixed type. The common liver cancer process is divided into four stages: cirrhosis, precancerous stage, early stage and advanced stage. Liver cancer has an insidious onset and develops rapidly. Most patients are already in the advanced stage when they seek medical treatment.

[0004] At present, the research on liver cancer mainly relies on cell lines and animal models. As a two-dimensional culture, cell lines are difficult to simulate the three-dimensional structure of in vivo tissues, cannot be expanded stably for a long time, and lack the interaction between cells and cells and cells and matrix. Three-dimensional tumor organoids can simulate the genome and histological structure and tumorigenesis mechanism of parent tumors to the greatest extent, but the success rate of liver cancer organoid culture methods is low, only 20%-40%, and its establishment procedures and quality control have not been standardized worldwide, and the experimental reproducibility between different laboratories is low. In addition, the model lacks an immune microenvironment.

[0005] Although animal models can simulate certain physiological environments for tissue growth, their construction process is complex and there are large differences between species. In preclinical studies, the correlation between drug screening using CDX models and clinical outcomes is less than 5%; after applying PDX models, the correlation is as high as 90%, but as the number of passages increases, only a single clone that adapts to in vitro culture conditions is retained in the original tumor tissue, thus losing clonal heterogeneity, and the tumor microenvironment is gradually replaced by the extracellular matrix, so there is a certain limit to the number of passages.

[0006] In summary, liver cancer shows high heterogeneity in its pathogenesis, tissue morphology, tumor microenvironment and molecular level. Therefore, there is an urgent need to develop a liver cancer research model that can stably maintain the in vivo characteristics of tumor cells for use in personalized precision diagnosis and treatment research of liver cancer. Summary of the invention

[0007] The purpose of the present invention is to improve a culture medium and a culture method for culturing a liver cancer micro-tumor model. The special serum-free culture medium prepared by the present invention uses a suspension culture system to allow various types of cells isolated from liver cancer tissue to form cell clusters with a 3D spherical structure through self-assembly and proliferation mechanisms, which is called a "liver cancer micro-tumor model".

[0008] In a first aspect, the present invention claims a culture medium for culturing a liver cancer microtumor model.

[0009] The culture medium for culturing a liver cancer microtumor model claimed in the present invention is composed of three antibacterial and antifungal agents, HEPES, GlutaMax, human recombinant protein EGF, human recombinant protein FGF-10, human recombinant protein HGF, human recombinant protein IL-2, human recombinant protein IL-15, human recombinant protein R-spondin 1, Forskolin (an adenylate cyclase agonist), Gastrin, A83-01, N-acetyl-L-cysteine, Nicotinamide, N2 supplement, B27 and basal culture medium; wherein the three antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B.

[0010] In the culture medium, the final concentration of HEPES is 8-12 mM (such as 10 mM); the final concentration of GlutaMax is 0.8-1.2% (such as 1%) by volume; the final concentration of the human recombinant protein EGF is 10-100 ng / mL (such as 40 ng / mL); the final concentration of the human recombinant protein FGF-10 is 10-50 ng / mL (such as 20 ng / mL); the final concentration of the human recombinant protein HGF is 5-25 ng / mL (such as 20 ng / mL); the final concentration of the human recombinant protein IL-2 is 10-100 ng / mL (such as 20 ng / mL); the final concentration of the human recombinant protein IL-15 is 10-100 ng / mL (such as 20 ng / mL); the final concentration of the human recombinant protein R-spondin The final concentration of 1 is 250-500 ng / mL (such as 400 ng / mL); the final concentration of forskolin is 2-10 μM (such as 5 μM); the final concentration of gastrin is 2-10 nM (such as 5 nM); the concentration of A83-01 is 0.25-1.25 μM (such as 1 μM); the concentration of N-acetyl-L-cysteine ​​is 0.5-2 mM (such as 2 mM); the concentration of nicotinamide is 5-10 mM (such as 10 mM); the final concentration of N2 additive is 1% by volume; the final concentration of B27 is 1.5-2.5% (such as 2%) by volume.

[0011] Furthermore, the GlutaMax is an advanced cell culture additive that can directly replace L-glutamine in cell culture medium. The GlutaMax is "GlutaMAX™ Supplement" (such as Gibco#35050061, or other products with the same composition). The ingredient of the "GlutaMAX™ Supplement" is L-alanyl-L-glutamine, which is a substitute for L-glutamine, with a concentration of 200nM and a solvent of 0.85% NaCl solution. The A83-01 is "3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide" (such as Tocris#2939, or other products with the same composition). The B27 is "B-27™ Supplement (50X), minus vitamin A" (such as Gibco#12587010, or other products with the same composition). The “B-27™Supplement (50X), minus vitamin A” contains biotin, DL-α-tocopherol acetate, DL-α-tocopherol, BSA (fatty acidfree fraction V), catalase, human recombinant insulin, human transferrin, superoxide dismutase, corticosterone, D-galactose, ethanolamine hydrochloride, glutathione (reduced), L-carnitine hydrochloride, linoleic acid, linolenic acid, progesterone, putrescine2HCl, sodium selenite, triiodothyronine (T3 (triodo-I-thyronine)). The N-2 additive is "N-2 Supplement (100X)" (such as Gibco #17502001, or other products with the same composition).The "N-2 Supplement (100X)" contains human transferrin (Holo) at a final concentration of 1 mM, 500 mg / L of recombinant insulin full chain (Insulin Recombinant Full Chain), 0.63 mg / L of progesterone (Progesterone), 10 mM of putrescine (Putrescine), and 0.52 mg / L of selenite (Selenite).

[0012] Furthermore, the basic culture medium may be Advanced DMEM / F12 culture medium.

[0013] In the culture medium, the final concentration of penicillin in the antibacterial and antifungal agents is 100-200 U / mL (such as 100 U / mL), the final concentration of streptomycin is 100-200 μg / mL (such as 100 μg / mL), and the final concentration of amphotericin B is 200-250 ng / mL (such as 250 ng / mL).

[0014] Further, the antibacterial and antifungal triple antibiotic is "Antibiotic-Antimycotic, 100X" (such as Gibco #15240062, or other products with the same composition). The "Antibiotic-Antimycotic, 100X" contains 10,000 units of penicillin (base), 10,000 μg of streptomycin (base) and 25 μg of amphotericin B per milliliter, and uses penicillin G (sodium salt), streptomycin sulfate and amphotericin B in the form of 0.85% saline solution as antifungal agents.

[0015] Furthermore, the culture medium may exist in two forms:

[0016] First, the culture medium is a solution formed by mixing the antibacterial and antifungal agents, the triple antibody, the HEPES, the GlutaMax, the human recombinant protein EGF, the human recombinant protein FGF-10, the human recombinant protein HGF, the human recombinant protein IL-2, the human recombinant protein IL-15, the human recombinant protein R-spondin 1, the forskolin, the gastrin, the A83-01, the N-acetyl-L-cysteine, the nicotinamide, the N2 additive, the B27 and the basal culture medium.

[0017] After the culture medium is prepared, it needs to be filtered and sterilized using a 0.22 μM syringe filter (Millipore SLGP033RS) and can be stored at 4° C. for two weeks.

[0018] Secondly, each component in the culture medium exists independently and is prepared according to a formula when used.

[0019] Furthermore, human recombinant protein EGF, human recombinant protein FGF-10, human recombinant protein HGF, human recombinant protein IL-2, human recombinant protein IL-15, and human recombinant protein R-spondin 1 can be present in the form of a stock solution (mother solution), specifically a 1000-fold stock solution (mother solution). Forskolin, Gastrin, N-acetyl-L-cysteine, and Nicotinamide can be present in the form of a stock solution (mother solution), specifically a 1000-fold stock solution (mother solution). A83-01 can be present in the form of a stock solution (mother solution), specifically a 100,000-fold stock solution (mother solution).

[0020] The 1000× human recombinant protein EGF stock solution is composed of human recombinant protein EGF, BSA and PBS, wherein the final concentration of the human recombinant protein EGF is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0021] The 1000× human recombinant protein FGF-10 stock solution is composed of human recombinant protein FGF-10, BSA and PBS, wherein the final concentration of the human recombinant protein FGF-10 is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0022] The 1000× human recombinant protein HGF stock solution consists of human recombinant protein HGF, BSA and PBS, wherein the final concentration of the human recombinant protein HGF is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0023] The 1000× human recombinant protein IL-2 stock solution consists of human recombinant protein IL-2, BSA and PBS, wherein the final concentration of the human recombinant protein IL-2 is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0024] The 1000× human recombinant protein IL-15 stock solution consists of human recombinant protein IL-15, BSA and PBS, wherein the final concentration of the human recombinant protein IL-15 is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0025] The 1000× human recombinant protein R-spondin 1 stock solution consists of human recombinant protein R-spondin 1, BSA and PBS, wherein the final concentration of the human recombinant protein R-spondin 1 is 250 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0026] Among the above six 1000-fold stock solutions, the BSA can be present in the form of a 100-fold stock solution (mother solution) (prepared and used immediately), which is specifically composed of BSA and PBS, wherein the final concentration of BSA (Sigma#A1933) is 0.1 g / mL, and the remainder is PBS.

[0027] In addition, 1000×Forskolin consists of Forskolin and DMSO, wherein the final concentration of Forskolin is 10 mM, and the remainder is DMSO.

[0028] The 1000×Gastrin stock solution consists of Gastrin and ultrapure water, wherein the final concentration of Gastrin is 10 μM, and the remainder is ultrapure water.

[0029] The 1000×N-acetyl-L-cysteine ​​stock solution consists of N-acetyl-L-cysteine ​​and ultrapure water, wherein the concentration of the N-acetyl-L-cysteine ​​is 0.5 M, and the remainder is ultrapure water.

[0030] The 1000× Nicotinamide stock solution consists of Nicotinamide and ultrapure water, wherein the concentration of Nicotinamide is 5M and the remainder is ultrapure water.

[0031] The 100000×A83-01 stock solution consists of A83-01 and DMSO, wherein the concentration of A83-01 is 25 mM and the remainder is DMSO.

[0032] In a second aspect, the present invention claims a set of reagents for culturing a liver cancer microtumor model.

[0033] The reagent set for culturing a liver cancer microtumor model claimed in the present invention is composed of the culture medium described in the first aspect above and all or part of the following: sample dissociation solution, sample preservation solution, sample cleaning solution, cell digestion solution, digestion termination solution and cell freezing solution.

[0034] Furthermore, 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); and the remainder is PBS.

[0035] The unit U of collagenase (the collagenase I or the collagenase II or the collagenase IV) is defined by the enzymatic activity of the protease: at 37°C and pH 7.5, 1 U of protease is used to treat the collagenase (the collagenase I or the collagenase II or the collagenase IV) for 5 hours to release 1 μmol of L-leucine.

[0036] Furthermore, the sample preservation solution is composed of fetal bovine serum, three antibacterial and antifungal agents, HEPES and HBSS; wherein the three 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% (such as 2%) by volume; the final concentration of penicillin in the three antibacterial and antifungal agents is 100-200U / mL (such as 100U / mL), the final concentration of streptomycin is 100-200μg / mL (such as 100μg / mL), and the final concentration of amphotericin B is 200-250ng / mL (such as 250ng / mL); the final concentration of HEPES is 8-12mM (such as 10 mM); and the remainder is HBSS.

[0037] Furthermore, the sample cleaning solution is composed of three antibacterial and antifungal agents and PBS; wherein the three antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B; in the sample cleaning solution, the final concentration of penicillin in the three antibacterial and antifungal agents is 100-200U / mL (such as 100U / mL), the final concentration of streptomycin is 100-200μg / mL (such as 100μg / mL), and the final concentration of amphotericin B is 200-250ng / mL (such as 250ng / mL); the remainder is PBS.

[0038] Furthermore, the cell digestion solution has the following composition: every 10 mL of the cell digestion solution contains 4-6 mL (eg, 5 mL) Accutase, EDTA with a final concentration of 5 mM, 1.5-2.5 mL (eg, 2 mL) TrypLE Express, and the remainder is PBS.

[0039] Wherein, the Accutase is "StemPro™ Accutase™ Cell Dissociation Reagent" (such as Gibco#A11105-01, or other products with the same composition). The Accutase is a single-component enzyme that is dissolved in D-PBS, 0.5mM EDTA solution. The TrypLE Express is "TrypLE™ Express Enzyme (1X), no phenol red" (such as Gibco#12604013, or other products with the same composition). The "TrypLE™ Express Enzyme (1X), no phenol red" contains 200mg / L KCl, 200mg / L KH2PO4, 8000mg / L NaCl, 2160mg / L Na2HPO4·7H2O, 457.6mg / L EDTA; and also contains recombinant protease.

[0040] Furthermore, the digestion stop solution is composed of fetal bovine serum, three antifungal antibodies and DMEM culture medium; wherein 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% (such as 10%) by volume; the final concentration of penicillin in the three antibacterial and antifungal antibodies is 100-200U / mL (such as 100U / mL), the final concentration of streptomycin is 100-200μg / mL (such as 100μg / mL), and the final concentration of amphotericin B is 200-250ng / mL (such as 250ng / mL); the remainder is DMEM culture medium.

[0041] Furthermore, the cell freezing solution consists of Advanced DMEM / F12 culture medium, DMSO and 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM / F12 culture medium, the DMSO and the 1% methylcellulose solution is 20:2:(0.8-1.2), such as 20:2:1; and the 1% methylcellulose solution is a methylcellulose aqueous solution with a concentration of 1 g / 100 ml.

[0042] In a third aspect, the present invention claims the use of the culture medium described in the first aspect or the set of reagents described in the second aspect in constructing a liver cancer microtumor model.

[0043] In a fourth aspect, the present invention claims a method for constructing a liver cancer microtumor model.

[0044] The method for constructing a liver cancer microtumor model claimed in the present invention may include the following steps:

[0045] (a1) dissociating liver cancer solid tumor tissue using the sample dissociation solution described in the second aspect above;

[0046] (a2) Using the culture medium described in the first aspect above to suspend and culture the single cells dissociated in step (a1) to form cell clusters, thereby obtaining a liver cancer microtumor model.

[0047] In step (a1), the liver cancer solid tumor tissue can be dissociated using the sample dissociation solution according to a method comprising the following steps: using 1 mL of the sample dissociation solution to dissociate no more than 0.5 mg of tissue, the minced liver cancer solid tumor tissue is dissociated using the sample dissociation solution at 37° C. for a dissociation time of 15 minutes to 2 hours (e.g., 1 hour).

[0048] In step (a2), the cells dissociated from (a1) can be 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 the cells under conditions of 37° C. and 5% CO2.

[0049] The initial seeding density can be 10 5 Pieces / cm 2 The bottom area of ​​the container, taking a six-well plate as an example, is 10 per well. 6 The cells were plated at a density of 10 cells.

[0050] Furthermore, the culturing time in step (a2) is 2-3 days.

[0051] Furthermore, before step (a1), the following steps may be included for pre-dissociation treatment of the liver cancer solid tumor tissue: washing the surface of the liver cancer solid tumor tissue sample with 70-75% by volume ethanol for 10-30 seconds; washing the liver cancer solid tumor tissue sample with the sample washing solution described in the second aspect above for 5-10 times (e.g., 5 times), and washing the liver cancer solid tumor tissue sample with a sterile PBS solution for 5-10 times (e.g., 5 times); and then removing impurities, connective tissue, adipose tissue, necrotic tissue and other components that affect micro-tumor culture in the liver cancer solid tumor tissue sample.

[0052] The step of pre-dissociation treatment of the liver cancer solid tumor tissue needs to be performed on ice, and the entire operation step needs to be completed within 10 minutes.

[0053] Furthermore, in step (a1), after the liver cancer solid tumor tissue is dissociated with the sample dissociation solution, the following steps are also included: terminating the dissociation reaction with 8-15 times (such as 10 times) the volume of the digestion stop solution described in the second aspect above, and collecting the cell suspension; filtering the cell suspension with a 100μm or 40μm sterile cell filter to remove tissue debris and adherent cells; centrifuging at 800-1000g (such as 800g) at room temperature for 10-15 minutes (such as 10 minutes), and discarding the supernatant; then resuspending the cells with 3-5 mL (such as 5mL) of sterile PBS; centrifuging again at 800-1000g (such as 800g) at room temperature for 10-15 minutes (such as 10 minutes), and discarding the supernatant; and then resuspending the cell pellet with the culture medium described in the first aspect above.

[0054] The liver cancer solid tumor tissue sample subjected to the pre-dissociation treatment is isolated from the body within 12 hours, and is kept in the sample preservation solution described in the second aspect before the pre-dissociation treatment.

[0055] In a fifth aspect, the present invention claims protection for a liver cancer microtumor model constructed using the method described in the fourth aspect above.

[0056] In a sixth aspect, the present invention claims a method for obtaining primary liver cancer solid tumor cells.

[0057] The method for obtaining primary liver cancer solid tumor cells claimed in the present invention is to separate primary liver cancer solid tumor cells from the liver cancer microtumor model constructed by the method described in the fourth aspect above.

[0058] Furthermore, the method may include the following steps:

[0059] (b1) digesting the liver cancer microtumor model using the cell digestion solution described in the second aspect above to obtain single cells;

[0060] Furthermore, the method further comprises the step of terminating the digestion, such as using the digestion terminating solution as described above to terminate the digestion.

[0061] (b2) sorting out CD326 positive cells from the single cells obtained in step (b1) to obtain the primary liver cancer solid tumor cells.

[0062] Furthermore, CD326 positive cells can be sorted using CD326 magnetic beads.

[0063] In the above-mentioned related aspects, the liver cancer may be primary liver cancer or secondary liver cancer metastasis of various pathological types, and the surgical specimen weight is more than 20 mg. In an embodiment of the present invention, the liver cancer is primary hepatocellular carcinoma.

[0064] In all the above aspects,

[0065] In the present invention, all of the above PBS can be 1×PBS, pH 7.3-7.5, and its specific composition is as follows: the solvent is water, and the solute and concentration are: KH2PO4 144 mg / L, NaCl 9000 mg / L, Na2HPO4·7H2O 795 mg / L.

[0066] The present invention provides a method for extracting and culturing a liver cancer microtumor model from fresh liver cancer solid tumor tissue and a supporting reagent. The method has the following advantages:

[0067] 1. Small amount of tissue sample is required, only about 20 mg of liver cancer surgical sample is needed;

[0068] 2. The cultivation cycle is short, and 10 can be obtained in just 2-3 days. 4 -10 6 Order of magnitude liver cancer microtumor model;

[0069] 3. High culture stability. The success rate of in vitro culture of qualified liver cancer surgical specimens using this method is as high as 85%;

[0070] 4. Rich cell types. The liver cancer microtumor model can preserve multiple cell types such as tumor cells, stromal cells and immune cells in the original lesion, and reproduce the tumor microenvironment well;

[0071] 5. The liver cancer microtumor model can accurately reproduce the pathological subtype of the original lesion;

[0072] 6. The liver cancer microtumor model can accurately reproduce the genetic background of the original lesion.

[0073] The liver cancer micro-tumor model obtained by the method of the present invention can accurately reflect the various characteristics of the original lesion of the patient, and is a good scientific research experimental model and preclinical experimental model in the field of tumor precision diagnosis and treatment. It can be foreseen that this culture method has broad application prospects in the research and clinical diagnosis and treatment of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 This is a bright field image of the liver cancer microtumor model formation process (0-48h). The scale bar is 200μm, 100 times magnification.

[0075] Figure 2 This is a comparison of the HE staining effects of the liver cancer microtumor model and its corresponding original lesion tissue sample. The scale is 50μm, 400 times magnification.

[0076] Figure 3 This is a comparison of the multiple immunofluorescence staining effects of the liver cancer microtumor model. The scale is 50μm, 400 times magnification.

[0077] Figure 4 This is a bright field image of the isolated and purified primary liver cancer cells. The scale bar is 100 μm, and the magnification is 400 times.

[0078] Figure 5 Comparison results of copy number variation analysis (CNV) of liver cancer microtumor models cultured in three culture media of Schemes A, B, and C and their corresponding original lesion tissue samples. DETAILED DESCRIPTION

[0079] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0080] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0081] Example 1: Preparation of reagents for culturing liver cancer microtumor models

[0082] 1. Sample preservation solution

[0083] The specific formula of the sample preservation solution (100 mL) is shown in Table 1.

[0084] Table 1. Sample storage solution (100 mL)

[0085]

[0086] After the sample preservation solution is prepared, it is divided into 15mL centrifuge tubes, 5mL per tube. After division, it can be stored at 4℃ for 1 month.

[0087] 2. Sample cleaning solution

[0088] The specific formula of the sample cleaning solution (100 mL) is shown in Table 2.

[0089] Table 2. Sample cleaning solution (100 mL)

[0090]

[0091] The sample cleaning solution must be prepared and used immediately.

[0092] 3. Sample dissociation solution

[0093] The specific formula of the sample dissociation solution (10 mL) is shown in Table 3.

[0094] Table 3. Sample dissociation solution (10 mL)

[0095]

[0096] Note: The sample dissociation solution should be prepared and used immediately.

[0097] In Table 3, the preparation of collagenase stock solution is shown in Tables 4 to 6.

[0098] Table 4. 10× Collagenase I stock solution (100 mL)

[0099]

[0100] After the 10× collagenase I stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored at -20°C for a long time.

[0101] Table 5. 10× Collagenase II stock solution (100 mL)

[0102]

[0103] After the 10× collagenase II stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored at -20°C for a long time.

[0104] Table 6. 10× Collagenase IV stock solution (100 mL)

[0105]

[0106] After the 10× collagenase IV stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored at -20°C for a long time.

[0107] In Tables 4, 5 and 6, the unit U of collagenase (the collagenase I or the collagenase II or the collagenase IV) is defined by the enzymatic activity of the protease: at 37° C., pH 7.5, 1 U of protease is used to treat the collagenase (the collagenase I or the collagenase II or the collagenase IV) for 5 hours to release 1 μmol of L-leucine.

[0108] 4. Cell digestion fluid

[0109] The specific formula of the cell digestion solution (10 mL) is shown in Table 7.

[0110] Table 7. Cell digestion solution (10 mL)

[0111]

[0112] The cell digestion solution is prepared and used immediately.

[0113] 5. Digestion stop solution

[0114] The specific formula of the digestion stop solution (100 mL) is shown in Table 8.

[0115] Table 8. Digestion stop solution (100 mL)

[0116]

[0117] After the digestion stop solution is prepared, it can be stored at 4°C for one month.

[0118] 6. Liver cancer microtumor model culture medium

[0119] The specific formula of the liver cancer microtumor model culture medium (100 mL) is shown in Table 9.

[0120] Table 9. Liver cancer microtumor model culture medium (100 mL)

[0121]

[0122] After the liver cancer microtumor model culture medium is prepared, it is filtered and sterilized using a 0.22 μM syringe filter (Millipore SLGP033RS) and can be stored at 4°C for two weeks.

[0123] In Table 9, the preparation of human recombinant protein stock solutions is shown in Tables 11 to 16 (the preparation of BSA stock solution is shown in Table 10), the preparation of Forskolin stock solution is shown in Table 17, the preparation of Gastrin stock solution is shown in Table 18, the preparation of A83-01 stock solution is shown in Table 19, the preparation of N-acetyl-L-cysteine ​​stock solution is shown in Table 20, and the preparation of Nicotinamide stock solution is shown in Table 21.

[0124] Table 10, 100× BSA solution (1 mL)

[0125]

[0126] 100× BSA solution is prepared and used immediately.

[0127] Table 11. 1000× human recombinant protein EGF stock solution (5 mL)

[0128]

[0129] After the 1000× human recombinant protein EGF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0130] Table 12. 1000× human recombinant protein FGF-10 stock solution (5 mL)

[0131]

[0132] After the 1000× human recombinant protein FGF-10 stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0133] Table 13. 1000× human recombinant protein HGF stock solution (5 mL)

[0134]

[0135] After the 1000× human recombinant protein HGF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0136] Table 14. 1000× human recombinant protein IL-2 stock solution (5 mL)

[0137]

[0138] After the 1000× human recombinant protein IL-2 stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0139] Table 15. 1000× human recombinant protein IL-15 stock solution (5 mL)

[0140]

[0141] After the 1000× human recombinant protein IL-15 stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0142] Table 16. 1000× human recombinant protein R-spondin 1 stock solution (4 mL)

[0143]

[0144] After the 1000× human recombinant protein R-spondin stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0145] Table 17, 1000× Forskolin stock solution (2.44 mL)

[0146]

[0147] After the 1000× Forskolin stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0148] Table 18, 1000×Gastrin stock solution (48 mL)

[0149]

[0150] After the 1000× Gastrin stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0151] Table 19, 100000×A83-01 stock solution (1.05 mL)

[0152]

[0153] After the 100000×A83-01 stock solution is prepared, it is packaged in 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0154] Table 20, 1000×N-acetyl-L-cysteine ​​stock solution (5 mL)

[0155]

[0156] After the 1000× N-acetyl-L-cysteine ​​stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0157] Table 21. 1000×Nicotinamide stock solution (4 mL)

[0158]

[0159] After the 1000× Nicotinamide stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0160] 7. Cell Cryopreservation Solution

[0161] The specific formula of the cell freezing solution is shown in Table 22.

[0162] Table 22. Cell cryopreservation solution

[0163]

[0164] Cell freezing solution is prepared and used immediately.

[0165] In Table 22, the preparation of 1% methylcellulose solution is shown in Table 23.

[0166] Table 23. 1% methylcellulose solution (10 mL)

[0167]

[0168] 1% methylcellulose solution can be stored at 4°C for a long time after preparation.

[0169] Example 2: Acquisition of specimens after liver cancer surgery

[0170] 1. Cooperate with tertiary hospitals to obtain samples through researcher-initiated clinical research, and the cooperation has passed formal medical ethics review.

[0171] 2. The attending physician selects patients for inclusion in the study according to the clinical indications specified in the medical guidelines, and selects appropriate specimens for in vitro culture based on the intraoperative clinical indications. The sample selection criteria are: liver cancer, specimen weight exceeding 20 mg.

[0172] 3. All included cases are uniformly replaced by experimental numbers. The naming principle of the experimental number is the eight-digit date of sample collection + the last four digits of the patient's hospitalization number. When the hospital provides samples, it also provides the patient's pathological staging, clinical diagnosis and other basic clinical information. The naming principle of the experimental number is the eight-digit date of sample collection + the last four digits of the patient's hospitalization number. For example, for samples provided on January 1, 2020, the patient's hospitalization number is T001537474, then the sample experimental number is 202001017474.

[0173] 4. After the tumor tissue is removed from the body during surgery, a sample collection specialist collects fresh specimens in the sterile environment of the operating room. Samples should be collected from areas with rich fresh blood vessels, avoiding areas with poor cell activity such as necrotic tissue, adipose tissue, and fibrotic tissue. The collected samples are placed in a sample preservation solution (see Example 1) pre-cooled at 4°C. The sample preservation tube containing the sample is temporarily stored on ice and transported to the laboratory for the next step within 12 hours. The temperature during transportation is controlled at 2-8°C.

[0174] Example 3: Pre-dissociation treatment of liver cancer tissue samples

[0175] The following operations need to be performed on ice and the entire operation should be completed within 10 minutes.

[0176] The surgical instruments used in the following operations must be sterilized with high-temperature steam (120°C, 20 minutes) and dried before use.

[0177] 1. After weighing the sample, clean the sample surface with medical alcohol (75% by volume) for 10 to 30 seconds.

[0178] 2. Wash the sample 5 times with sample cleaning solution (see Example 1), and wash the sample 5 times with sterile PBS solution.

[0179] 3. Use ophthalmic scissors, ophthalmic tweezers, scalpel and other instruments to carefully peel off the fat tissue, connective tissue and necrotic tissue in the sample.

[0180] Example 4: Dissociation of liver cancer tissue samples

[0181] The surgical instruments used in the following embodiments must be sterilized by high-temperature steam (120°C, 20 minutes) and dried before use.

[0182] 1. Use ophthalmic scissors to cut the tissue into 0.5mm pieces. 3 Small pieces around.

[0183] 2. Treat the tissue with sample dissociation solution (see Example 1). For tissues with a sample size not exceeding 0.5 mg, use 1 mL of sample dissociation solution. For tissues with a sample size exceeding 0.5 mg, add 0.1 mL of sample dissociation solution for every 0.1 mg increase in tissue weight. The sample dissociation solution treatment condition is 37°C, and the dissociation time is 1 hour. During the dissociation process, observe the dissociation of the sample under a microscope every 15 minutes until most cells are observed to fall off from the tissue.

[0184] 3. Use 10 times the volume of digestion stop solution (see Example 1) to terminate the dissociation reaction. Filter the cell suspension through a 100 μm sterile cell strainer to remove tissue debris and adherent cells, centrifuge at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0185] 4. Resuspend the cells in 5 mL of sterile PBS, centrifuge at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0186] 5. Resuspend the cell pellet in the liver cancer microtumor model culture medium (see Table 9 of Example 1), count the cells, and determine the cell viability by trypan blue staining. If the viability of the isolated cells is greater than 70%, the cells can be inoculated and cultured.

[0187] Example 5: Cultivation of liver cancer microtumor model

[0188] 1. A vessel with a low adsorption surface was used to perform suspension culture of a liver cancer microtumor model. The culture medium used was the liver cancer microtumor model culture medium shown in Table 9 of Example 1 (wherein the final concentration of the antibacterial and antifungal agent triple antibody was 1% by volume; the final concentration of the HEPES was 10 mM; the final concentration of the GlutaMax was 1% by volume; the final concentration of the human recombinant protein EGF was 40 ng / mL; the final concentration of the human recombinant protein FGF-10 was 20 ng / mL; the final concentration of the human recombinant protein HGF was 20 ng / mL; the final concentration of the human recombinant protein IL-2 was 20 ng / mL; the final concentration of the human recombinant protein IL-15 was 20 ng / mL; the human recombinant protein R-spondin was The final concentration of 1 is 400ng / mL; the final concentration of forskolin is 5μM; the final concentration of gastrin is 5nM; the concentration of A83-01 is 1μM; the concentration of N-acetyl-L-cysteine ​​is 2mM; the concentration of nicotinamide is 10mM; the final concentration of N2 additive is 1% by volume; the final concentration of B27 is 2% by volume; the rest is Advanced DMEM / F12 medium), taking a six-well plate as an example, 10 per well. 6 The cells were plated at a density of 10 cells / well, with 2-3 mL of culture medium used per well. The inoculated cells were cultured in a cell culture incubator at 37 °C and 5% CO2.

[0189] 2. Observe the cell status every day until the cells form clumps with a diameter of about 100-300μm. Then replace the culture medium every 2-3 days to maintain the growth state of liver cancer microtumors.

[0190] like Figure 1 As shown in Figure 2, within the first 48 hours of culture, various types of cells from cancer tissues spontaneously aggregated and self-assembled into cell clusters of 100-300 μm in size, which we call microtumor models. The total number of microtumor cell clusters can reach 10 4 -10 6 This method has been tested on a large number of samples, and the success rate of culturing microtumor models of different liver cancer surgical samples can reach 85%.

[0191] Example 6. Comparative identification of hepatocellular carcinoma microtumor model and primary lesion by HE staining

[0192] Description of reagents and consumables used in the following examples:

[0193] Hematoxylin and eosin (HE) staining kit (Beijing Solebow Biotechnology Co., Ltd., #G1120);

[0194] 1. Freshly collected samples, fixed, routinely paraffin-embedded, and sliced ​​to 3-8 μm;

[0195] 2. Dewax in xylene twice, 5-10 min each time, rehydrate in a series of ethanol (100%, 95%, 85%, 75%), 3 min each gradient, and soak in distilled water for 2 min;

[0196] 3. Dye with hematoxylin solution for 2-20 minutes, and wash away the floating color with distilled water;

[0197] 4. Differentiate in differentiation solution for 10-60 seconds, add tap water or soak twice, 3-5 minutes each time;

[0198] 5. Place in eosin stain solution for 30s-2min, pour off excess stain solution and dehydrate quickly;

[0199] 6. Dehydration, transparency, and sealing: 75% ethanol, 85% ethanol, 95% ethanol, and 100% ethanol (I) were used for 2-3 seconds each, 100% ethanol (II) was used for 1 minute, xylene was used for transparency twice, each time for 1 minute, and neutral resin was used for sealing;

[0200] 7. Observe the staining effect under the microscope and take photos.

[0201] Figure 2 A comparison of the HE staining effects of the liver cancer microtumor model and its corresponding original lesion tissue sample cultured in Example 5 is shown. It can be seen that the microtumor can reproduce the pathological tissue structure of the original lesion to the greatest extent.

[0202] Example 7. Multiple immunofluorescence staining of liver cancer microtumor model and primary lesion

[0203] The reagents used in the following examples are described:

[0204] Paraformaldehyde (Beijing Chemical Reagent Company, analytical grade) was prepared by dissolving paraformaldehyde powder in ultrapure water to prepare a 4% (4 g / 100 mL) paraformaldehyde solution;

[0205] Methanol and dimethyl sulfoxide (Beijing Chemical Reagent Company, analytical grade);

[0206] Hydrogen peroxide (Beijing Chemical Reagent Company, 35%);

[0207] Methanol, dimethyl sulfoxide, and 35% hydrogen peroxide were mixed in a ratio of 4:4:1 (volume ratio) to prepare Dan's rinse solution;

[0208] Bovine serum albumin (Sigma, #A1933) was dissolved in PBS to prepare a 3% (3 g / 100 mL) BSA solution;

[0209] Primary antibodies for immunofluorescence (anti-PanCK antibody: Abcam#ab215838; anti-Vim antibody: abclonal#A19607; anti-CD3 antibody: Biolegend#300434);

[0210] Secondary antibodies for immunofluorescence (CST#4408s, CST#8889s);

[0211] Hoechst stain (Beijing Solebow Biotechnology Co., Ltd., #C0021);

[0212] Using the paraffin sections obtained in Example 6 as materials, multiple immunofluorescence staining was performed according to the following steps. The three antibodies respectively labeled pan-CK to characterize epithelial tumor cells, Vimentin to label stromal cells, and CD3 to label T cells:

[0213] 1. After dewaxing and rehydration of paraffin sections, wash them with PBS and treat them with pre-cooled methanol solution for 1 hour.

[0214] 2. Treat with Dan's rinse solution at room temperature for 2 hours, and then treat with 75%, 50%, and 25% (volume percentage) methanol solution diluted with PBS in sequence, each time for 10 minutes.

[0215] 3. Block with 3% BSA solution at room temperature for 2 hours.

[0216] 4. Incubate the primary antibody in mixed dilution solution (3% BSA solution) at 4°C overnight.

[0217] 5. Wash the sections with PBS solution 5 times, 20 minutes each time.

[0218] 6. Add secondary antibody mixed dilution (diluted with 3% BSA solution at a ratio of 1:2000) and incubate the secondary antibody at room temperature for 2 hours.

[0219] 7. Wash the sections with PBS solution 5 times, 20 minutes each time.

[0220] 8. Hoechst staining solution, staining at room temperature for 20 minutes.

[0221] 9. Seal the slides with quenching sealing medium and use a laser confocal microscope to observe the staining and take pictures.

[0222] Figure 3The effect diagram of multiple immunofluorescence staining of the cultured liver cancer microtumor model is shown. It can be seen that the liver cancer microtumor model not only retains the panCK-positive (green fluorescence) tumor cells in the original lesion, but also preserves the Vimentin-positive stromal cells (white) and immune cells (T cells red), reproducing the cell diversity and microenvironment of the original lesion to the greatest extent. It confirms the high similarity in cell composition between the microtumor model cultured by this method and the original lesion. We performed multiple immunofluorescence staining identification on 10 liver cancer (hepatocellular carcinoma) microtumor samples and their corresponding original lesion tissue blocks. The statistical results are shown in Table 24.

[0223] Table 24. Multiple immunofluorescence staining identification of liver cancer microtumor model and its corresponding primary lesion tissue samples

[0224]

[0225] Example 8: Isolation of primary liver cancer tumor cells from a liver cancer microtumor model

[0226] The cell magnetic bead sorting process mentioned in the following examples uses the Miltenyi CD326 magnetic bead positive selection kit (Miltenyi #130-061-101).

[0227] 1. Collect the liver cancer microtumor model cultured in suspension in Example 5, centrifuge at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0228] 2. Wash the cell pellet once with sterile PBS solution, centrifuge at 800g for 10 minutes at room temperature, and discard the supernatant.

[0229] 3. Reselect the cell pellet with cell digestion solution (see Example 1) and digest at 37°C for 5-30 minutes. During the digestion process, observe the digestion of cell clumps under a microscope every 5 minutes until most cell clumps are digested into single cells.

[0230] 4. Terminate the digestion with 10 times the volume of the digestion stop solution (see Example 1), centrifuge the cell suspension at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0231] 5. The single cell suspension obtained by digestion was sorted with Miltenyi CD326 magnetic bead positive selection kit to obtain CD326 positive tumor cells: resuspend the cell pellet with sorting buffer (formula is Table 25), and the cell amount is less than 10 7Add 20 μL CD326 magnetic beads to the system and incubate on ice for 30 minutes. Wash the cell pellet with 2 mL sorting buffer. Wash the sorting column with 5 mL sorting buffer and place the sorting column on the magnetic rack. Pass the cell suspension through the cell sorting column and rinse the sorting column three times with 3 mL sorting buffer to elute the CD326 negative cells. Remove the sorting column from the magnetic rack and rinse the sorting column once with 5 mL sorting buffer to elute the CD326 positive cells.

[0232] Table 25, Sorting Buffer

[0233]

[0234] Note: Prepare and use immediately.

[0235] 6. Centrifuge the sorted CD326 positive cells at 800g for 10 minutes at room temperature and discard the supernatant. 5 / 3.5cm cell culture dish for inoculation and adherence culture.

[0236] Example 9: Passaging of primary liver cancer cells

[0237] 1. For adherent cultured primary liver cancer cells, remove the culture medium and wash the cells with sterile PBS solution.

[0238] 2. Digest the cells with 0.05% trypsin at room temperature for 30-300 seconds, and observe the cell status during the process until most of the cells are digested into spherical shapes.

[0239] 3. Terminate the digestion reaction with 10 times the volume of DMEM medium containing 10% serum, collect the cell suspension and centrifuge at 800g for 10 minutes at room temperature, and discard the supernatant.

[0240] 4. Centrifuge at 800g for 10 minutes at room temperature and discard the supernatant.

[0241] 5. Resuspend the cell pellet in DMEM medium containing 10% serum and count the cells. 5 The cells were inoculated and cultured at a density of 1 / 3.5 cm cell culture dish (using the culture medium in Table 9 plus 10% FBS).

[0242] After the purification step of Example 8 and the subculture of Example 9, high-purity primary liver cancer tumor cells ( Figure 4 ).

[0243] Example 10: Cryopreservation of primary liver cancer cells

[0244] The primary liver cancer tumor cells purified and cultured in Example 8 can be cryopreserved after 2-3 passages and amplification:

[0245] 1. For adherent cultured primary liver cancer cells, remove the culture medium and wash the cells with sterile PBS solution.

[0246] 2. Digest the cells with 0.05% trypsin at room temperature for 30-300 seconds, and observe the cell status during the process until most of the cells are digested into spherical shapes.

[0247] 3. Terminate the digestion reaction with 10 times the volume of DMEM medium containing 10% serum, collect the cell suspension and centrifuge at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0248] 4. Centrifuge at 800g for 10 minutes at room temperature and discard the supernatant.

[0249] 5. Use cell freezing solution (see Example 1) at 10 6 Resuspend the cell pellet at a density of 1 mL / mL and add 1 mL of cell suspension to each 2 mL cryovial. Cryopreservate in a gradient cooling box overnight and then transfer to liquid nitrogen for long-term storage.

[0250] Example 11: Recovery of primary liver cancer cells

[0251] Primary liver cancer cells stored in liquid nitrogen can be revived:

[0252] 1. Prepare 37℃ sterile water five minutes in advance.

[0253] 2. Take out the cryovial from liquid nitrogen and thaw the cells quickly in sterile water at 37°C.

[0254] 3. Transfer the cells in the cryopreservation tube to a 15 ml centrifuge tube, add ten times the volume of DMEM medium containing 10% serum and mix thoroughly, centrifuge at 800g for 10 minutes at room temperature, and discard the supernatant.

[0255] 4. Resuspend the cell pellet in DMEM medium containing 10% serum and count the cells. 5 / 3.5cm cell culture dish for inoculation and adherence culture.

[0256] Example 12 Comparison of the ability of different culture media to form liver cancer microtumor structures

[0257] The operation method and process of primary culture of all samples in this embodiment are completely consistent (refer to the above description), and only the culture medium formula is different. The various culture media tested are shown in Table 26. Among them, Scheme A is the formula used in the present invention, and the details are shown in Table 9.

[0258] Table 26. Test medium formula (100 mL)

[0259]

[0260] Twenty liver cancer solid tumor tissue samples (primary hepatocellular carcinoma) were treated with each of the five culture medium schemes. The sample processing and culture operations were performed according to the methods described in Examples 3, 4, and 5. After 3 days of culture, the formation of cell cluster structures (i.e., microtumor models) in the culture system was observed as shown in Table 27.

[0261] Table 27. Cultivation results of different culture media

[0262]

[0263] It can be seen that the liver cancer micro-tumor model can be cultured using the three culture media of Schemes A, B, and C, while the culture media of Schemes D and E are not suitable for the formation of liver cancer micro-tumor models. The liver cancer micro-tumor model formed in the culture medium of Scheme A is larger in volume, which is slightly better than the culture media of Schemes B and C.

[0264] Example 13. Comparison of genetic backgrounds of liver cancer microtumor models formed in different culture media

[0265] The DNA extraction process mentioned in the following examples was performed using the Tiangen Blood / Tissue / Cell Genomic Extraction Kit (DP304).

[0266] The library construction process mentioned in the following examples was performed using the NEB DNA sequencing library construction kit (E7645).

[0267] The high-throughput sequencing mentioned in the following examples refers to the Illumina HiSeq X-ten sequencing platform.

[0268] The present invention obtained liver cancer microtumor models and liver cancer primary lesion samples cultured in three culture media of Schemes A, B, and C obtained in Example 12, and performed DNA extraction, whole genome sequencing, and copy number variation analysis methods as follows:

[0269] 1. Take 10 mg of liver cancer lesion tissue and culture it with three culture media of Scheme A, B and C obtained in Example 12 to obtain liver cancer microtumor model of about 10 mg. 5 DNA was extracted from a number of cell clusters, and libraries were constructed and whole genome high-throughput sequenced (WGS) was performed with a sequencing depth of 30×.

[0270] 2. The sequencing results of the two groups of original lesions and liver cancer microtumor models were analyzed for copy number variation (CNV) respectively, and the copy number variation between the primary liver cancer tissue samples and the liver cancer microtumor models of each generation was compared, such as Figure 5 The results showed that the liver cancer micro-tumor models cultured in the three culture media A, B, and C can well retain the copy number variation characteristics of the primary liver cancer lesions.

[0271] Example 14 Comparison of cell composition of liver cancer microtumor models formed in different culture media

[0272] The present invention obtained liver cancer (primary hepatocellular carcinoma) microtumor models and liver cancer primary lesion samples cultured in three culture media of Schemes A, B, and C obtained in Example 12, and performed multiple immunofluorescence staining to identify their cell composition (methods are shown in Example 7). The results are shown in Table 28.

[0273] Table 28. Comparison of cell components in liver cancer microtumor models formed in different culture media

[0274]

[0275] It can be seen that culture media B and C can well preserve the tumor cells in the original lesions, and the liver cancer microtumor model formed by them is formed with tumor cells of higher purity. In addition to maintaining the activity of tumor cells in the original lesions, culture media A can also well preserve the fibroblasts and T cells in the tissue, and has greater advantages than culture media B and C in maintaining cell diversity.

[0276] Therefore, the liver cancer solid tumor micro-tumor model culture medium (Table 9) used in the present invention can protect the various cell types in the original lesion to the greatest extent, and promote them to spontaneously form a multi-cellular structure under suspension conditions, thereby constructing an in vitro cell model that is extremely close to the characteristics of the original lesion.

[0277] Example 15: Comparison of the success rate of culture in different sample dissociation solutions

[0278] The operation method and process of primary culture of all samples in this embodiment are completely consistent (refer to the above description), and only the sample dissociation solution formula is different. The various sample dissociation solutions tested are shown in Table 29, among which Scheme D is the formula used in the present invention, as shown in Table 3.

[0279] Table 29. Test sample dissociation solution formula (10mL)

[0280]

[0281] The sample dissociation solution is prepared and used immediately.

[0282] 10 samples of liver cancer solid tumor tissue blocks weighing more than 100 mg were selected and divided into four equal parts. The four sample dissociation solutions were used respectively to perform sample processing and culture operations according to the methods described in Examples 3, 4, and 5. After 3 days of culture, the formation of cell cluster structures in the culture system was observed as shown in Tables 30 and 31:

[0283] Table 30. Statistics of the number of liver cancer microtumors formed in different sample dissociation solutions

[0284]

[0285] Table 31. Statistics of liver cancer micro-tumor cell cluster sizes in different sample dissociation solutions (measured by long diameter, unit μm)

[0286]

[0287] It can be seen that the sample dissociation solution formula has a certain influence on the formation ability of the liver cancer micro-tumor model and the size of the micro-tumor cell cluster. The sample dissociation solution (Table 3) used in the present invention can gently dissociate cells in the tissue, maintain cell activity to the greatest extent, and improve the formation efficiency and quality of the micro-tumor model.

[0288] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A culture medium for culturing a liver cancer microtumor model, characterized in that: The culture medium is composed of three antibiotics of antibacterial and antifungal agents, HEPES, GlutaMax, human recombinant protein EGF, human recombinant protein FGF-10, human recombinant protein HGF, human recombinant protein IL-2, human recombinant protein IL-15, human recombinant protein R-spondin 1, forskolin, gastrin, A83-01, N-acetyl-L-cysteine, nicotinamide, N2 additive, B27 and basic culture medium; wherein the three antibiotics of antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B; In the culture medium, the final concentration of HEPES is 8-12 mM; the final concentration of GlutaMax is 0.8-1.2% by volume; the final concentration of human recombinant protein EGF is 10-100 ng / mL; the final concentration of human recombinant protein FGF-10 is 10-50 ng / mL; the final concentration of human recombinant protein HGF is 5-25 ng / mL; the final concentration of human recombinant protein IL-2 is 10-100 ng / mL; the final concentration of human recombinant protein IL-15 is 10-100 ng / mL; the final concentration of human recombinant protein R-spondin is 10-100 ng / mL; The final concentration of 1 is 250-500ng / mL; the final concentration of forskolin is 2-10μM; the final concentration of gastrin is 2-10nM; the concentration of A83-01 is 0.25-1.25μM; the concentration of N-acetyl-L-cysteine ​​is 0.5-2mM; the concentration of nicotinamide is 5-10mM; the final concentration of N2 additive is 1% by volume; the final concentration of B27 is 1.5-2.5% by volume; The basic culture medium is Advanced DMEM / F12 culture medium.

2. The culture medium according to claim 1, characterized in that: In the culture medium, the final concentration of penicillin in the antibacterial and antifungal agents is 100-200 U / mL, the final concentration of streptomycin is 100-200 μg / mL, and the final concentration of amphotericin B is 200-250 ng / mL.

3. A set of reagents for culturing a liver cancer microtumor model, comprising the culture medium and the sample dissociation solution according to claim 1 or 2; The sample dissociation solution is composed of collagenase I, collagenase II, collagenase IV and PBS; wherein, The final concentration of the collagenase I is 150-250 U / mL; the final concentration of the collagenase II is 150-250 U / mL; the final concentration of the collagenase IV is 150-250 U / mL; and the remainder is PBS.

4. The reagent set according to claim 3, characterized in that: The complete set of reagents also includes all or part of the following: sample preservation solution, sample cleaning solution, cell digestion solution, digestion termination solution and cell freezing solution.

5. The reagent set according to claim 4, characterized in that: The sample preservation solution is composed of fetal bovine serum, three antibiotics of antibacterial and antifungal agents, HEPES and HBSS; wherein 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 is 100-200μg / mL, and the final concentration of amphotericin B is 200-250ng / mL; the final concentration of HEPES is 8-12mM; the remainder is HBSS; and / or The sample cleaning solution is composed of three antibiotics of antibacterial and antifungal agents and PBS; wherein 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 is 100-200μg / mL, and the final concentration of amphotericin B is 200-250ng / mL; the rest is PBS; and / or The cell digestion solution is composed of: 4-6 mL Accutase, EDTA with a final concentration of 5 mM, 1.5-2.5 mL TrypLE Express, and the balance is PBS per 10 mL of the cell digestion solution; and / or The digestion stop solution is composed of fetal bovine serum, three antifungal antibodies and DMEM culture medium; wherein 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% by volume; the final concentration of penicillin in the three antibacterial and antifungal antibodies is 100-200 U / mL, the final concentration of streptomycin is 100-200 μg / mL, and the final concentration of amphotericin B is 200-250 ng / mL; the remainder is DMEM culture medium; and / or The cell freezing solution consists of Advanced DMEM / F12 culture medium, DMSO and 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM / F12 culture medium, the DMSO and the 1% methylcellulose solution is 20:2:(0.8-1.2); and the 1% methylcellulose solution is a methylcellulose aqueous solution with a concentration of 1g / 100ml.

6. Use of the culture medium according to claim 1 or 2 or the reagent set according to any one of claims 3 to 5 in constructing a liver cancer microtumor model.

7. A method for constructing a liver cancer microtumor model, comprising the following steps: (a1) dissociating liver cancer solid tumor tissue using the sample dissociation solution described in claim 3; (a2) Using the culture medium described in claim 1 or 2 to suspend and culture the single cells dissociated in step (a1) to form cell clusters, thereby obtaining a liver cancer microtumor model.

8. A method for obtaining primary liver cancer solid tumor cells, characterized in that: The method is to separate primary liver cancer solid tumor cells from the liver cancer microtumor model constructed by the method of claim 7.

9. The method according to claim 8, characterized in that: The method comprises the following steps: (b1) digesting the liver cancer microtumor model using the cell digestion solution described in claim 4 or 5 to obtain single cells; (b2) sorting out CD326 positive cells from the single cells obtained in step (b1) to obtain the primary liver cancer solid tumor cells.

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