CN112760280A

CN112760280A - Method for culturing primary cells of gallbladder cholangiocarcinoma

Info

Publication number: CN112760280A
Application number: CN201911065539.4A
Authority: CN
Inventors: 尹申意; 张函槊
Original assignee: Beijing Genex Health Technology Co ltd
Current assignee: Beijing Genex Health Technology Co ltd
Priority date: 2019-11-04
Filing date: 2019-11-04
Publication date: 2021-05-07

Abstract

本发明公开了一种胆囊胆管癌原代细胞的培养方法。本发明提供了胆囊胆管癌原代细胞培养方法及配套试剂，该技术核心是：用温和细胞解离试剂处理胆囊胆管癌实体瘤组织，最大程度保证了组织中癌细胞活力；直接从胆囊胆管癌胆汁样本中分离出肿瘤细胞，最大限度排除胆汁中胆红素结晶和正常细胞的干扰；配制特殊无血清培养基，利用悬浮培养体系对胆囊胆管癌来源的肿瘤细胞进行体外培养，保证癌细胞正常扩增的同时最大限度的排除正常细胞的干扰。利用本发明方法得到的胆囊胆管癌原代细胞培养物可用于多种细胞水平体外实验、二代测序、构建动物模型、构建细胞系等等。可预见，这种培养方法在胆囊胆管癌的研究和临床诊断治疗领域具有广泛的应用前景。The invention discloses a method for culturing primary cells of gallbladder cholangiocarcinoma. The invention provides a method for culturing primary cells of gallbladder and cholangiocarcinoma and supporting reagents. The core of the technology is: treating solid tumor tissue of gallbladder and cholangiocarcinoma with mild cell dissociation reagents to ensure the vitality of cancer cells in the tissue to the greatest extent; Tumor cells are isolated from bile samples, and the interference of bilirubin crystals in bile and normal cells is eliminated to the greatest extent; special serum-free medium is prepared, and the tumor cells derived from gallbladder and cholangiocarcinoma are cultured in vitro using a suspension culture system to ensure that the cancer cells are normal. While expanding, the interference of normal cells is excluded to the greatest extent. The primary cell culture of gallbladder and cholangiocarcinoma obtained by the method of the present invention can be used for various cell-level in vitro experiments, second-generation sequencing, construction of animal models, construction of cell lines and the like. It is foreseeable that this culture method has broad application prospects in the research and clinical diagnosis and treatment of gallbladder and cholangiocarcinoma.

Description

Method for culturing primary cells of gallbladder cholangiocarcinoma

Technical Field

The invention relates to the technical field of biology, in particular to a method for culturing primary cells of gallbladder cholangiocarcinoma.

Background

Gallbladder and bile duct cancer is common malignant tumor of digestive system at gallbladder, bile duct and intrahepatic bile duct parts, and comprises gallbladder cancer, bile duct cell cancer and the like. The gallbladder and bile duct related malignant tumor has a total incidence rate of about 3% in China, wherein bile duct cancer accounts for 2%, and is the 5 th highest in digestive tract malignant tumor in China. Although the incidence rate is not high, the cancers related to gallbladder and bile duct are all very malignant, and the bile duct cancer is even called as 'king of liver cancer' or 'king of cancer'. For unresectable gallbladder cancer, median survival is only 8 months.

Although scientific research and medical institutions in various countries around the world have great investment in the research on the etiology and development process of gallbladder-bile duct cancer, human beings still have little knowledge of the disease. The gallbladder bile duct cancer is a complex disease, the occurrence and the development of which are dynamic processes, and the interaction of a plurality of signal molecules is involved, so that a complex molecular regulation network is formed and is influenced by external environmental factors. The etiology, occurrence and development process of gallbladder bile duct cancer are highly different among individuals and cannot be generally known. And part of patients with cholangiocarcinoma are not easy to obtain operation/biopsy tissues for pathological diagnosis, so that the timely treatment of the disease is delayed. Therefore, the trend of performing individual accurate research on primary cell cultures of gallbladder, bile duct cancer solid tumors or bile and the like as models is the research field of gallbladder, bile duct cancer and even the diagnosis and treatment field of gallbladder, bile duct cancer.

The existing primary tumor cell culture technology mainly comprises 2D culture, 3D culture, reprogramming culture and the like, and the methods all face the problems of extremely long culture period, low culture success rate, difficult removal of mixed cells and the like in different degrees.

Disclosure of Invention

In order to effectively solve the technical problems, the invention provides a novel primary cell culture technology and a matched reagent for gallbladder cancer, solid tumor operation samples of bile duct cancer or biopsy puncture tissue samples or bile samples of gallbladder cancer and bile duct cancer, and the core of the technology is as follows: (1) the gallbladder cancer, the solid tumor of the bile duct cancer or the biopsy puncture tissue are treated by using a mild cell dissociation reagent, so that the activity of cancer cells in the tissue is ensured to the greatest extent; (2) tumor cells are directly separated from bile samples of gallbladder cancer and bile duct cancer, and the interference of bilirubin crystals and normal cells in the bile is eliminated to the maximum extent; (3) preparing a special serum-free culture medium, and culturing primary tumor cells of the gallbladder cancer and the bile duct cancer in vitro by using a suspension culture system, so that the interference of normal cells is eliminated to the maximum extent while the normal amplification of the cancer cells is ensured.

In a first aspect, the invention claims a method of culturing primary cells of gallbladder cholangiocarcinoma.

The method for culturing primary cells of gallbladder bile duct cancer, which is claimed by the invention, can comprise the following steps:

suspending and culturing primary gallbladder cholangiocarcinoma cells by using a primary gallbladder cholangiocarcinoma cell culture medium;

the primary cell culture medium for cholangiocarcinoma of gall bladder is prepared from antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B), HEPES, GlutaMax, Non-essential amino acids (Non-essential amino acids), 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, SB202190(4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole), A83-01(3- (6-Methyl-2-pyridinyl) -N-phenyl-4- (4-quinolinyl) -1H-pyrazozole-1-carbothioamide), and Primocin^TMN-acetyl-L-cysteine (N-acetyl-L-cysteine), nicotine (Nicotinamide), N-2Supplement, cortisol, B27, ITS-X (Insulin, Transferrin, Selenium, ethanol Solution), gastrin (Gastin 1), Y-27632 and Advanced DMEM/F12. Wherein the final concentration of penicillin in the three-antibody of the antibacterial antifungal agent is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the three-antibody of the antibacterial antifungal agent is 100-200 mu g/mL (such as 100 mu g/mL); the final concentration of amphotericin B in the three-antibody of the antibacterial antifungal agent is 250ng/mL (such as 250 ng/mL); the final concentration of HEPES is 8-12mM (e.g., 10 mM); the final concentration of GlutaMax is 0.8-1.2% (e.g., 1%,% represents volume percent); the concentration of glycine in the non-essential amino acid is 80-120 μ M; the concentration of L-alanine in the non-essential amino acids is 80-120. mu.M (e.g., 100. mu.M); the concentration of L-asparagine in the non-essential amino acid is 80-120. mu.M (e.g., 100. mu.M); the concentration of L-aspartic acid in the non-essential amino acid is 80-120. mu.M (e.g., 100. mu.M); the concentration of L-glutamic acid in the non-essential amino acid is 80-120. mu.M (e.g., 100. mu.M); the concentration of L-proline in the non-essential amino acids is 80-120. mu.M (e.g., 100. mu.M); the concentration of L-serine in the non-essential amino acid is 80-120. mu.M (e.g., 100. mu.M); the final concentration of the human recombinant protein EGF is 10-100 ng/mL; what is needed isThe 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 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 the human recombinant protein Noggin is 100-200 ng/mL; the final concentration of the SB202190 is 5-10 μ M; the final concentration of the A83-01 is 0.25-1.25 mu M; the final concentration of the Primocin is 1% (volume percentage); the final concentration of the N-acetyl-L-cysteine (N-acetyl-L-cysteine) is 0.5-2 mM; the final concentration of nicotine (Nicotinamide) is 5-10 mM; the final concentration of the N-2Supplement is 1 percent (volume percentage); the final concentration of the cortisol is 20-50 ng/mL; the final concentration of B27 is 1.5-2.5% (e.g., 2%,% indicates volume percent); the final concentration of ITS-X is 0.8-1.2% (e.g. 1%,% represents volume percent); the final concentration of said Gastrin (Gastrin1) is 8-12nM (e.g., 10 nM); the final concentration of the Y-27632 is 5-20 mu M; the balance is Advanced DMEM/F12 medium.

Further, the composition of the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is as follows: each ml contains 10000 units of penicillin (base), 10000. mu.g of streptomycin (base) and 25. mu.g of amphotericin B. The antimicrobial antifungal agent triantibody (penicillin-streptomycin-amphotericin B) is "antibacterial-antibacterial, 100X" (e.g., Gibco #15240062, or other products of the same composition). The "Antibiotic-Antibiotic, 100X" contained 10000 units of penicillin (base), 10000. mu.g of streptomycin (base) and 25. mu.g of amphotericin B per ml, using penicillin G (sodium salt), streptomycin sulfate and amphotericin B in the form of 0.85% saline as the active ingredients

An antifungal agent. The GlutaMAX is GlutaMAX^TMSupplement "(e.g., Gibco #35050061, or other products of the same composition). The "GlutaMAX^TMThe Supplement "was composed of L-allyl-L-glutamine as a substitute for L-glutamine at a concentration of 200nM in a 0.85% NaCl solution. The non-essential amino acids are composed as follows: the non-essential amino acids comprise 750 μ g glycine, 890 μ g L alanine per mlAcid, 1320 mu g L-asparagine, 1330 mu g L-aspartic acid, 1470 mu g L-glutamic acid, 1150 mu g L-proline, 1050 mu g L-serine, and water as a solvent (the concentration of each amino acid involved in the above is 10mM per ml of the non-essential amino acid). The Primocin is an antibacterial agent (such as Invivogen # ant-pm-1 or other products with the same composition) for primary cells, is an antibiotic for protecting the primary cells from being polluted by microorganisms, and has killing effects on gram-positive bacteria, gram-negative bacteria, mycoplasma and fungi. The N-2Supplement is "N-2 Supplement (100X)" (e.g., Gibco #17502001, or other products of the same composition). The "N-2 Supplement (100X)" contained Human total Transferrin (Human Transferrin (Holo)) at a final concentration of 1mM, 500mg/L Recombinant Insulin Full Chain (Insulin Recombinant Full Chain), 0.63mg/L Progesterone (Progesterone), 10mM Putrescine (Putrescine), and 0.52mg/L Selenite (Selenite). The B27 is' B-27^TMSupplement (50X), minus vitamin A "(e.g., Gibco #12587010, or other products of the same composition). Said "B-27^TMExample 50X, minus vitamin A "contains Biotin (Biotin), DL-Alpha-tocopheryl Acetate (DL Alpha-tocopheryl Acetate), DL-Alpha-Tocopherol (DL Alpha-tocopheryl), BSA (fat acid fragment V), Catalase (Catalase), Human Recombinant Insulin (Human Recombinant Insulin), Human Transferrin (Human Transferrin), Superoxide Dismutase (Superoxide Dismutase), Corticosterone (Cortisosterone), D-Galactose (D-Galactose), ethanolamine hydrochloric Acid (Ethanolamine HCl), reduced glutathione (reduced)), L-Carnitine HCl (L-Carnitine HCl), Linoleic Acid (Linoleic Acid), Linolenic Acid (Linolenic Acid), Progesterone (Progesterone), Putrescine (Putrescine 2HCl), Sodium Selenite (Sodium Selenite), triiodothyronine (T3 (triodo-I-thyronine)). The ITS-X solvent is EBSS solution (Earle's balanced salt solution), and the solutes and the concentrations are as follows: 1g/L of insulin; 0.55g/L of transferrin; 0.00067g/L sodium selenite; ethanolamine 0.2 g/L. The GlutaMAX is a high-grade cell culture additive and can directly replace L-glutamine in a cell culture medium. The GlutaMAX is GlutaMAX^TMSupplement”(e.g., Gibco #35050061, or other products of the same composition). Y-27632 is "Y-27632 dihydrochloride (an ATP-competitive ROCK-I and ROCK-II inhibitor with Ki of 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 antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is under the brand code Gibco # 15240062; the brand of HEPES is Gibco # 15630080; the brand name of GlutaMAX is Gibco # 35050061; the nonessential amino acids are under the brand code Gibco # 11140050; the brand of the human recombinant protein EGF is Peprotech AF-100-15-100; the brand of the human recombinant protein bFGF is Peprotech AF-100-18B-50; the brand of the human recombinant protein HGF is Peprotech AF-100-39-100; the brand of the human recombinant protein FGF-10 is Peprotech AF-100-26-100; the brand of the human recombinant protein Wnt-3a is R&D5036-WN-500; the brand goods number of the human recombinant protein Noggin is Shanghai near shore # C018; the brand of the SB202190 is Sigma # S7067; the brand name of A83-01 is Tocris # 2939; the Primocin^TMThe brand of (1) is Invivogen # ant-pm-1; the brand and cargo number of the N-acetyl-L-cysteine is Sigma # A9165; the brand of Nicotinamide is Sigma # N0636; the brand goods number of the N-2Supplement is Gibco # 17502001; the brand of cortisol is Sigma # H0888; the brand name of B27 is Gibco # 12587010; the ITS-X brand has a goods number of Gibco # 51500056; the brand name of the gastrin is NJpeptide # Pep 12307; the brand goods number of the Y-27632 is MCE # 129830-38-2; the brand of the Advanced DMEM/F12 medium is Gibco # 12634010.

The primary gallbladder cholangiocarcinoma cells can be primary gallbladder cholangiocarcinoma solid tumor cells or primary gallbladder cholangiocarcinoma bile sample tumor cells.

When the primary gallbladder cholangiocarcinoma cells are primary gallbladder cholangiocarcinoma solid tumor cells, the primary gallbladder cholangiocarcinoma solid tumor cells can be obtained by dissociating a gallbladder cholangiocarcinoma solid tumor tissue with a sample dissociation solution.

The sample dissociation liquid consists of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of the collagenase I in the dissociation solution of the sample is 150-250U/mL (such as 200U/mL); the final concentration of the collagenase II in the dissociation solution of the sample is 150-250U/mL (such as 200U/mL); the final concentration of the collagenase IV in the dissociation solution of the sample is 50-150U/mL (such as 100U/mL); the balance being PBS.

Wherein the unit U of collagenase (said collagenase I, said collagenase II or said collagenase IV) is defined by the enzymatic activity of a protease: 1 μmol of L-leucine can be released by treating collagenase (said collagenase I, said collagenase II or said collagenase IV) with 1U of protease at 37 ℃ and pH 7.5 for 5 hours.

In a specific embodiment of the present invention, the brand name of collagenase I is Gibco # 17100-017; the brand of collagenase II is Gibco # 17101-015; the brand goods number of the collagenase IV is Gibco # 17104-; the PBS was branded under Gibco # 21-040-CVR.

Further, the gallbladder bile duct cancer solid tumor tissue can be dissociated by the sample dissociation liquid according to the method comprising the following steps: cutting the cut gallbladder bile duct cancer solid tumor tissue (for example, into 0.8-1.2 mm) according to the dosage of 0.1-0.3mL (for example, 0.1mL) of the sample dissociation liquid per mg of tissue³Small pieces of (a) were treated with the sample dissociation solution preheated at 37 ℃ in advance, and sample dissociation was performed at 37 ℃ for 15 minutes to 3 hours. The dissociation of the samples was observed under the microscope every 15 minutes until a large number of single cells were observed.

When the primary gallbladder cholangiocarcinoma cells are primary gallbladder cholangiocarcinoma bile sample tumor cells, the primary gallbladder cholangiocarcinoma bile sample tumor cells can be obtained by separating the gallbladder cholangiocarcinoma bile sample with a cell separation buffer solution.

The cell separation buffer solution consists of double-antibody P/S (penicillin-streptomycin), heparin sodium and PBS; wherein the final concentration of penicillin in the double-resistant P/S (penicillin-streptomycin) is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the double-resistant P/S (penicillin-streptomycin) is 100-200 mug/mL (such as 100 mug/mL); the final concentration of the heparin sodium is 10 IU/mL; the balance being PBS.

In a specific embodiment of the invention, the bis-anti P/S (penicillin-streptomycin) is under the brand code Gibco # 15140122; the brand code of the heparin sodium is Solarbio # H8270; the PBS was branded under Gibco # 21-040-CVR.

Further, the bile sample of cholecystic and cholangiocarcinoma can be separated by the separation buffer according to the method comprising the following steps: suspending the cells in the bile sample of the bile duct cancer by using the cell separation buffer solution, and then obtaining primary tumor cells of the bile sample of the bile duct cancer by density gradient centrifugation (by using Ficoll lymphocyte separation solution).

Still further, before the separation of the bile sample of gallbladder, bile duct cancer by the separation buffer solution, the method may further comprise a step of performing separation pretreatment on the bile sample of gallbladder, bile duct cancer: and removing impurities, coagulated blood clots and other components which influence the cell density gradient separation in the bile sample of the gallbladder, bile duct cancer.

In the method, the primary cholecystcholangiocarcinoma cells may be cultured in suspension with the primary cholecystcholangiocarcinoma cell culture medium according to a method comprising: using a cell culture container M to culture the primary gallbladder cholangiocarcinoma cells in a suspension manner by using the primary gallbladder cholangiocarcinoma cell culture medium at 37 ℃ and 5% CO₂Culturing is carried out under conditions in which the medium is changed every 2 to 4 days (e.g., 3 days) until the cells form a mass of 50 to 80 μm (e.g., 80 μm) in diameter.

Wherein the initial seeding density may be 10⁵Per cm²Bottom area of the container, e.g. six-well plate, 10 per well⁶Density of individual cells was plated.

Wherein, the cell culture container M can be any one of the following: (I) a cell culture container made of polystyrene, a cell culture container made of polycarbonate, a cell culture container made of polymethyl methacrylate, a cell culture container made of COC resin, a cell culture container made of cyclic olefin polymer, or a cell culture container with a low adsorption surface; (II) subjecting the cell culture vessel of (I) to CYTOP modification.

Further, the cell culture vessel is a cell culture dish, a cell culture well plate, or a microplate chip for cell culture (such as the microplate chip shown in fig. 5 in example 12) or the like.

In the (II), the cell culture vessel in the (I) may be subjected to CYTOP modification according to a method comprising the steps of: carrying out pure oxygen etching on the cell culture container in the step (I), wherein the etching condition is that the power is 20W, and the etching time is 3 minutes; the cell culture vessel surface was then covered with a 1% CYTOP solution and the CYTOP modification was completed by air drying the 1% CYTOP solution.

Wherein the composition of the 1% CYTOP solution is as follows: each 100mL of the 1% CYTOP solution contained 1mL of CYTOP, with the balance being fluoro oil.

Wherein the CYTOP is perfluoro (1-butenylvinylether) polymer. The fluoro oil may be a fluoro oil of brand 3M # FC40, or other product of the same composition.

In a particular embodiment of the invention, the CYTOP brand code is specifically Asashi glass # CTL-809M; the brand goods number of the fluorine oil is specifically 3M # FC 40.

Further, the method can also comprise the following step of carrying out dissociation pretreatment on the gallbladder bile duct cancer solid tumor tissue: washing the surface of the gallbladder, bile duct cancer and solid tumor tissue sample with 70-75% (such as 75%) ethanol by volume for 10-30 seconds; washing the gallbladder bile duct cancer solid tumor tissue sample 10-20 times (such as 10 times) with a sample washing solution, and washing the gallbladder bile duct cancer solid tumor tissue sample 5-10 times (such as 5 times) with a sterile PBS solution; and then removing impurities, connective tissues, adipose tissues, necrotic tissues and other components which influence the culture of the primary cells in the gallbladder bile duct cancer solid tumor tissue sample.

Wherein the sample cleaning solution consists of an antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) and PBS; wherein the final concentration of penicillin in the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the antibacterial antifungal agent triantibody (penicillin-streptomycin-amphotericin B) is 100-200. mu.g/mL (such as 100. mu.g/mL); the final concentration of amphotericin B in the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is 250-500ng/mL (such as 250 ng/mL); the balance being PBS.

An antifungal agent.

In a specific embodiment of the invention, the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is under the brand code Gibco # 15240062; the PBS was branded under Gibco # 21-040-CVR.

The step of pretreatment for dissociation of the gallbladder bile duct cancer solid tumor tissue needs to be operated on ice, and the whole operation step needs to be completed within 10 minutes.

Furthermore, the gallbladder bile duct cancer solid tumor tissue sample subjected to the dissociation pretreatment needs to be separated in less than 2 hours, and is preserved in a sample preservation solution before the dissociation pretreatment.

Wherein the sample preservation solution consists of fetal calf serum, three antibiotics (penicillin-streptomycin-amphotericin B), HEPES and HBSS (Hank's balanced salt solution); wherein the final concentration of fetal calf serum is 1-5% (such as 2%,% represents volume percentage content); the final concentration of penicillin in the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the antibacterial antifungal agent triantibody (penicillin-streptomycin-amphotericin B) is 100-200. mu.g/mL (such as 100. mu.g/mL); the final concentration of amphotericin B in the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is 250-500ng/mL (such as 250 ng/mL); the final concentration of HEPES is 8-12mM (e.g., 10 mM); the balance being HBSS.

An antifungal agent.

In a specific embodiment of the invention, the brand of fetal bovine serum is Gibco # 16000-; the brand code of the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is Gibco # 15240062; the brand of HEPES is Gibco # 15630080; the HBSS is sold under the brand name Gibco # 14170161.

Further, in the method, after the dissociation treatment of the gallbladder bile duct cancer solid tumor tissue by the sample dissociation liquid, the method further comprises the following steps: terminating the dissociation reaction with 8-15 (e.g., 10) times the volume of the digestion stop solution, and collecting the cell suspension; filtering the cell suspension with a 100 μm or 40 μm sterile cell strainer to remove tissue debris and adherent cells; 800-1000g (e.g., 800g) of the suspension is centrifuged at room temperature for 10-15 minutes (e.g., 10 minutes), and the supernatant is discarded; then resuspend the cells in 3-5mL (e.g., 5mL) sterile PBS; centrifuging at room temperature for 10-15 min (such as 10min) again at 800-; then, the primary cell culture medium of the cholecystic and cholangiocarcinoma solid tumor is used for resuspending the cell sediment, and the cell state is observed under a microscope for cell counting.

Wherein the digestion stop solution consists of fetal calf serum, antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) and DMEM culture medium; wherein the final concentration of fetal calf serum is 8-12% (such as 10%,% represents volume percentage content); the final concentration of penicillin in the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the antibacterial antifungal agent triantibody (penicillin-streptomycin-amphotericin B) is 100-200. mu.g/mL (such as 100. mu.g/mL); the final concentration of amphotericin B in the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is 250-500ng/mL (such as 250 ng/mL); the balance is DMEM medium.

An antifungal agent.

In a specific embodiment of the invention, the brand of fetal bovine serum is Gibco # 16000-; the brand code of the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is Gibco # 15240062; the DMEM medium is sold under the brand name Gibco # 11965-092.

Further, in the method, the following steps may be further included: and (3) when the primary gallbladder cholangiocarcinoma cells form a lump with the diameter of 50-80 μm (such as 80 μm), carrying out passage on the primary gallbladder cholangiocarcinoma cells.

Wherein, the cell digestive juice adopted when the passage is carried out consists of the following components: each 10mL of the cell digest contained 4-6mL (e.g., 5mL) of Accutase, a final concentration of 5mM EDTA (i.e., 10. mu.L of 0.5M EDTA), 1.5-2.5mL (e.g., 2mL) of TrypLE Express, and the balance PBS.

Further, the Accutase is StemPro^TMAccutase^TMCell discovery Reagent "(e.g., Gibco # A11105-01, or other products of the same composition). The Accutase is a single-component enzyme, and is dissolved in D-PBS, 0.5mM EDTA solution. The TrypLE Express is' TrypLE^TMExpress Enzyme (1X), no phenol red "(e.g., Gibco #12604013, or other products of the same composition). The TrypLE^TMExpress Enzyme (1X), no phenol red "contains 200mg/L KCl and 200mg/L KH₂PO₄8000mg/L NaCl, 2160mg/L Na₂HPO₄·7H₂O, 457.6mg/L EDTA; also contains recombinant protease.

In a specific embodiment of the invention, the brand name of the Accutase is Gibco # A11105-01; the brand name of the 0.5M EDTA is Invitrogen # AM 9261; the brand goods number of the TrypLE Express is Gibco # 12604013; the PBS was branded under Gibco # 21-040-CVR.

Further, the digestion temperature used for the passage was 37 ℃.

Further, the digestion stop solution used in the passage is the digestion stop solution described above.

More specifically, the step of performing said passaging is carried out: collecting cell mass to be passaged, centrifuging, washing the cell mass with sterile PBS solution, centrifuging, suspending the cell mass with the cell digestive juice, digesting at 37 deg.C until the cell mass is digested into single cell, stopping digestion reaction with the digestion stopping solution (the dosage can be 5-10 times, such as 10 times of volume), and collecting cell suspension; resuspending the cell pellet with the cholecystcholangiocarcinoma primary cell culture medium after centrifugation, counting, and then suspension culturing the cells using a culture vessel with a low adsorption surface (initial seeding density may be 10)⁵Per cm²Bottom area of the container, e.g. six-well plate, 10 per well⁶Density plating of individual cells), culture conditions were 37 ℃ and 5％CO₂. All the centrifugation in the above-mentioned passaging step may be specifically 800-1000g (e.g., 800g) at room temperature for 10-20 minutes (e.g., 10 minutes).

Further, the method can also comprise the step of performing cryopreservation and/or resuscitation on the primary gallbladder cholangiocarcinoma cells after passage expansion for 2-3 times.

Wherein the cell freezing solution adopted during freezing is composed of Advanced DMEM/F12 culture medium, DMSO and 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM/F12 culture medium to the DMSO to the 1% methylcellulose solution is 20:2 (0.8-1.2), such as 20:2: 1; the 1% methylcellulose solution is an aqueous solution of methylcellulose having a concentration of 1g/100 ml.

In a specific embodiment of the invention, the Advanced DMEM/F12 medium is under the brand code Gibco # 12634010; the brand code of the DMSO is Sigma # D2438; the brand of methylcellulose is Sigma # M7027.

Further, the specific steps of the cryopreservation are as follows: collecting cell mass to be frozen, centrifuging, washing the cell mass with sterile PBS solution, centrifuging, suspending the cell mass with the cell digestive juice, digesting at 37 deg.C until the cell mass is digested into single cell, terminating the digestion reaction with the digestion terminating solution (the dosage can be 5-10 times, such as 10 times of volume), and collecting cell suspension; centrifuging, and freezing the cells at 0.5-2 × 10⁶/mL (e.g., 10)⁶mL), and transferring the cell sediment to liquid nitrogen for long-term storage after the cell sediment is frozen and stored overnight by a gradient cooling box. All the centrifugation in the above freezing step may be specifically 800-1000g (e.g., 800g) at room temperature for 10-20 minutes (e.g., 10 minutes).

Further, the specific steps of performing the resuscitation are: taking out the freezing tube containing the cells to be rescued from the liquid nitrogen, and rapidly thawing the cells in sterile water at 37-39 deg.C (such as 37 deg.C); suspending the cell pellet with the primary cell culture medium of cholecystcholangiocarcinoma after centrifugation (e.g. 800-Can be 10⁵Per cm²Bottom area of container), cells per tube (10)⁶Respectively) reviving to 3.5cm culture dish), culturing at 37 deg.C and 5% CO₂。

In a second aspect, the invention claims a kit for culturing primary cells of cholecystochhol carcinoma.

The kit for culturing primary gallbladder cholangiocarcinoma cells provided by the invention specifically comprises the primary gallbladder cholangiocarcinoma cell culture medium and at least one of the following reagents: the sample dissociation solution, the sample preservation solution, the cell separation buffer solution, the cell digestion solution, the sample washing solution, the digestion stop solution, the cell cryopreservation solution and the 1% CYTOP solution described above.

The sample preservation solution can be used for temporarily preserving a sample after the sample is separated, and can maintain the activity of cells in the sample in a short time after the sample is separated. The sample preservation solution can be preserved for 1 month at 4 ℃ after being prepared.

The sample washing solution can be used for washing and disinfecting a sample. The sample cleaning solution needs to be ready for use.

The sample dissociation liquid can be used for dissociation of a sample, and primary cells of cholecystcholangiocarcinoma solid tumors in the sample can be dissociated from tissues. The sample dissociation solution is prepared, wherein collagenase I, collagenase II and collagenase IV can be stored in a stock solution (mother solution) at-20 ℃ for a long time, and specifically 10 or 20 times of the stock solution (mother solution). The 10 × collagenase I stock consists of the collagenase I and PBS; wherein the final concentration of collagenase I is 2000U/mL; a 10 × collagenase II stock solution consists of the collagenase II and PBS; wherein the final concentration of collagenase II is 2000U/mL; the balance being PBS; 20 × collagenase IV stock consists of the collagenase IV and PBS; wherein the final concentration of collagenase IV is 2000U/mL; the balance being PBS. The enzyme activities of collagenase I, collagenase II and collagenase IV are defined above.

The cell separation buffer is used for suspending cells in a bile sample. After the preparation of the cell separation buffer solution is finished, the cell separation buffer solution can be stored at 4 ℃ for 1 month.

The cell digestive juice can be used for digesting and passaging cell masses and can digest tumor masses of gallbladder, bile duct cancer into single cells. The cell digestive juice is required to be prepared immediately.

The digestion stop solution can be used for stopping the dissociation of the sample or the digestion process of the cells. The prepared digestion stop solution can be stored for one month at 4 ℃.

The primary gallbladder cholangiocarcinoma cell culture medium can be used for culturing primary gallbladder cholangiocarcinoma cells. After the primary cell culture medium for gallbladder cholangiocarcinoma, the primary cell culture medium needs to be filtered and sterilized by a 0.22 mu M needle filter (Millipore SLGP033RS), and can be stored for two weeks at 4 ℃. 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 and the human recombinant protein Noggin can be stored in a stock solution (mother solution) form at-80 ℃ for a long time, and particularly can be stored in a stock solution (mother solution) of 1000 times. SB202190, N-acetyl-L-cysteine, Nicotinamide, cortisol, gastrin and Y-27632 can be stored in stock solution (mother solution) form at-20 deg.C for a long period, specifically 1000 times of stock solution (mother solution). A83-01 can be stored in stock solution (mother liquor) at-20 deg.C for a long period, specifically 100000 times of stock solution (mother liquor). The 1000 Xhuman recombinant protein EGF stock solution consists of human recombinant protein EGF, BSA and PBS, wherein the final concentration of the human recombinant protein EGF is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS. The stock solution of 1000 Xhuman recombinant protein bFGF consists of human recombinant protein bFGF, BSA and PBS, wherein the final concentration of the human recombinant protein bFGF is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS. The 1000 Xhuman recombinant protein HGF stock solution consists of human recombinant proteins HGF, BSA and PBS, wherein the final concentration of the human recombinant proteins HGF is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS. The 1000 Xhuman recombinant protein FGF-10 stock solution consists of human recombinant protein FGF-10, BSA and PBS, wherein the final concentration of the human recombinant protein FGF-10 is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS. The 1000 Xhuman recombinant protein Wnt-3a stock solution 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 multiplied human 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 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS. In the five 1000-fold stock solutions, the BSA can be present (ready for formulation) in the form of 100-fold stock solution (mother liquor), and specifically consists of BSA and PBS, wherein the final concentration of BSA (Sigma # A1933) is 0.1g/mL, and the balance is PBS. Additionally, the 1000 × SB202190 stock consisted of SB202190 and DMSO, with the final concentration of SB202190 being 10mM, the balance being DMSO. The 100000 XA 83-01 stock solution 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. The 1000 Xcortisol stock solution consists of cortisol, absolute ethyl alcohol and ultrapure water, wherein the final concentration of the cortisol is 25 mu g/mL, the final concentration of the absolute ethyl alcohol is 5% (volume percentage content), and the balance is the ultrapure water. 1000 x gastrin stock solution consists of gastrin and ultrapure water, wherein the concentration of the gastrin is 10 mu M, and the balance is the ultrapure water. The 1000 XY-27632 stock solution consists of Y-27632 and ultrapure water, wherein the final concentration of Y-27632 is 10mM, and the balance is ultrapure water.

The cell freezing medium needs to be prepared at present. Wherein the 1% methylcellulose solution can be stored for a long period of time at 4 ℃.

In a third aspect, the invention claims the use of the kit as described hereinbefore for culturing primary cells of cholecystochhol carcinoma.

In the above aspects, the gallbladder bile duct cancer may be primary gallbladder cancer or bile duct cancer. The pathological type is gallbladder bile duct cancer or gallbladder bile duct cancer metastasis focus. The clinical stages are stage II, stage III or stage IV (according to TNM).

In the aspects, the primary gallbladder cholangiocarcinoma cell may be a primary gallbladder cholangiocarcinoma solid tumor cell or a primary gallbladder cholangiocarcinoma bile sample tumor cell.

In the aspects, the primary gallbladder cholangiocarcinoma cells may be isolated from a surgical sample (being a solid tumor sample), a puncture sample (being a solid tumor sample) or a bile sample of a cholecystic cholangiocarcinoma patient. Wherein, the weight of the solid tumor tissue specimen of the gallbladder bile duct cancer obtained from the operation sample is preferably more than 20 mg. The bile sample is preferably not less than 10 mL. Not less than 4 puncture samples (solid tumor samples).

In the present invention, all of the above PBS's may be 1 XPBS, pH 7.3-7.5. The concrete composition is as follows: the solvent is water, and the solute and the concentration are as follows: KH (Perkin Elmer)₂PO₄ 144mg/L，NaCl 9000mg/L，Na₂HPO₄·7H₂O 795mg/L。

The invention provides a method for extracting and culturing primary tumor cells of gallbladder cancer and bile duct cancer from fresh gallbladder cancer and solid tumor operation samples or biopsy puncture tissue samples or gallbladder cancer and bile duct cancer samples and a matched reagent, and the method has the following advantages:

1. the dosage of the tissue sample is less, and only about 20mg of operation sample or about 10-20mL of bile sample is needed;

2. the method can be used for culturing primary tumor cells of primary tumors of gallbladder cancer and bile duct cancer, and can also be used for culturing primary tumor cells of metastatic lesions of gallbladder cancer and bile duct cancer;

3. the culture period is short, and only 3-10 days are needed to obtain 10⁶-10⁷An order of magnitude of primary tumor cells;

4. the culture stability is high, and the success rate of in vitro culture of qualified gallbladder cancer, bile duct cancer operation specimens or biopsy puncture specimens by using the method is up to 70 percent;

5. the purity of the cells is high, the ratio of the cancer cells in the primary cell culture of the gallbladder cancer and the bile duct cancer obtained by the method can reach 60-95 percent, and the interference of the mixed cells is less.

The primary cell culture of the gallbladder cancer and the bile duct cancer obtained by the method can be used for in vitro experiments, next generation sequencing, animal model construction, cell line construction and the like of various cell levels. The culture method can be expected to have wide application prospect in the research and clinical diagnosis and treatment fields of gallbladder cancer and bile duct cancer.

Drawings

FIG. 1 shows a single cell obtained after treatment of cholangiocarcinoma tissue. The scale is 100 μm, 100 times magnification.

FIG. 2 shows the cell masses obtained after primary culture of cholangiocarcinoma tissues. The scale is 100 μm, 100 times magnification.

FIG. 3 is a staining chart of bile duct cancer cell mass section HE obtained after primary culture of bile duct cancer tissue. The scale is 100 μm, 200 times magnification.

FIG. 4 is an immunohistochemical staining chart of a paraffin section of a cancer cell mass obtained after primary culture of cholangiocarcinoma tissue. The scale is 100 μm, 200 times magnification.

FIG. 5 is a diagram of a microplate chip design of the invention.

FIG. 6 shows single cells obtained after bile sample of bile duct cancer is processed. The scale is 100 μm, 100 times magnification.

FIG. 7 shows cell masses obtained after primary culture of bile samples of bile duct cancer. The scale is 100 μm, 100 times magnification.

FIG. 8 is a staining chart of bile duct cancer cell mass section HE obtained after primary culture of bile duct cancer bile samples. The scale is 100 μm, 100 times magnification.

FIG. 9 is an immunohistochemical staining chart of a paraffin section of a cancer cell mass obtained after primary culture of bile samples of cholangiocarcinoma. The scale is 50 μm, 100 times magnification.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of reagents for culturing Primary cells of gallbladder carcinoma and solid tumor of bile duct carcinoma

1. Sample preservation solution (100mL)

The specific formulation of the specimen preservation solution (100mL) is shown in table 1.

TABLE 1 sample preservation solution (100mL)

After the preparation of the sample preservation solution is completed, the sample preservation solution is subpackaged by 15mL centrifuge tubes, and each tube is 5 mL. Can be stored at 4 deg.C for 1 month after subpackaging.

2. Sample cleaning solution (100mL)

The specific formulation of the sample rinse (100mL) is shown in table 2.

TABLE 2 sample rinse (100mL)

The sample cleaning solution needs to be prepared for use.

3. Sample dissociation liquid (10mL)

The specific formulation of the sample dissociation solution (10mL) is shown in table 3.

TABLE 3 sample dissociation solution (10mL)

Note: the sample dissociation liquid is prepared for use.

In Table 3, the formulation of collagenase stocks is shown in tables 4-6.

TABLE 410 collagenase I stock solution (100mL)

After preparing the 10 Xcollagenase I stock solution, the solution was dispensed into 1.5mL sterile centrifuge tubes, 1mL each. The stock solution can be stored at-20 deg.C for a long period.

TABLE 510 collagenase II stock solution (100mL)

After preparing the 10 Xcollagenase II stock solution, the solution was dispensed into 1.5mL sterile centrifuge tubes, 1mL each. The stock solution can be stored at-20 deg.C for a long period.

TABLE 620 collagenase IV stock solution (100mL)

After preparing 20 Xcollagenase IV stock solution, the solution was dispensed into 1.5mL sterile centrifuge tubes, 1mL each. The stock solution can be stored at-20 deg.C for a long period.

In tables 4, 5 and 6, the unit U of collagenase (said collagenase I or said collagenase IV) is defined by the enzymatic activity of the protease: 1 μmol of L-leucine can be released by treating collagenase (said collagenase I or said collagenase IV) with 1U of protease at 37 ℃ and pH 7.5 for 5 hours.

4. Cell digestive juice (10mL)

The specific formulation of the cell digest (10mL) is shown in Table 7.

TABLE 7 cell digest (10mL)

The cell digestive juice is prepared for use.

5. Digestive stop solution (100mL)

The specific formulation of the digestion-stopping solution (100mL) is shown in Table 8.

TABLE 8 digestive stop solution (100mL)

The digestion stop solution can be stored for one month at 4 ℃ after being prepared.

6. Primary cell culture medium (100mL) for gallbladder cancer and bile duct cancer solid tumors

The specific formula of the primary cell culture medium (100mL) for gallbladder cancer and cholangiocarcinoma solid tumors is shown in Table 9.

TABLE 9 gallbladder carcinoma, cholangiocarcinoma solid tumor Primary cell culture Medium (100mL)

After the preparation of the primary cell culture medium for gallbladder cancer and cholangiocarcinoma solid tumors, the cells were sterilized by filtration using a 0.22 μ M syringe filter (Millipore SLGP033RS) and stored at 4 ℃ for two weeks.

In Table 9, the formulation of human recombinant protein stocks is shown in tables 11 to 16, the formulation of SB202190 stock is shown in Table 17, the formulation of A83-01 stock is shown in Table 18, the formulation of N-acetyl-L-cysteine stock is shown in Table 19, the formulation of Nicotinamide stock is shown in Table 20, the formulation of cortisol stock is shown in Table 21, the formulation of gastrin stock is shown in Table 22, and the formulation of Y-27632 stock is shown in Table 23. The 100 × BSA solutions required to formulate these stock solutions are shown in table 10.

TABLE 10100 XBSA solution (1mL)

The 100 × BSA solution is ready for use.

TABLE 111000 × stock solution of human recombinant protein EGF (5mL)

After 1000 Xhuman recombinant protein EGF stock solution is prepared, the stock solution is subpackaged by a sterile centrifuge tube with 1.5mL, and the stock solution can be preserved at the temperature of minus 80 ℃ for a long time.

TABLE 121000 × stock solution of human recombinant protein bFGF (25mL)

After 1000 Xhuman recombinant protein bFGF stock solution is prepared, the stock solution is subpackaged by a sterile centrifuge tube with the volume of 1.5mL, and the stock solution can be preserved at the temperature of minus 80 ℃ for a long time.

TABLE 131000 Xhuman recombinant protein HGF stock solution (5mL)

1000 Xthe human recombinant protein HGF stock solution is prepared and subpackaged by a sterile centrifuge tube of 1.5mL, and the stock solution can be preserved for a long time at the temperature of minus 80 ℃.

TABLE 141000 × stock solution of human recombinant protein FGF-10 (5mL)

After 1000 Xhuman recombinant protein FGF-10 stock solution is prepared, the stock solution is subpackaged by a sterile centrifuge tube with the volume of 1.5mL, and the stock solution can be preserved at the temperature of minus 80 ℃ for a long time.

TABLE 151000 Xhuman recombinant protein Wnt-3a stock solution (2.5mL)

1000 Xthe human recombinant protein Wnt-3a stock solution is prepared and then subpackaged by a sterile centrifuge tube with 1.5mL, and the stock solution can be preserved for a long time at the temperature of minus 80 ℃.

TABLE 161000 × stock solution of human recombinant protein Noggin (5mL)

1000 times of human recombinant protein Noggin stock solution is prepared and then subpackaged by a 1.5mL sterile centrifuge tube, and the stock solution can be stored for a long time at the temperature of minus 80 ℃.

TABLE 171000 XSB 202190 stock solution (1.51mL)

After preparing the stock solution of 1000 XSB 202190, the stock solution can be stored for a long time at-20 ℃ by subpackaging with a 0.5mL sterile centrifuge tube.

TABLE 18100000 XA 83-01 stock solution (1.05mL)

After preparing a stock solution of 1000 XA 83-01, the stock solution can be stored for a long time at-20 ℃ by dispensing with a 0.5mL sterile centrifuge tube.

TABLE 191000 XN-acetyl-L-cysteine stock solutions (5mL)

After preparing a stock solution of 1000 XN-acetyl-L-cysteine, subpackaging the stock solution by using a sterile centrifuge tube with the volume of 0.5mL, and storing the stock solution at the temperature of 20 ℃ below zero for a long time.

TABLE 201000 XNicotinamide stock solutions (4mL)

1000 XNicotinamide stock solution is prepared and then subpackaged by a sterile centrifuge tube of 0.5mL, and the stock solution can be stored for a long time at the temperature of minus 20 ℃.

TABLE 211000 cortisol stock solution (100mL)

1000 Xcortisol stock solution is prepared and then subpackaged with 1.5mL sterile centrifuge tubes, and the stock solution can be stored at-20 ℃ for a long time.

TABLE 221000 × Gastrin stock solution (48mL)

1000 Xgastrin stock solution is prepared and then subpackaged by a sterile centrifuge tube of 0.5mL, and the stock solution can be preserved for a long time at the temperature of minus 20 ℃.

TABLE 231000 XYY-27632 stock solution (3.125mL)

After preparing the stock solution of 1000 XY-27632, the stock solution is subpackaged by a sterile centrifuge tube of 0.5mL and can be stored for a long time at the temperature of minus 20 ℃.

7. Cell cryopreservation liquid

The specific formulation of the cell culture medium is shown in Table 24.

TABLE 24 cell cryopreservation solution

The cell frozen stock solution is prepared for use at present.

In table 24, the preparation of the 1% methylcellulose solution is shown in table 25.

TABLE 251% methylcellulose solution (10mL)

The 1% methyl cellulose solution can be stored for a long time at 4 ℃ after being prepared.

8. 1% CYTOP solution

TABLE 261% CYTOP solution (100mL)

After the 1% CYTOP solution is prepared, the product can be stored for a long time at normal temperature.

Example 2 acquisition of postoperative/biopsy puncture specimen for gallbladder cancer and cholangiocarcinoma

1. In cooperation with the Hospital, the cooperative development passed a formal medical ethical examination.

2. The attending physician selects patients for inclusion in the cohort according to clinical indications prescribed by medical guidelines and selects appropriate samples for in vitro culture based on the intraoperative clinical indication. The selection criteria of the surgical specimen are: primary gallbladder cancer or bile duct cancer, pathological stages are II stage, III stage or IV stage, various pathological typing gallbladder cancer or bile duct cancer metastasis focuses, and samples with the weight of operation specimens exceeding 20 mg. Selection criteria of biopsy puncture samples are as follows: primary gallbladder cancer or bile duct cancer, pathological stages are II stage, III stage or IV stage, various pathological typing gallbladder cancer or bile duct cancer metastasis focuses, and more than 4 samples of puncture specimens are obtained.

3. The primary physician provides basic clinical information such as sex, age, medical history, family history, smoking history, pathological staging, clinical diagnosis, etc. of the patient. The name, the identification card number and other information of the patient related to the privacy of the patient are hidden and replaced by a uniform experiment number, and the naming principle of the experiment number is eight-digit numerical date of the collected sample plus four digits after the patient is hospitalized. For example, if the sample is provided on 1/2018, the hospitalization number of the patient is T001512765, and the sample experiment number is 201801012765.

4. During surgery, a surgeon collects fresh surgical specimens in a sterile operating room environment and places the specimens in a prepared specimen preservation solution (see example 1). The samples were kept temporarily on ice after being isolated and transported to the laboratory within two hours for further processing.

5. The puncture surgeon collects a fresh puncture specimen in a sterile environment of a puncture operating room and places the specimen in a specimen preservation solution (see example 1) prepared in advance. The samples were kept temporarily on ice after being isolated and transported to the laboratory within two hours for further processing.

Example 3 pretreatment for dissociation of gallbladder cancer and cholangiocarcinoma samples

The following operations required working on ice and the entire procedure required completion within 10 minutes.

The surgical instruments used in the following operations all need to be sterilized in advance at high temperature and high pressure and can be used after being dried.

1. The samples were weighed.

2. The sample surface was rinsed with 75% (volume percent) ethanol for 10 to 30 seconds.

3. The samples were washed 10 times with sample wash and 5 times with sterile PBS solution.

4. The fat tissue, connective tissue and necrotic tissue in the sample are carefully stripped off with the aid of an ophthalmic scissors, an ophthalmic forceps, a scalpel and the like.

Example 4 gallbladder cancer, biliary duct cancer tissue sample dissociation

The surgical instruments used in the following examples were sterilized at high temperature and high pressure in advance and dried before use.

1. Cutting the tissue into pieces of 1mm by using an ophthalmic scissors³The left and right small blocks.

2. The minced tissue samples were treated with a sample dissociation solution preheated at 37 ℃ in advance at a dose of 0.1mL of the sample dissociation solution (see example 1) per mg of tissue, and dissociation was carried out at 37 ℃ for 15 minutes to 3 hours. The dissociation of the samples was observed under the microscope every 15 minutes until a large number of single cells were observed.

3. The dissociation reaction was stopped with 10 volumes of a digestion stop solution (see example 1) and the cell suspension was collected.

4. The cell suspension was filtered through a 40 μm sterile cell strainer to remove tissue debris and adherent cells.

5. 800g were centrifuged at room temperature for 10 minutes and the supernatant discarded.

6. The cells were resuspended in 5mL sterile PBS, centrifuged at 800g for 10 minutes at room temperature, and the supernatant discarded.

7. Resuspend the cell pellet with gallbladder cancer, cholangiocarcinoma solid tumor primary cell culture medium (see example 1), observe the cell state under microscope, and count the cells.

As shown in FIG. 1, the dissociated single cell suspension contains a large amount of various types of cells, such as erythrocytes, lymphocytes, and fibroblasts, in addition to tumor cells. One of the advantages of the method is that in the subsequent culture process, only cancer cells can be greatly amplified, and the proportion of other cells is gradually reduced or even disappears, so that the primary tumor cells of gallbladder cancer and cholangiocarcinoma with higher purity are finally obtained.

Example 5 Primary cell culture for gallbladder cancer and cholangiocarcinoma

1. The suspension culture of the primary solid tumor cells of the gallbladder cancer and the bile duct cancer is carried out by using a low-adsorption surface (low-adsorption surface), namely, the culture medium of the primary solid tumor cells of the gallbladder cancer and the bile duct cancer in example 1 (wherein, the final concentration of human recombinant protein EGF is 50ng/mL, the final concentration of human recombinant protein bFGF is 20ng/mL, the final concentration of human recombinant protein HGF is 20ng/mL, the final concentration of human recombinant protein FGF-10 is 20ng/mL, the final concentration of human recombinant protein Wnt-3a is 250ng/mL, the final concentration of human recombinant protein Noggin is 100ng/mL, the final concentration of SB202190 is 10 muM, the final concentration of A83-01 is 0.5 muM, the final concentration of N-acetyl-L-cysteine is 1mM, the final concentration of Nicotinamide is 10mM, the concentration of cortisol is 25ng/mL, the final concentration of Y-27632 muM), taking a six-hole plate as an example, according to 10 holes⁶Individual cells were plated at 37 ℃ in density with 5% CO₂The culture was carried out in a cell culture incubator under the conditions.

2. The cell status was observed every day, and the medium was changed every 3 days until the cells formed clumps of about 80 μm in diameter.

As shown in FIG. 2, after 3-10 days of culture, cancer cells are greatly expanded to form cell masses with the diameter of 80 μm, and the total number of tumor cells can exceed 10⁷The number of other types of cells is significantly reduced or even eliminated. Through a large number of sample tests, the success rate of the in vitro culture of primary tumor cells of the gallbladder cancer and the bile duct cancer can reach 80 percent.

Example 6 Primary cell passage of gallbladder carcinoma, cholangiocarcinoma solid tumors

1. The cell pellet was collected from the dish, centrifuged at 800g at room temperature for 10 minutes, and the supernatant was discarded.

2. The cell pellet was washed with sterile PBS solution, centrifuged at 800g at room temperature for 10 minutes, and the supernatant was discarded.

3. The cell pellet was resuspended in cell digest (see example 1) and digested at 37 ℃. The digestion of the cell pellet was observed under a microscope every 5 minutes until the cell pellet was digested into single cells.

4. The dissociation reaction was stopped with 10 volumes of a digestion stop solution (see example 1) and the cell suspension was collected.

6. Resuspending the cell pellet with primary cell culture medium for gallbladder cancer and bile duct cancer, and counting the cells.

7. Using a low-adsorption surface (low-adsorption-surface) to culture primary gallbladder cancer and cholangiocarcinoma cells, wherein the culture medium is the culture medium for the primary gallbladder cancer and cholangiocarcinoma solid tumors in example 1, and taking a six-well plate as an example, 10 cells are cultured in each well⁶Individual cells were plated at 37 ℃ in density with 5% CO₂The culture was carried out in a cell culture incubator under the conditions.

Example 7 cryopreservation of solid tumor Primary cells from gallbladder cancer and cholangiocarcinoma

After carrying out passage amplification for 2-3 times on the primary cells of the gallbladder cancer and the bile duct cancer solid tumors cultured in a suspension manner, freezing and storing can be carried out:

3. The cell pellet was resuspended in cell digest (see example 1) and digested at 37 ℃. The digestion of the cell pellet was observed under a microscope every 15 minutes until the cell pellet was digested into single cells.

4. The dissociation reaction was stopped with 10 volumes of digestion stop solution (see example 1), and the cell suspension was collected and counted.

6. Cell cryopreservation (see example 1) at 10⁶Resuspending the cell sediment at a density of/mL, freezing 1mL of cell suspension in each tube of a 2mL freezing tube, freezing overnight by using a gradient cooling box, and transferring the cell sediment into liquid nitrogen for long-term storage.

Example 8 Resuscitation of solid tumor Primary cells of gallbladder cancer and cholangiocarcinoma

The primary gallbladder cancer and cholangiocarcinoma solid tumor cells stored in liquid nitrogen can be recovered:

1. sterile water at 37 ℃ was prepared five minutes in advance.

2. The vial was removed from the liquid nitrogen and the cells were rapidly thawed in sterile water at 37 ℃.

3. 800g were centrifuged at room temperature for 10 minutes and the supernatant discarded.

4. Resuspending the cell pellet with gallbladder cancer and cholangiocarcinoma solid tumor primary cell culture medium (see example 1), culturing the gallbladder cancer and cholangiocarcinoma solid tumor primary cells with low adsorption surface, resuscitating each tube of cells into a 3.5cm culture dish at 37 deg.C and 5% CO₂The culture was carried out in a cell culture incubator under the conditions.

Example 9 HE staining identification of Primary cells of gallbladder carcinoma, cholangiocarcinoma solid tumors

The reagent consumables used in the following examples are illustrated:

HE staining kit (beijing solibao biotechnology limited, # G1120);

cation anticreep slide (Beijing China fir Jinqiao Biotech limited);

xylene, methanol, acetone (Beijing chemical reagent company, analytical pure);

neutral resin adhesive (fine chemicals, GmbH, Beijing).

1. 800g of gallbladder cancer and cholangiocarcinoma solid tumor primary cell mass cultured by the primary cell culture medium for gallbladder cancer and cholangiocarcinoma solid tumor in example 1 (wherein, the final concentration of human recombinant protein EGF is 20ng/mL, the final concentration of human recombinant protein bFGF is 20ng/mL, the final concentration of human recombinant protein HGF is 20ng/mL, the final concentration of human recombinant protein FGF-10 is 20ng/mL, the final concentration of human recombinant protein Wnt-3a is 200ng/mL, the final concentration of human recombinant protein Noggin is 100ng/mL, the final concentration of SB202190 is 5 muM, the final concentration of A83-01 is 1 muM, the final concentration of N-acetyl-L-cysteine is 1mM, the final concentration of Nicotinamide is 10mM, the final concentration of cortisol is 25ng/mL, the final concentration of Y-27632 is 10 muM), fix with 4% paraformaldehyde. The pellet of cells was embedded in paraffin and sliced to a thickness of 5 μm.

2. Paraffin sections were incubated in xylene solution for 5 minutes at room temperature for deparaffinization, repeated 3 times, and the sections were rinsed 2 times with deionized water.

3. Sections were incubated in absolute ethanol for 10min at room temperature, repeated twice.

4. Ginger slices were incubated in 95% ethanol for 10 minutes at room temperature, and after repeating twice, the slices were rinsed twice with deionized water.

5. When the water on the slide is slightly dry, 100 mu L of hematoxylin staining solution is added for staining for 1 mins.

6. The hematoxylin stain was aspirated and the slides were washed 3 times with tap water.

7. 100 mu L of differentiation solution is added dropwise for differentiation for 1 mins.

8. The differentiation medium was aspirated off, and the slides were washed sequentially 2 times with tap water and 1 time with distilled water.

9. The water on the surface of the slide is sucked off, and 200 mu L of eosin dye solution is dripped to stain the slide for 40 s.

10. Absorbing eosin dye solution, rinsing and dehydrating with 75%, 80%, 90% and 100% ethanol for 20s, 40s and 40 s.

11. After the ethanol was dried, 50. mu.L of xylene was added dropwise for cell permeation.

12. After xylene is completely dried, a drop of neutral resin adhesive is added dropwise, and the piece is mounted by a cover glass, observed under a microscope and photographed.

Fig. 3 shows the HE staining effect of bile duct cancer primary tumor cells obtained by in vitro culture, and it can be seen that these cells generally have the characteristics of cancer cells such as high nuclear-mass ratio, deep nuclear staining, chromatin condensation in nuclei, multinucleate, and uneven cell size, and dozens to hundreds of tumor cells aggregate to form tumor cell masses with certain three-dimensional structures.

Example 10 immunohistochemical staining identification of Primary cells of gallbladder carcinoma, cholangiocarcinoma solid tumors

The reagents used in the following examples are illustrative:

paraformaldehyde (Beijing chemical reagent company, analytical pure) was dissolved in ultrapure water to prepare a 4% (4g/100mL) paraformaldehyde solution;

hydrogen peroxide (beijing chemicals, 35%);

blocking with normal goat serum (Solarbio, SL 038);

immunohistochemical primary anti-antibodies (Abcam, ab 215838);

immunohistochemical secondary antibodies (Abcam, ab 205719);

EDTA repair solution (Abcam, ab 93684);

DAB color-developing liquid (

DAB Substrate Kit，8059S)

The gallbladder cancer and bile duct cancer solid tumor cell mass obtained by culturing the gallbladder cancer and bile duct cancer solid tumor primary cell culture medium (wherein, the final concentration of human recombinant protein EGF is 50ng/mL, the final concentration of human recombinant protein bFGF is 25ng/mL, the final concentration of human recombinant protein HGF is 25ng/mL, the final concentration of human recombinant protein FGF-10 is 25ng/mL, the final concentration of human recombinant protein Wnt-3a is 300ng/mL, the final concentration of human recombinant protein Noggin is 200ng/mL, the final concentration of SB202190 is 10 muM, the final concentration of A83-01 is 0.5 muM, the final concentration of N-acetyl-L-cysteine is 1mM, the final concentration of Nicotinamide is 10mM, the final concentration of cortisol is 25ng/mL, the final concentration of Y-27632 is 10 muM) in example 1 is collected and paraffin sections are cut, and pan-CK antibody is used according to the following steps, cells of epithelial origin were characterized.

1. The slices were sequentially immersed in xylene I for 10min and xylene II (10 min).

2. Soaking in anhydrous ethanol I (5min) -anhydrous ethanol II (5min) -95% ethanol (5min) -80% ethanol (5min) -70% ethanol (5min), and washing with deionized water for 2 times, each for 2 min.

3. The tissue slices were placed in a repair box, and then a suitable amount of diluted EDTA repair solution (pH 9.0) was added, the surface of the solution being submerged in the tissue.

4. Microwave medium-grade repair for 10min (time is started when liquid boils), during which time no tissue dry-slices are allowed.

5. The repair box is taken out of the microwave oven, naturally cooled and cooled, when the repair liquid is cooled to room temperature, the slide is taken out, and washed with PBS (pH7.4) for 3 times and 3min each time (the tissue is not washed against the tissue during the washing process so as to avoid breaking the tissue).

6. Prepared 3% hydrogen peroxide (30% hydrogen peroxide diluted with deionized water) was added dropwise to the sliced tissue to block endogenous peroxidase, incubated at room temperature for 15min, and washed 3 times with PBS, 3min each.

7. The PBS was blotted on absorbent paper, 10% goat serum (from the same or similar source as the secondary antibody species) was added dropwise to the slide, and the slide was blocked at 37 ℃ for 60 min.

8. The liquid surrounding the slide tissue was wiped dry with absorbent paper, a circle was drawn around the tissue with an oil pen, then diluted primary antibody was added dropwise and incubated overnight in a wet box at 4 ℃.

And 9, washing the slices with PBS for 3 times, each time for 3min, wiping the slices with absorbent paper, dripping horseradish peroxidase-labeled secondary antibody, and incubating at room temperature for 60 min.

And (10) washing the slices with PBS for 3 times, 3min each time, throwing away PBS liquid, wiping the slices with absorbent paper, dripping a freshly prepared DAB color developing solution into each slice, observing under a microscope, and washing the slices with tap water after positive signals to stop color development.

11. And (3) performing hematoxylin counterstaining for 1min, washing with water, then differentiating with an acidic ethanol differentiation solution, and washing with tap water to turn blue.

12. Placing the slices into water for washing, and then sequentially placing the slices into: dehydrating 70% ethanol-80% ethanol-90% ethanol-95% ethanol-absolute ethanol I-absolute ethanol II-xylene I-xylene II, standing each reagent for 2min, and air drying in a fume hood.

13. The slides were mounted using neutral gum and covered with a coverslip. Placing in a fume hood for air drying.

14. The dried sections can be viewed under a microscope or photographed.

FIG. 4 shows the effect of immunohistochemical staining of bile duct cancer primary tumor cell mass cultured in vitro, and it can be seen that the cells constituting the cell mass are all pan-CK positive and are of epithelial origin, confirming that the tumor cells cultured by the method are of higher purity. Immunohistochemical staining identification is carried out on the primary cultures of the 5 gallbladder cancer and bile duct cancer samples, and statistical results show that the proportion of tumor cells in the primary cells of the gallbladder cancer and the bile duct cancer obtained by the method reaches 84-95 percent (Table 27).

Table 27 gallbladder cancer, bile duct cancer primary culture immunohistochemical staining identification

Example 11 cultivation of Primary tumor cells of gallbladder carcinoma, solid tumor of biliary duct carcinoma Using CYTOP-modified cell culture consumables

In this example, the procedures of all primary cultures were identical (see the above description), the CYTOP modification method was identical, and only the materials of the cell culture consumables were different (table 28).

The CYTOP modification method comprises the following steps: firstly, pure oxygen etching is carried out on the cell culture container, the etching condition is 20W, and the etching time is 3 minutes. Then, the surface of the culture dish or the culture plate is covered with an appropriate amount of 1% CYTOP solution (taking a 96-well plate as an example, 20 mu L of each well, and the appropriate amount refers to the condition of completely covering the bottom of the culture dish), and the CYTOP solution can be used after being completely dried.

TABLE 28 Effect of CYTOP-modified consumables on gallbladder carcinoma, cholangiocarcinoma solid tumor primary tumor cell culture

Note: polystyrene (Polystyrene, abbreviated PS).

As can be seen from table 28: it can be seen that the success rate of sample culture can be greatly improved after CYTOP modification.

Example 12 microplate chip processing

In this embodiment, a method of injection molding is used, and PMMA material (or PS, PC, COC, COP, LAS, etc.) is used to process the microplate chip for culturing primary cells of gallbladder cancer and cholangiocarcinoma solid tumors of the present invention. The chip can be used for primary gallbladder cancer and cholangiocarcinoma cell culture and in-vitro drug sensitivity detection experiments. The microplate chip design is shown in FIG. 5.

In the practical application process, the PMMA material (or PS, PC, COC, COP, LAS and other materials) is used to prepare the structure of the microplate chip shown in the design drawing of FIG. 5, and then the surface of the microplate chip is subjected to CYTOP modification by the CYTOP modification method (see example 11), so that the microplate chip which can be used for culturing primary cells of gallbladder cancer and cholangiocarcinoma is obtained.

Example 13 preparation of reagents for culturing Primary cells of bile samples for gallbladder cancer, bile duct cancer

1. Cell separation buffer (100mL)

The specific formulation of cell isolation buffer (100mL) is shown in Table 29:

TABLE 29 cell isolation buffer (100mL)

After the preparation of the cell separation buffer, the cells can be stored at 4 ℃ for 1 month.

In table 29, the preparation of the heparin sodium solution is shown in table 30.

TABLE 301000 Xheparin sodium (1mL)

1000 Xheparin sodium solution is prepared for use.

2. Cell digestive juice (10mL)

The specific formulation (Table 7) and formulation method of the cell digest (10mL) are shown in example 1, step 4.

3. Digestive stop solution (100mL)

The specific formulation (Table 8) and formulation method of the digestion-stopping solution (100mL) are shown in step 5 of example 1.

4. Primary cell culture medium (100mL) of bile sample of gallbladder cancer and bile duct cancer

Specific formula (table 9) and preparation method of primary cell culture medium (100mL) of gallbladder cancer and bile duct cancer bile samples are shown in step 6 of example 1.

5. Cell cryopreservation liquid

The specific formulation of the cell culture medium (Table 24) and the preparation method are shown in step 7 of example 1.

EXAMPLE 14 acquisition of bile samples for gallbladder cancer and bile duct cancer

2. The attending physician selects patients for inclusion in the cohort according to clinical indications prescribed by medical guidelines and selects appropriate samples for in vitro culture based on the intraoperative clinical indication. The selection criteria of the surgical specimen are: primary gallbladder cancer or bile duct cancer, the pathological stage is stage II, stage III or stage IV, and the volume of the bile sample is more than 20 mL.

4. The doctor is in charge of the patient, and the sterile equipment is used for collecting more than 10mL of fresh bile specimen. The samples were kept temporarily on ice after being removed from the body and transported to the laboratory for further processing within 48 hours.

Example 15 pretreatment of bile samples for gallbladder cancer and bile duct cancer

1. Standing the bile sample on ice for about 30 minutes to allow the coagulated blood clots and large insoluble solids in the sample to settle to the bottom of the sample tube;

2. carefully transferring the supernatant into a 50mL sterile centrifuge tube, adding one volume of precooled PBS and mixing uniformly;

3. 2000g, centrifuging for 5 minutes at 4 ℃, and removing supernatant;

4. resuspending the cell pellet in cell isolation buffer (see example 13), centrifuging at 2000g and 4 ℃ for 5 minutes, and discarding the supernatant;

5. resuspending the cell pellet with cell isolation buffer (see example 13) and adjusting the cell concentration to 10⁷/mL。

Example 16 gallbladder cancer, bile duct cancer bile sample Density gradient centrifugation

1. An equal volume of Ficoll cell separation (MP #50494) was taken from the cell suspension using a 50mL sterile centrifuge tube.

2. The cell suspension is carefully applied to the upper layer of the cell separation medium, so that a clear interface is formed between the two.

3. 2000g of the suspension were centrifuged horizontally at room temperature for 20 minutes.

4. Sucking the middle layer white film into a new tube.

5. The cell pellet was resuspended in 20mL sterile PBS, 1500g was centrifuged at RT for 10min, and the supernatant was discarded.

6. The cell pellet was resuspended in bile culture medium (see example 13) of bile samples of gallbladder and bile duct cancer, and the cell status was observed under a microscope for cell counting.

As shown in FIG. 6, the single cell suspension obtained by separation contains a large amount of various types of other cells, such as erythrocytes, lymphocytes, fibroblasts, etc., in addition to tumor cells. One of the advantages of the method is that in the subsequent culture process, only cancer cells can be greatly amplified, and the proportion of other cells is gradually reduced or even disappears, so that the primary tumor cells of gallbladder cancer and cholangiocarcinoma with higher purity are finally obtained.

Example 17 gallbladder cancer, bile duct cancer Primary cell culture

1. Suspension culture of bile sample primary cells of gallbladder cancer and bile duct cancer is carried out by using a low-adsorption surface (low-adsorption surface), namely the culture medium of bile sample primary cells of gallbladder cancer and bile duct cancer in example 13 (wherein the final concentration of human recombinant protein EGF is 50ng/mL, the final concentration of human recombinant protein bFGF is 20ng/mL, the final concentration of human recombinant protein HGF is 20ng/mL, the final concentration of human recombinant protein FGF-10 is 20ng/mL, the final concentration of human recombinant protein FGF-3 a is 250ng/mL, the final concentration of human recombinant protein Noggin is 100ng/mL, the final concentration of SB202190 is 10 μ M, the final concentration of A83-01 is 0.5 μ M, the final concentration of N-acetyl-L-cysteine is 1mM, the final concentration of Nicotinamide is 10mM, the final concentration of cortisol is 25ng/mL, and the final concentration of Y-27632 μ M), taking a six-hole plate as an example, according to 10 holes⁶Individual cells were plated at 37 ℃ in density with 5% CO₂The culture was carried out in a cell culture incubator under the conditions.

As shown in FIG. 7, after 3-10 days of culture, cancer cells are greatly expanded to form cell masses with the diameter of 80 μm, and the total number of tumor cells can exceed 10⁷The number of other types of cells is significantly reduced or even eliminated. Through a large number of sample tests, the success rate of in-vitro culture of primary tumor cells of gallbladder cancer and bile duct cancer bile samples can reach 70%.

Example 18 passage of bile samples for gallbladder and biliary tract cancer

3. The cell pellet was resuspended in cell digest (see example 13) and digested at 37 ℃. The digestion of the cell pellet was observed under a microscope every 5 minutes until the cell pellet was digested into single cells.

4. The dissociation reaction was stopped with 10 volumes of a digestion stop solution (see example 13) and the cell suspension was collected.

7. Using a low-adsorption surface (low-adsorption-surface) to culture bile cells of bile samples of gallbladder cancer and bile duct cancer, wherein the culture medium is the culture medium of the bile samples of gallbladder cancer and bile duct cancer in example 13, taking a six-well plate as an example, and each well is 10⁶Individual cells were plated at 37 ℃ in density with 5% CO₂The culture was carried out in a cell culture incubator under the conditions.

EXAMPLE 19 cryopreservation of bile cells from gallbladder cancer and cholangiocarcinoma samples

After 2-3 passages of the suspension cultured bile cell of the gallbladder cancer and bile duct cancer bile sample are amplified, the primary cells can be frozen:

3. The cell pellet was resuspended in cell digest (see example 13) and digested at 37 ℃. The digestion of the cell pellet was observed under a microscope every 15 minutes until the cell pellet was digested into single cells.

4. The dissociation reaction was stopped with 10 volumes of digestion stop solution (see example 13), and the cell suspension was collected and counted.

6. Cell cryopreservation (see example 13) as per 10⁶Resuspending the cell sediment at a density of/mL, freezing 1mL of cell suspension in each tube of a 2mL freezing tube, freezing overnight by using a gradient cooling box, and transferring the cell sediment into liquid nitrogen for long-term storage.

Example 20 Resuscitation of bile cells from gallbladder cancer and bile duct cancer samples

The bile sample primary cells of gallbladder cancer and bile duct cancer preserved in liquid nitrogen can be recovered:

1. sterile water at 37 ℃ was prepared five minutes in advance.

4. Resuspending the cell pellet with gallbladder cancer, bile duct cancer bile sample primary cell culture medium (see example 13), culturing the gallbladder cancer, bile duct cancer bile sample primary cells with low adsorption surface, resuscitating each tube of cells in a 3.5cm culture dish at 37 deg.C and 5% CO₂The culture was carried out in a cell culture incubator under the conditions.

Example 21 HE staining identification of bile cells in gallbladder cancer and bile duct cancer bile samples

The reagent consumables used in the following examples are illustrated:

HE staining kit (beijing solibao biotechnology limited, # G1120);

cation anticreep slide (Beijing China fir Jinqiao Biotech limited);

xylene, methanol, acetone (Beijing chemical reagent company, analytical pure);

neutral resin adhesive (fine chemicals, GmbH, Beijing).

1. 800g of gallbladder cancer and bile duct cancer primary cell mass cultured by the primary cell culture medium of bile samples of gallbladder cancer and bile duct cancer in example 13 (wherein, the final concentration of human recombinant protein EGF is 20ng/mL, the final concentration of human recombinant protein bFGF is 20ng/mL, the final concentration of human recombinant protein HGF is 20ng/mL, the final concentration of human recombinant protein FGF-10 is 20ng/mL, the final concentration of human recombinant protein Wnt-3a is 200ng/mL, the final concentration of human recombinant protein Noggin is 100ng/mL, the final concentration of SB202190 is 5 muM, the final concentration of A83-01 is 1 muM, the final concentration of N-acetyl-L-cysteine is 1mM, the final concentration of Nicotinamide is 10mM, the final concentration of cortisol is 25ng/mL, the final concentration of Y-27632 is 10 muM), fix with 4% paraformaldehyde. The pellet of cells was embedded in paraffin and sliced to a thickness of 5 μm.

Fig. 8 shows the HE staining effect of bile duct cancer bile sample primary tumor cells obtained by in vitro culture, and it can be seen that these cells generally have the characteristics of high nuclear mass ratio, deep nuclear staining, chromatin condensation in the nucleus, multinucleate, uneven cell size and other cancer cells, and dozens to hundreds of tumor cells aggregate to form tumor cell masses with certain three-dimensional structures.

Example 22 immunohistochemical staining identification of bile cells from gallbladder cancer, bile duct cancer bile samples

The reagents used in the following examples are illustrative:

hydrogen peroxide (beijing chemicals, 35%);

blocking with normal goat serum (Solarbio, SL 038);

immunohistochemical primary anti-antibodies (Abcam, ab 215838);

immunohistochemical secondary antibodies (Abcam, ab 205719);

EDTA repair solution (Abcam, ab 93684);

DAB color-developing liquid (

DAB Substrate Kit，8059S)

The gall bladder cancer bile sample cell mass obtained by culturing the gall bladder cancer bile sample primary cell culture medium (wherein the final concentration of human recombinant protein EGF is 50ng/mL, the final concentration of human recombinant protein bFGF is 25ng/mL, the final concentration of human recombinant protein HGF is 25ng/mL, the final concentration of human recombinant protein FGF-10 is 25ng/mL, the final concentration of human recombinant protein Wnt-3a is 300ng/mL, the final concentration of human recombinant protein Noggin is 200ng/mL, the final concentration of SB202190 is 10 muM, the final concentration of A83-01 is 0.5 muM, the final concentration of N-acetyl-L-cysteine is 1mM, the final concentration of Nicotinamide is 10mM, the final concentration of cortisol is 25ng/mL, and the final concentration of Y-27632 is 10 muM) in example 13 is collected and is sliced into paraffin wax, cells of epithelial origin were characterized with pan-CK antibody according to the following procedure.

14. The dried sections can be viewed under a microscope or photographed.

FIG. 9 shows the effect of immunohistochemical staining of bile duct cancer primary tumor cell mass cultured in vitro, and it can be seen that the cells constituting the cell mass are all pan-CK positive and are of epithelial origin, confirming that the tumor cells cultured by the method are of higher purity. Immunohistochemical staining identification is carried out on the primary cultures of the 5 gallbladder cancer and bile duct cancer samples, and statistical results show that the proportion of tumor cells in the primary cells of the gallbladder cancer and bile duct cancer bile samples obtained by the method reaches 64% -80% (Table 31).

Table 31 gallbladder cancer, bile duct cancer primary culture immunohistochemical staining identification

Claims

1. A method for culturing primary cells of gallbladder cholangiocarcinoma comprises the following steps: suspending and culturing primary gallbladder cholangiocarcinoma cells by using a primary gallbladder cholangiocarcinoma cell culture medium;

the primary cell culture medium for cholecystcholangiocarcinoma consists of three antibacterial antifungal agents, HEPES, GlutaMax, nonessential amino acids, 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, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, nicotine, N-2Supplement, cortisol, B27, ITS-X, gastrin, Y-27632 and Advanced DMEM/F12 culture medium; wherein the final concentration of penicillin in the three-antibody of the antibacterial antifungal agent is 100-200U/mL; the final concentration of streptomycin in the three-antibody of the antibacterial antifungal agent is 100-; the final concentration of amphotericin B in the three-antibody of the antibacterial antifungal agent is 250 ng/mL; the final concentration of the HEPES is 8-12 mM; the final concentration of the GlutaMax is 0.8-1.2% (volume percentage); the concentration of glycine in the non-essential amino acid is 80-120 μ M; the concentration of L-alanine in the non-essential amino acid is 80-120 mu M; the concentration of L-asparagine in the non-essential amino acid is 80-120. mu.M; the concentration of L-aspartic acid in the non-essential amino acid is 80-120 μ M; the concentration of L-glutamic acid in the non-essential amino acid is 80-120 mu M; the concentration of L-proline in the non-essential amino acids is 80-120 mu M; the concentration of L-serine in the non-essential amino acid is 80-120 μ M; 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 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 the human recombinant protein Noggin is 100-200 ng/mL; the final concentration of the SB202190 is 5-10 μ M; the final concentration of the A83-01 is 0.25-1.25 mu M; the final concentration of the Primocin is 1% (volume percentage); the final concentration of the N-acetyl-L-cysteine is 0.5-2 mM; the final concentration of nicotine is 5-10 mM; the final concentration of the N-2Supplement is 1 percent (volume percentage); the final concentration of the cortisol is 20-50 ng/mL; the final concentration of B27 is 1.5-2.5% (volume percentage); the final concentration of ITS-X is 0.8-1.2% (volume percentage content); the final concentration of the gastrin is 8-12 nM; the final concentration of the Y-27632 is 5-20 mu M; the balance is Advanced DMEM/F12 medium.

2. The method of claim 1, wherein: the primary gallbladder cholangiocarcinoma cells are primary gallbladder cholangiocarcinoma solid tumor cells or primary gallbladder cholangiocarcinoma bile sample tumor cells.

3. The method of claim 2, wherein: the primary gallbladder cholangiocarcinoma solid tumor cells are obtained by dissociating a gallbladder cholangiocarcinoma solid tumor tissue with a sample dissociation solution;

the sample dissociation liquid consists of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of the collagenase I in the sample dissociation liquid is 150-250U/mL; the final concentration of the collagenase II in the sample dissociation liquid is 150-250U/mL; the final concentration of the collagenase IV in the dissociation liquid of the sample is 50-150U/mL; the balance being PBS;

further, the gallbladder bile duct cancer solid tumor tissue is dissociated by the sample dissociation liquid according to the method comprising the following steps: treating the sheared solid tumor tissue of the gallbladder, bile duct cancer by using the sample dissociation solution preheated at 37 ℃ in advance according to the dosage of 0.1-0.3mL of the sample dissociation solution per mg of tissue, and dissociating the sample at 37 ℃ for 15 minutes to 3 hours;

and/or

The primary tumor cells of the bile sample of the gallbladder, bile duct cancer are obtained by separating the bile sample of the gallbladder, bile duct cancer by using a cell separation buffer solution;

the cell separation buffer solution consists of double-antibody P/S, heparin sodium and PBS; wherein the final concentration of penicillin in the double-resistant P/S is 100-200U/mL; the final concentration of streptomycin in the double-antibody P/S is 100-200 mug/mL; the final concentration of the heparin sodium is 10 IU/mL; the balance being PBS;

further, the bile sample of the gallbladder bile duct cancer is separated by the separation buffer according to the method comprising the following steps: suspending the cells in the bile sample of the gallbladder, bile duct cancer by using the cell separation buffer solution, and then obtaining primary tumor cells of the bile sample of the gallbladder, bile duct cancer by density gradient centrifugation.

4. A method according to any one of claims 1-3, characterized in that: the method comprises the following steps of performing suspension culture on the primary gallbladder cholangiocarcinoma cells by using the primary gallbladder cholangiocarcinoma cell culture medium: using a cell culture container M to culture the primary gallbladder cholangiocarcinoma cells in a suspension manner by using the primary gallbladder cholangiocarcinoma cell culture medium at 37 ℃ and 5% CO₂Culturing under the condition, and replacing the culture medium every 2-4 days;

the cell culture vessel M is any one of: (I) a cell culture container made of polystyrene, a cell culture container made of polycarbonate, a cell culture container made of polymethyl methacrylate, a cell culture container made of COC resin, a cell culture container made of cyclic olefin polymer, or a cell culture container with a low adsorption surface; (II) subjecting the cell culture vessel of (I) to CYTOP modification;

further, the cell culture container is a cell culture dish, a cell culture pore plate or a micropore plate chip for cell culture; and/or

Further, in the (II), the cell culture vessel in the (I) is subjected to CYTOP modification according to a method comprising the following steps: carrying out pure oxygen etching on the cell culture container in the step (I), wherein the etching condition is that the power is 20W, and the etching time is 3 minutes; then covering the surface of the cell culture container with 1% CYTOP solution, and airing the 1% CYTOP solution to finish the CYTOP modification;

still further, the composition of the 1% CYTOP solution is as follows: each 100mL of the 1% CYTOP solution contained 1mL of CYTOP, with the balance being fluoro oil.

5. The method according to any one of claims 2-4, wherein: the method also comprises the following step of carrying out dissociation pretreatment on the solid tumor tissue of the gallbladder bile duct cancer: cleaning the surface of the gallbladder, bile duct cancer and solid tumor tissue sample by using ethanol with the volume percentage of 70-75%; sequentially cleaning the gallbladder bile duct cancer solid tumor tissue samples by using a sample cleaning solution and a sterile PBS solution;

specifically, the sample cleaning solution consists of an antibacterial antifungal agent triantion and PBS; wherein the final concentration of penicillin in the three-antibody of the antibacterial antifungal agent is 100-200U/mL; the final concentration of streptomycin in the three-antibody of the antibacterial antifungal agent is 100-; the final concentration of amphotericin B in the three-antibody of the antibacterial antifungal agent is 250-500 ng/mL; the balance being PBS.

6. The method of claim 5, wherein: the in vitro time of the gallbladder bile duct cancer solid tumor tissue sample subjected to the dissociation pretreatment is less than 2 hours, and the gallbladder bile duct cancer solid tumor tissue sample is preserved in a sample preservation solution before the dissociation pretreatment;

specifically, the sample preservation solution consists of fetal calf serum, three antibiotics of antibacterial and antifungal agents, HEPES and HBSS; wherein the final concentration of the fetal calf serum is 1-5% (volume percentage content); the final concentration of penicillin in the three-antibody of the antibacterial antifungal agent is 100-200U/mL; the final concentration of streptomycin in the three-antibody of the antibacterial antifungal agent is 100-; the final concentration of amphotericin B in the three-antibody of the antibacterial antifungal agent is 250-500 ng/mL; the final concentration of the HEPES is 8-12 mM; the balance being HBSS.

7. The method according to any one of claims 1-6, wherein: in the method, after the dissociation treatment of the gallbladder bile duct cancer solid tumor tissue by using the sample dissociation solution, the method further comprises the following steps: terminating the dissociation reaction by using a digestion termination solution, and collecting cell suspension; filtering the cell suspension to remove tissue debris and adherent cells; resuspending the cells with sterile PBS after centrifugation; re-centrifuging, and then re-suspending the cell pellet with the primary gallbladder cholangiocarcinoma cell culture medium;

specifically, the digestion stop solution consists of fetal calf serum, an antibacterial antifungal agent three-antibody and a DMEM culture medium; wherein the final concentration of the fetal calf serum is 8-12% (volume percentage content); the final concentration of penicillin in the three-antibody of the antibacterial antifungal agent is 100-200U/mL; the final concentration of streptomycin in the three-antibody of the antibacterial antifungal agent is 100-; the final concentration of amphotericin B in the three-antibody of the antibacterial antifungal agent is 250-500 ng/mL; the balance is DMEM medium.

8. The method according to any one of claims 1-7, wherein: in the process of culturing the primary gallbladder cholangiocarcinoma cells by using the primary gallbladder cholangiocarcinoma fine culture medium, the method further comprises the following steps: when the primary gallbladder cholangiocarcinoma cells form a lump with the diameter of 50-80 mu m, carrying out passage on the primary gallbladder cholangiocarcinoma cells;

specifically, the cell digest used for the passage was composed as follows: every 10mL of the cell digestive juice contains 4-6mL of Accutase, EDTA with the final concentration of 5mM, 1.5-2.5mL of TrypLE Express and the balance of PBS; and/or

The use of a digestion stopping solution for said passaging, which is the digestion stopping solution according to claim 7;

and/or

The method also comprises the step of performing cryopreservation and/or resuscitation on the primary gallbladder cholangiocarcinoma cells after passage expansion for 2-3 times;

specifically, the cell cryopreservation solution adopted in the cryopreservation process consists of an Advanced DMEM/F12 culture medium, DMSO and a 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM/F12 culture medium to the DMSO to the 1% methylcellulose solution is 20:2 (0.8-1.2); the 1% methylcellulose solution is an aqueous solution of methylcellulose having a concentration of 1g/100 ml.

9. A kit for culturing primary cells of cholecystcholangiocarcinoma, which consists of the primary cell culture medium of cholecystcholangiocarcinoma according to any one of claims 1 to 8 and at least one of the following reagents: the sample dissociation solution, the sample preservation solution, the cell separation buffer solution, the cell digestion solution, the sample washing solution, the digestion stop solution, the cell cryopreservation solution and the 1% CYTOP solution according to any one of claims 1 to 8.

10. Use of a kit according to claim 9 for culturing primary cells of cholecystochhol carcinoma.