CN112760284A

CN112760284A - Culture medium for culturing primary gallbladder cholangiocarcinoma cells

Info

Publication number: CN112760284A
Application number: CN201911065661.1A
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

The invention discloses a culture medium for culturing primary cells of gallbladder cholangiocarcinoma. The culture medium for culturing primary cells of gallbladder cholangiocarcinoma provided by the invention is a special serum-free culture medium, and the culture medium is used for in vitro suspension culture of tumor cells derived from the gallbladder cholangiocarcinoma, so that normal amplification of the cancer cells can be ensured, and interference of normal cells can be eliminated to the maximum extent. The primary gallbladder cholangiocarcinoma cell culture obtained by the method can be used for in vitro experiments of various cell levels, next generation sequencing, animal model construction, cell line construction and the like. The culture method has wide application prospect in the fields of research on gallbladder bile duct cancer and clinical diagnosis and treatment.

Description

Culture medium for culturing primary gallbladder cholangiocarcinoma cells

Technical Field

The invention relates to the technical field of biology, in particular to a culture medium 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 gallbladder cholangiocarcinoma cell culture technology and a matched reagent.

In a first aspect, the invention claims a medium for culturing primary cells of cholecystochhol carcinoma.

The culture medium for culturing primary cells of cholecystocholene carcinoma provided by the invention comprises antibacterial antifungal agents of trinexazine (penicillin-streptomycin-amphotericin B), HEPES, GlutaMax, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein FGF-10, human recombinant protein R-spondin, 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-pyridyl) -N-phenyl-4- (4-quinolyl) -1H-pyrazole-1-carbothioamide), Primocin, N-acetyl-L-cysteine (N-acetyl-L-cysteine), Nicotine (Nicotinamide), N-2Supplement, Cholera Toxin (Cholera Toxin), B27, Y-27632, CHIR99021 and Advanced DMEM/F12 medium.

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 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 R-spondin is 250-500 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, for example, 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 Cholera Toxin (Cholera Toxin) is 0.1-1 nM; the final concentration of B27 is 1.5-2.5% (e.g., 2%,% indicates volume percent); the final concentration of the Y-27632 is 5-20 mu M; the final concentration of the CHIR99021 is 1-5 mu M; the balance is Advanced DMEM/F12 medium. The final concentrations of the substances are the final concentrations in the culture medium for culturing primary cells of cholecystcholangiocarcinoma.

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 three-resistant (penicillin-streptomycin-amphotericin B) of the antibacterial antifungal agent is' Antibiotic-Antimycin, 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 Primocin^TMAntibiotics used for protecting primary cells from microbial contamination, which are antibacterial agents for primary cells (such as Invivogen # ant-pm-1, or other products having the same composition), have 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 (Cortisonine), D-Galactose (D-Galactose), Ethanolamine hydrochloric acid (Ethanolamine HCl), glutathione (glutathione), Carnitine L-hydrochloric acid (L-Carnitine HCl), linoleic acid (L-Carnitine HCl), and L-Alpha-Tocopherol Acetate (DL Alpha-Tocopherol Acetate), DL-Alpha-Tocopherol (DL-Tocopherol), and L-beta-cyclodextrin (L-Carnitine L-Tocopherol, L-Carnitine, L-Tocopherol, D-Galactose, L-Galactose, and L-Galactose (L-Galactose, L-GalactoseLinoleic Acid), Linolenic Acid (Linolenic Acid), Progesterone (Progesterone), Putrescine (Putrescine 2HCl), Sodium Selenite (Sodium Selenite), triiodothyronine (T3 (triodo-I-thyronine)). 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). The CHIR-99021 is specifically 'CHIR-99021 (CT99021) HCl' (such as Selleck # S2924, or other products with the same composition). The CHIR-99021(CT99021) HCl is hydrochloride of the CHIR-99021, is a GSK-3 alpha/beta inhibitor and has an IC50 of 10nM/6.7 nM. It has selectivity on GSK-3 over Cdc2 and ERK2 by over 500 times.

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 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 goods number of the human recombinant protein R-spondin is Shanghai nearshore # CD 83; the brand and commodity number of the human recombinant protein Wnt-3a is R & D5036-WN-500; the brand goods number of the human recombinant protein Noggin is Shanghai near shore # C018; the brand of the SB202190 is Sigma # S7067; the brand name of A83-01 is Tocris # 2939; the brand name of the Primocin 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 name of Cholera Toxin is Listlab # 100B; the brand name of B27 is Gibco # 12587010; the brand goods number of the Y-27632 is MCE # 129830-38-2; the brand name of the CHIR99021 is Selleck # S2924; the brand of the Advanced DMEM/F12 medium is Gibco # 12634010.

Further, the culture medium for culturing primary cholecystcholangiocarcinoma cells may exist in two forms:

the medium for culturing primary cells of cholecystcholangiocarcinoma is a solution containing the antibacterial antifungal agent tris (penicillin-streptomycin-amphotericin B), the HEPES, the GlutaMax, 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 R-spondin, the human recombinant protein Wnt-3a, the human recombinant protein Noggin, the SB202190, the a83-01, the Primocin, the N-acetyl-L-cysteine, the nicotine, the N-2Supplement, the cholera toxin, the B27, the Y-27632, the CHIR99021, and the Advanced DMEM/F12 medium.

The media was prepared and sterilized by filtration through a 0.22 μ M needle filter (Millipore SLGP033RS) and stored at 4 ℃ for two weeks.

Secondly, the components of the culture medium for culturing primary gallbladder cholangiocarcinoma cells exist independently and are prepared according to a formula when in use.

Furthermore, the human recombinant protein EGF, the human recombinant protein bFGF, the human recombinant protein HGF, the human recombinant protein FGF-10, the human recombinant protein R-spondin, the human recombinant protein Wnt-3a and the human recombinant protein Noggin can exist in a stock solution (mother solution) form, can be stored for a long time at the temperature of minus 80 ℃, and can be specifically 1000 times of the stock solution (mother solution). SB202190, N-acetyl-L-cysteine, Nicotinamide and Y-27632 can exist in stock solution (mother liquor) form (long-term preservation at-20 deg.C), specifically 1000 times of stock solution (mother liquor). Cholera Toxin and CHIR99021 can exist in stock solution (mother liquor) form (-20 ℃ for long-term storage), and specifically can be 10000 times of stock solution (mother liquor). A83-01 can exist in stock solution (mother liquor) form (long-term storage at-20 deg.C), specifically 100000 times 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 R-spondin stock solution is composed of human recombinant protein R-spondin, BSA and PBS, wherein the final concentration of the human recombinant protein R-spondin is 250 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.

10000 XCholera Toxin stock solution consists of Cholera Toxin and Cholera Toxin solution, wherein the final concentration of Cholera Toxin is 10 μ M, and the rest is Cholera Toxin solution. The Cholera Toxin dissolving solution comprises the following components: each 10mL of the Cholera Toxin solution contained Tris (1M) pH 7.00.05M, NaCl 0.2M, sodium azide 3mM, EDTA (0.5M) pH 8.01 mM, and the balance 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.

10000 XCHIR 99021 stock solution consists of CHIR99021 and sterile water, wherein the final concentration of CHIR99021 is 10mM, and the balance is sterile water.

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

The kit for culturing primary cells of cholecystcholangiocarcinoma provided by the invention comprises the culture medium and at least one of the following reagents: digestion stop solutions and the cell cryopreservation solution described below.

In a third aspect, the invention claims the application of the culture medium or the reagent set in the culture of primary cells of cholecystephiobile duct cancer.

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

The method for culturing primary cells of gallbladder cholangiocarcinoma provided by the invention specifically comprises the following steps: using a culture container with a low-adsorption surface (low-adsorption surface), culturing the primary gallbladder cholangiocarcinoma cells in suspension by using the 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 be10⁵Per cm²Bottom area of the container, e.g. six-well plate, 10 per well⁶Density of individual cells was plated.

Further, the method may further comprise the steps of: 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 digestion stop solution adopted during the passage (can be stored for one month at 4 ℃ after being prepared) consists of fetal calf serum, three antibiotics (penicillin-streptomycin-amphotericin B) of an antibacterial antifungal agent and a DMEM medium; wherein the final concentration of the fetal calf serum in the digestion stop solution 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) in the digestion stop solution is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the antibacterial antifungal agent triantibody (penicillin-streptomycin-amphotericin B) in the digestion stop solution 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) in the digestion stop solution is 250-500ng/mL (such as 250 ng/mL); the balance is DMEM 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.

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.

More specifically, the step of performing said passaging is carried out: collecting cell masses to be passaged, washing the cell masses by using a sterile PBS solution after centrifugation, then centrifuging, then re-suspending the cell masses by using a cell digestion solution, digesting at 37 ℃ until the cell masses are digested into single cells, stopping digestion reaction by using the digestion stop solution (the dosage can be 5-10 times, for example, 10 times of the volume), and collecting cell suspension; after centrifugation, the cell pellet is resuspended with the medium, counted, and cells are then suspension cultured using a culture vessel with a low adsorption surface (initial seeding density can be 10⁵Per 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 frozen stock solution (which is used as the frozen stock solution) adopted during the 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, resuspending the cell mass with the cell digestive juice, and feeding at 37 deg.CDigesting until the cell mass is digested into single cells, terminating the digestion reaction with the digestion stop solution (which can be used in an amount of 5-10 times, for example, 10 times, the volume), and collecting a 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); after centrifugation (e.g., 800-⁵Per cm²Bottom area of container), cells per tube (10)⁶Respectively) reviving to 3.5cm culture dish), culturing at 37 deg.C and 5% CO₂。

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 culture medium and a culture method for extracting and culturing primary tumor cells of gallbladder cancer and bile duct cancer from fresh gallbladder cancer, solid tumor operation samples or biopsy puncture tissue samples of bile duct cancer and bile duct cancer, 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 shows single cells obtained after bile sample of bile duct cancer is processed. The scale is 100 μm, 100 times magnification.

FIG. 6 shows cell masses obtained after primary culture of bile samples of cholangiocarcinoma. The scale is 100 μm, 100 times magnification.

FIG. 7 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, 200 times magnification.

FIG. 8 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, 400 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 preparation of human recombinant protein stocks is shown in tables 11 to 17, the preparation of SB202190 stock is shown in Table 18, the preparation of A83-01 stock is shown in Table 19, the preparation of N-acetyl-L-cysteine stock is shown in Table 20, the preparation of Nicotinamide stock is shown in Table 21, the preparation of Cholera Toxin stock is shown in Table 22, the preparation of Y-27632 stock is shown in Table 23, and the preparation of CHIR99021 stock is shown in Table 25. 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 (2.5mL)

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 R-spondin stock solutions (4mL)

After 1000 Xhuman recombinant protein R-spondin 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 161000 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 171000 × human recombinant protein Noggin stock solution (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 181000 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 19100000 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 201000 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 211000 Nicotinamide 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 2210000 XCholera Toxin stock solution (1.17mL)

After preparing a stock solution of 1000 XCholera Toxin, subpackaging the stock solution by using a sterile centrifuge tube of 0.5mL, and storing the stock solution at the temperature of 20 ℃ below zero for a long time.

In table 22, the formulation of the Cholera Toxin dissolution solution is shown in table 23.

TABLE 23 Cholera Toxin solution (10mL)

The Cholera Toxin dissolving 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 241000 XY-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 ℃.

TABLE 2510000 XCHIR 99021 stock solutions (0.996mL)

10000 XCHIR 99021 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 20 ℃ below zero.

7. Cell cryopreservation liquid

The specific formulation of the cell culture medium is shown in Table 26.

TABLE 26 cell cryopreservation solution

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

In table 26, the preparation of the 1% methylcellulose solution is shown in table 27.

TABLE 271% methylcellulose solution (10mL)

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

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 gallbladder cancer and bile duct cancer solid tumor primary cells is carried out by using a low-adsorption surface (low-adsorption surface), namely the culture medium of the gallbladder cancer and bile duct cancer solid tumor primary cells 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 R-spondin is 400ng/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 SB 190 is 10 MuM, the final concentration of A83-01 is 0.5 MuM, the final concentration of N-acetyl-L-cycesine is 1mM, the final concentration of Nicotinamide is 10mM, the final concentration of Choline is 0.275 nM and the final concentration of Tolixin Toxin is 2025 nM 10 mu M; CHIR99021 final concentration of 3. mu.M), in six well plates, 10 per well, for example⁶Individual 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 culture medium (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 R-spondin is 400ng/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 Cholera Toxin is 0.5nM, the final concentration of Y-27632 is 10 muM, and the final concentration of CHIR99021 is 3 muM) obtained by centrifugal collection, Bile duct carcinoma solid tumor primary cell mass, fixed 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 cholangiocarcinoma 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 R-spondin is 400ng/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 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 Cholera Toxin is 0.5nM, the final concentration of Y-2710 μ M, the final concentration of CHIR99021 is 3 μ M) in example 1 was collected and cultured to obtain the gallbladder cancer, The cholangiocarcinoma cell pellets were paraffin sectioned and cells of epithelial origin were characterized with pan-CK antibody as follows.

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 89-96 percent (Table 28).

TABLE 28 immunohistochemical staining identification of primary culture of gallbladder cancer and bile duct cancer

Example 11 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 12 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 13 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 11), centrifuging at 2000g and 4 ℃ for 5 minutes, and discarding the supernatant;

5. resuspending the cell pellet with cell isolation buffer (see example 11) and adjusting the cell concentration to 10⁷/mL。

Example 14 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. Resuspend the cell pellet with bile sample primary cell culture medium (see example 11) of gallbladder cancer and bile duct cancer, observe the cell state under microscope, and count the cells.

As shown in FIG. 5, 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 15 Primary cell culture of bile samples for gallbladder cancer and cholangiocarcinoma

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 11 (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 R-spondin is 400ng/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 SB 190 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 Choline is 0.275 μ M, the final concentration of Tolixin Toxin is 2025 nM The degree is 10 mu M; CHIR99021 final concentration of 3. mu.M), in six well plates, 10 per well, for example⁶Individual 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. 6, 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 15 passage of bile samples for gallbladder cancer, bile duct cancer

3. The cell pellet was resuspended in cell digest (see example 11) 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 11) 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 16 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 11) 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 11), and the cell suspension was collected and counted.

6. Cell cryopreservation (see example 11) as described in 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 17 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 11), culturing the gallbladder cancer, bile duct cancer bile sample primary cell using low adsorption surface, resuscitating each tube of cells into 3.5cm culture dish, 37 deg.C, 5% CO₂The culture was carried out in a cell culture incubator under the conditions.

Example 18 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 bile sample primary cell culture medium (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 R-spondin is 400ng/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 μ M, the final concentration of A83-01 is 1 μ M, the final concentration of N-acetyl-L-cysteine is 1mM, the final concentration of Nicotinamide is 10mM, the final concentration of Cholera Toxin is 0.5nM, the final concentration of Y-27632 is 10 μ M, and the final concentration of CHIR99021 is 3 μ M) obtained by centrifugal collection, Bile duct carcinoma solid tumor primary cell mass, fixed with 4% paraformaldehyde. The pellet of cells was embedded in paraffin and sliced to a thickness of 5 μm.

Fig. 7 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 19 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 gallbladder cancer and bile duct cancer bile samples obtained in example 11 were cultured in primary cell culture medium (wherein the final concentration of human recombinant protein EGF was 50ng/mL, the final concentration of human recombinant protein bFGF was 25ng/mL, the final concentration of human recombinant protein HGF was 25ng/mL, the final concentration of human recombinant protein FGF-10 was 25ng/mL, the final concentration of human recombinant protein R-spondin was 400ng/mL, the final concentration of human recombinant protein Wnt-3a was 300ng/mL, the final concentration of human recombinant protein Noggin was 200ng/mL, the final concentration of SB202190 was 10. mu.M, the final concentration of A83-01 was 0.5. mu.M, the final concentration of N-acetyl-L-cysteine was 1mM, the final concentration of Nicotinamide was 10mM, the final concentration of Cholera Toxin was 0.5nM, the final concentration of Y-2710. mu.M, and the final concentration of CHIR99021 was 3. mu.M) to obtain gallbladder cancer cells, Cholangiocarcinoma bile samples primary cell pellets were paraffin sectioned and cells of epithelial origin were characterized with pan-CK antibody as follows.

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

FIG. 8 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 74-85% (Table 31).

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

Claims

1. A culture medium for culturing primary cells of cholecystcholangiocarcinoma, which is characterized in that: the culture medium consists of antibacterial antifungal agent three-antibody, HEPES, GlutaMax, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein FGF-10, human recombinant protein R-spondin, human recombinant protein Wnt-3a, human recombinant protein Noggin, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, nicotine, N-2Supplement, cholera toxin, B27, Y-27632, CHIR99021 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 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 R-spondin is 250-500 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 cholera toxin is 0.1-1 nM; the final concentration of B27 is 1.5-2.5% (volume percentage); the final concentration of the Y-27632 is 5-20 mu M; the final concentration of the CHIR99021 is 1-5 mu M; the balance is Advanced DMEM/F12 medium.

2. The culture medium according to claim 1, wherein: the culture medium is a solution containing the antimicrobial antifungal agent triaantibody, the HEPES, the GlutaMax, 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 R-spondin, the human recombinant protein Wnt-3a, the human recombinant protein Noggin, the SB202190, the A83-01, the Primocin, the N-acetyl-L-cysteine, the nicotine, the N-2Supplement, the cholera toxin, the B27, the Y-27632, the CHIR99021 and the Advanced DMEM/F12 culture medium.

3. The culture medium according to claim 1, wherein: the components of the medium are present separately.

4. The culture medium of claim 3, wherein: said human recombinant protein EGF, said human recombinant protein bFGF, said human recombinant protein HGF, said human recombinant protein FGF-10, said human recombinant protein R-spondin, said human recombinant protein Wnt-3a, said human recombinant protein Noggin, said SB202190, said A83-01, said N-acetyl-L-cysteine, said nicotine, said cholera toxin, said Y-27632, and said CHIR99021 are present in stock solution;

specifically, the mother liquor is 1000-100000 times of mother liquor;

the 1000 multiplied human 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 multiplied human recombinant protein bFGF consists of human recombinant protein bFGF, BSA and PBS, wherein the final concentration of the human recombinant protein bFGF is 20 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 protein 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;

1000 Xthe stock solution of the human recombinant protein R-spondin consists of the human recombinant protein R-spondin, BSA and PBS, wherein the final concentration of the human recombinant protein R-spondin is 250 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;

the 1000 × SB202190 stock consists of SB202190 and DMSO, wherein the final concentration of SB202190 is 10mM, the balance being DMSO;

the 100000 XA 83-01 stock solution consists of A83-01 and DMSO, wherein the concentration of the A83-01 is 25mM, and the balance is DMSO;

the 1000 XN-acetyl-L-cysteine stock solution consists of N-acetyl-L-cysteine and water, wherein the concentration of the N-acetyl-L-cysteine is 0.5M, and the balance is water;

1000 × nicotine stock solution is composed of nicotine and water, wherein the concentration of nicotine is 5M, and the balance is water;

10000 Xcholera toxin stock solution consists of cholera toxin and cholera toxin solution, wherein the final concentration of the cholera toxin is 10 μ M, and the rest is cholera toxin solution; the cholera toxin dissolving solution comprises the following components: tris (1M) pH 7.00.05M, NaCl 0.2M, sodium azide 3mM, EDTA (0.5M) pH 8.01 mM, the balance being water;

the 1000 XY-27632 stock solution consists of Y-27632 and water, wherein the final concentration of Y-27632 is 10mM, and the balance is water;

10000 XCHIR 99021 stock solution consists of CHIR99021 and water, wherein the final concentration of CHIR99021 is 10mM, and the balance is water.

5. The culture medium according to claim 4, wherein: in said stock of 1000 × human recombinant protein EGF, said stock of 1000 × human recombinant protein bFGF, said stock of 1000 × human recombinant protein HGF, said stock of 1000 × human recombinant protein FGF-10, said stock of 1000 × human recombinant protein R-spondin, said stock of 1000 × human recombinant protein Wnt-3a, and stock of 1000 × human recombinant protein Noggin, said BSA is present in a stock solution;

specifically, the BSA exists in the form of 100 times of mother liquor; 100 × BSA solution consisted of BSA and PBS; wherein the final concentration of BSA was 0.1g/mL, and the balance was PBS.

6. Kit of parts for culturing primary cells of cholecystcholangiocarcinoma comprising a medium according to any one of claims 1 to 5 and at least one of the following reagents: the digestion stopping solution and the cell culture solution according to claim 9.

7. Use of a medium according to any one of claims 1 to 5 or a kit of parts according to claim 6 for culturing primary cells of cholecystic cholangiocarcinoma.

8. A method for culturing primary cells of gallbladder cholangiocarcinoma comprises the following steps: using a culture vessel with a low adsorption surface, culturing the primary gallbladder cholangiocarcinoma cells in suspension by using the culture medium at 37 ℃ and 5% CO₂Culturing under the condition, and replacing the culture medium every 2-4 days.

9. The method of claim 8, wherein: the method further comprises the steps of: 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 digestion stop solution adopted during the passage consists of fetal calf serum, three antibiotics of an antibacterial antifungal agent and a DMEM medium; wherein the final concentration of the fetal calf serum is 8-12%; 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 rest is DMEM culture medium;

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.

10. A medium or kit of parts or a use or method according to any one of claims 1 to 9, wherein: the primary gallbladder cholangiocarcinoma cells are primary gallbladder cholangiocarcinoma solid tumor cells or primary gallbladder cholangiocarcinoma bile sample tumor cells.