WO2020206999A1 - 胃癌和胆囊胆管癌原代细胞培养方法及配套试剂 - Google Patents

胃癌和胆囊胆管癌原代细胞培养方法及配套试剂 Download PDF

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WO2020206999A1
WO2020206999A1 PCT/CN2019/115306 CN2019115306W WO2020206999A1 WO 2020206999 A1 WO2020206999 A1 WO 2020206999A1 CN 2019115306 W CN2019115306 W CN 2019115306W WO 2020206999 A1 WO2020206999 A1 WO 2020206999A1
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gallbladder
final concentration
cholangiocarcinoma
solution
gastric cancer
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PCT/CN2019/115306
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English (en)
French (fr)
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尹申意
张函槊
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北京基石生命科技有限公司
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Priority claimed from CN201910289073.XA external-priority patent/CN111808815A/zh
Priority claimed from CN201910289074.4A external-priority patent/CN111808816A/zh
Application filed by 北京基石生命科技有限公司 filed Critical 北京基石生命科技有限公司
Priority to US17/594,276 priority Critical patent/US20220177852A1/en
Priority to EP19924173.8A priority patent/EP3954764A4/en
Priority to JP2021559639A priority patent/JP7434359B2/ja
Priority to AU2019440405A priority patent/AU2019440405A1/en
Publication of WO2020206999A1 publication Critical patent/WO2020206999A1/zh

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Definitions

  • the invention relates to the field of biotechnology, in particular to a method for culturing primary cells of gastric cancer and gallbladder cholangiocarcinoma and supporting reagents.
  • Gastric cancer is one of the most common malignant tumors that seriously threaten human health.
  • my country is a country with a high incidence of gastric cancer.
  • the incidence and death of gastric cancer account for 42.6% and 45% of the global incidence and death respectively.
  • the incidence of gastric cancer in my country is 11.8%, which is the fourth place among all malignant tumors.
  • the mortality rate of gastric cancer is 22.0%, which is the fifth place among all malignant tumors.
  • the risk of gastric cancer recurrence and metastasis is high. More than 50% of gastric cancer patients will have different degrees of recurrence and metastasis within months to years after radical treatment.
  • Gallbladder cholangiocarcinoma is a common malignant tumor of the digestive system that occurs in the gallbladder, bile duct, and intrahepatic bile duct, including gallbladder cancer and cholangiocellular carcinoma.
  • the overall incidence of gallbladder and bile duct-related malignant tumors in my country is about 3%, of which cholangiocarcinoma accounts for 2%, ranking fifth in my country's digestive tract malignant tumors.
  • gallbladder and bile duct-related cancers are extremely malignant, and cholangiocarcinoma is even called the "king of liver cancer" and "king of cancer.”
  • the median survival time is only 8 months.
  • Gastric cancer and cholangiocarcinoma of the gallbladder are both complex diseases. Their occurrence and development are a dynamic process, involving the interaction of many signal molecules, forming a complex molecular regulatory network, and being affected by external environmental factors. There are strong individual differences in the etiology and development process of gastric cancer and gallbladder cholangiocarcinoma, which cannot be generalized. Therefore, using gastric cancer and gallbladder cholangiocarcinoma primary cell cultures as models for individualized and accurate research is a trend in the field of gastric cancer and cholangiocarcinoma research and even the diagnosis and treatment of gastric cancer.
  • the existing primary tumor cell culture technologies mainly include 2D culture, 3D culture, and reprogramming culture. These methods all face the problems of extremely long culture cycle, low culture success rate, and difficult removal of foreign cells to varying degrees.
  • the present invention provides a new primary cell culture method and supporting reagents for gastric cancer and cholangiocarcinoma of the gallbladder.
  • the core of the technology is: (1) Treat gastric cancer and cholangiocarcinoma with mild cell dissociation reagents Solid tumor tissues ensure the vitality of cancer cells in the tissues to the greatest extent; use a gentle method to isolate primary tumor cells of gallbladder cholangiocarcinoma in bile samples to ensure the vitality of cancer cells to the greatest extent; (2) Prepare a special serum-free medium , Use the suspension culture system to culture tumor cells derived from gastric cancer and gallbladder cholangiocarcinoma in vitro to ensure the normal expansion of cancer cells while eliminating the interference of normal cells to the greatest extent.
  • the present invention claims a medium for culturing primary cells of gastric cancer and/or gallbladder cholangiocarcinoma.
  • the medium for culturing primary cells of gastric cancer and/or gallbladder cholangiocarcinoma claimed by the present invention is composed of antibacterial and antifungal agents (penicillin-streptomycin-amphotericin B), HEPES, GlutaMax, and 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-fluoro Phenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole), A83-01(3-(6-Methyl-2-pyridinyl)-N-phenyl-4- (4-quinolinyl)-1H-pyrazole-1-carbothioami de), Primocin TM
  • the final concentration of penicillin in the antibacterial and antifungal agent is 100-200 U/mL (such as 100 U/mL); the final concentration of streptomycin in the antibacterial and antifungal agent is 100-200 ⁇ g/mL mL (such as 100 ⁇ g/mL); the final concentration of amphotericin B in the antibacterial and antifungal third antibody is 250-250ng/mL (such as 250ng/mL); the final concentration of HEPES is 8-12mM (such as 10mM); the final concentration of GlutaMax is 0.8-1.2% (such as 1%,% means volume percentage); the concentration of glycine in the non-essential amino acid is 80-120 ⁇ M; the L in the non-essential amino acid -The concentration of alanine is 80-120 ⁇ M (such as 100 ⁇ M); the concentration of L-asparagine in the non-essential amino acid is 80-120 ⁇ M (such as 100 ⁇ M); the L-asparagine in the
  • composition of the third antibacterial and antifungal agent is as follows: each milliliter contains 10,000 units of penicillin (base), 10,000 ⁇ g of streptomycin (base) and 25 ⁇ g of amphotericin B.
  • the third antibacterial and antifungal agent is "Antibiotic-Antimycotic, 100X” (such as Gibco#15240062, or other products with the same composition).
  • the "Antibiotic-Antimycotic, 100X” contains 10000 units penicillin (alkali), 10000 ⁇ g streptomycin (alkali) and 25 ⁇ g amphotericin B per milliliter, using penicillin G (sodium salt) and streptomyces sulfate in the form of 0.85% saline solution And amphotericin B as Antifungal agent.
  • the GlutaMAX is "GlutaMAX TM Supplement” (such as Gibco#35050061, or other products with the same composition).
  • the composition of the "GlutaMAX TM Supplement” is L-alanyl-L-glutamine, which is a substitute for L-glutamine, the concentration is 200 nM, and the solvent is 0.85% NaCl solution.
  • each milliliter of the non-essential amino acid contains 750 ⁇ g glycine, 890 ⁇ g L-alanine, 1320 ⁇ g L-asparagine, 1330 ⁇ g L-aspartic acid, 1470 ⁇ g L-glutamic acid, 1150 ⁇ g L -Proline, 1050 ⁇ g L-serine, and the solvent is water (the concentration of the various amino acids involved in the non-essential amino acids per ml is 10 mM).
  • the Primocin is an antibacterial agent for primary cells (such as Invivogene#ant-pm-1, or other products with the same composition). It is an antibiotic used to protect primary cells from microbial contamination.
  • the N-2 Supplement is "N-2 Supplement (100X)” (such as Gibco#17502001, or other products with the same composition).
  • the “N-2 Supplement (100X)” contains human transferrin (Holo) at a final concentration of 1 mM, 500 mg/L of recombinant insulin full chain (Insulin Recombinant Full Chain), 0.63 mg/L L progesterone (Progesterone), 10mM putrescine (Putrescine), 0.52mg/L selenite (Selenite).
  • the B27 is "B-27 TM Supplement (50X), minus vitamin A” (such as Gibco#12587010, or other products with the same composition).
  • the "B-27 TM Supplement (50X), minus vitamin A” contains biotin (Biotin), DL- ⁇ -tocopherol acetate (DL Alpha Tocopherol Acetate), DL- ⁇ -tocopherol (DL Alpha- Tocopherol), BSA (fatty acid free Fraction V), catalase (Catalase), human recombinant insulin (Human Recombinant Insulin), human transferrin (Human Transferrin), superoxide dismutase (Superoxide Dismutase), corticosterone (Corticosterone), D-Galactose (D-Galactose), Ethanolamine HCl, Glutathione (reduced), L-Carnitine HCl, Linoleic acid (Linoleic) Acid), Linolenic Acid, Progesterone, Putrescine 2HCl, Sodium Selenite, T3 (triodo-I-thyronine).
  • the solvent of the ITS-X is EBSS solution (Earle's balanced salt solution), and the solute and concentration are as follows: insulin 1g/L; transferrin 0.55g/L; sodium selenite 0.00067g/L; ethanolamine 0.2g/L.
  • the GlutaMAX is an advanced cell culture additive that can directly replace L-glutamine in cell culture media.
  • the GlutaMAX is "GlutaMAX TM Supplement" (such as Gibco#35050061, or other products with the same composition).
  • the Y-27632 is "Y-27632dihydrochloride (an ATP-competitive ROCK-I and ROCK-II inhibitor, Ki is 220nM and 300nM, respectively)" (such as MCE#129830-38-2, or its composition other products).
  • the brand product number of the third antibacterial and antifungal agent is Gibco#15240062; the brand product number of the HEPES is Gibco#15630080; The brand product number of GlutaMAX is Gibco#35050061; the brand product number of the non-essential amino acid is Gibco#11140050; the brand product number of the human recombinant protein EGF is Peprotech AF-100-15-100; the brand product number of the human recombinant protein bFGF It is Peprotech AF-100-18B-50; the brand product number of the human recombinant protein HGF is Peprotech AF-100-39-100; the brand product number of the human recombinant protein FGF-10 is Peprotech AF-100-26-100; The brand product number of the human recombinant protein Wnt-3a is R&D 5036-WN-500; the brand product number of the human recombinant protein Wnt-3a is R&D 5036-WN-500; the brand product number of the
  • the medium for culturing primary cells of gastric cancer and/or gallbladder cholangiocarcinoma can exist in two forms:
  • the medium for culturing primary cells of gastric cancer and/or gallbladder cholangiocarcinoma contains the antibacterial and antifungal agent, the third antibody, the HEPES, the GlutaMax, the non-essential amino acid, and the human recombinant Protein EGF, the human recombinant protein bFGF, the human recombinant protein HGF, the human recombinant protein FGF-10, the human recombinant protein Wnt-3a, the human recombinant protein Noggin, the SB202190, the A83- 01.
  • the Primocin TM the N-acetyl-L-cysteine, the nicotine, the N-2 Supplement, the cortisol, the B27, the ITS-X, the stomach A solution of secretin, the Y-27632 and the Advanced DMEM/F12 medium.
  • the medium After the medium is prepared, it needs to be filtered and sterilized with a 0.22 ⁇ M syringe filter (Millipore SLGP033RS), and can be stored at 4° C. for two weeks.
  • a 0.22 ⁇ M syringe filter (Millipore SLGP033RS)
  • each component in the culture medium for culturing primary cells of gastric cancer and/or gallbladder cholangiocarcinoma exists separately, and is prepared according to the formula during use.
  • human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein FGF-10, human recombinant protein Wnt-3a, and human recombinant protein Noggin can exist in the form of a stock solution (mother liquor) (-80 Stored at °C for a long time), specifically 1000 times the stock solution (mother liquor).
  • SB202190, N-acetyl-L-cysteine, Nicotinamide, Cortisol, Gastrin and Y-27632 can exist in the form of a stock solution (mother solution) (long-term storage at -20°C), and specifically can be a 1000 times stock solution (mother solution).
  • A83-01 can exist in the form of stock solution (mother liquor) (long-term storage at -20°C), specifically it can be 100,000 times stock solution (mother liquor).
  • the 1000 ⁇ human recombinant protein EGF stock solution is composed of human recombinant proteins EGF, BSA and PBS, wherein the final concentration of the human recombinant protein EGF is 20 ⁇ g/mL, the final concentration of the BSA is 0.01 g/mL, and the balance is both PBS.
  • the 1000 ⁇ human recombinant protein bFGF stock solution is composed of human recombinant protein bFGF, BSA and PBS, wherein the final concentration of the human recombinant protein bFGF is 20 ⁇ g/mL, the final concentration of the BSA is 0.01 g/mL, and the balance is both PBS.
  • the 1000 ⁇ human recombinant protein HGF stock solution is composed of human recombinant proteins HGF, BSA and PBS, wherein the final concentration of the human recombinant protein HGF is 20 ⁇ g/mL, the final concentration of the BSA is 0.01 g/mL, and the balance is both PBS.
  • the 1000 ⁇ human recombinant protein FGF-10 stock solution is composed of human recombinant proteins FGF-10, BSA and PBS, wherein the final concentration of the human recombinant protein FGF-10 is 20 ⁇ g/mL, and the final concentration of the BSA is 0.01 g/mL. mL, the balance is PBS.
  • the 1000 ⁇ human recombinant protein Wnt-3a stock solution is composed of human recombinant protein Wnt-3a, BSA and PBS, wherein the final concentration of the human recombinant protein Wnt-3a is 200 ⁇ g/mL, and the final concentration of the BSA is 0.01 g/mL. mL, the balance is PBS.
  • the 1000 ⁇ human recombinant protein Noggin stock solution is composed of human recombinant protein Noggin, BSA and PBS, wherein the final concentration of the human recombinant protein Noggin is 100 ⁇ g/mL, the final concentration of the BSA is 0.01 g/mL, and the balance is PBS.
  • the BSA can exist in the form of 100-fold stock solution (mother solution) (prepared for current use), specifically composed of BSA and PBS, and the final concentration of BSA (Sigma#A1933) is 0.1g /mL, the remainder is PBS.
  • the 1000 ⁇ SB202190 stock solution is composed of SB202190 and DMSO, wherein the final concentration of the SB202190 is 10 mM, and the balance is all DMSO.
  • the 100000 ⁇ A83-01 stock solution is composed of A83-01 and DMSO, wherein the concentration of A83-01 is 25 mM, and the balance is all DMSO.
  • the 1000 ⁇ N-acetyl-L-cysteine stock solution is composed of N-acetyl-L-cysteine and ultrapure water, wherein the concentration of the N-acetyl-L-cysteine is 0.5M, and the balance is all ultrapure water.
  • the 1000 ⁇ Nicotinamide stock solution is composed of Nicotinamide and ultrapure water, wherein the concentration of Nicotinamide is 5M, and the balance is all ultrapure water.
  • the 1000 ⁇ cortisol stock solution is composed of cortisol, absolute ethanol and ultrapure water, wherein the final concentration of the cortisol is 25 ⁇ g/mL, and the final concentration of the absolute ethanol is 5% (volume percentage), The balance is ultrapure water.
  • the 1000 ⁇ gastrin stock solution is composed of gastrin and ultrapure water.
  • the concentration of gastrin is 10 ⁇ M, and the balance is ultrapure water.
  • the 1000 ⁇ Y-27632 stock solution is composed of Y-27632 and ultrapure water.
  • the final concentration of Y-27632 is 10mM, and the balance is ultrapure water.
  • the present invention claims a kit of reagents for culturing primary cells of gastric cancer and/or gallbladder cholangiocarcinoma.
  • the set of reagents claimed in the present invention can be any of the following:
  • (A1) is composed of all or part of the culture medium described in the first aspect above: sample dissociation solution, sample preservation solution and sample cleaning solution.
  • (A2) is composed of the medium and cell separation buffer described in the first aspect above.
  • (A3) consists of (A1) and all or part of the following reagents: cell digestion solution, digestion stop solution and cell cryopreservation solution.
  • (A4) consists of (A2) and all or part of the following reagents: cell digestion solution, digestion termination solution and cell cryopreservation solution.
  • the sample dissociation solution is composed of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of the collagenase I is 150-250 U/mL (such as 200 U/mL); the collagenase II The final concentration of the collagenase IV is 150-250 U/mL (such as 200 U/mL); the final concentration of the collagenase IV is 50-150 U/mL (such as 100 U/mL); the balance is PBS.
  • the unit U of collagenase (the collagenase I, the collagenase II or the collagenase IV) is defined by the enzyme activity of the protease: at 37°C and pH 7.5, the collagenase is treated with 1 U protease (The collagenase I, the collagenase II, or the collagenase IV) can release 1 ⁇ mol of L-leucine in 5 hours.
  • the brand product number of the collagenase I is Gibco#17100-017.
  • the brand product number of the collagenase II is Gibco#17101-015;
  • the brand product number of the collagenase IV is Gibco#17104-019;
  • the brand product number of the PBS is Gibco#21-040-CVR.
  • the sample preservation solution is composed of fetal bovine serum, antibacterial and antifungal agents (penicillin-streptomycin-amphotericin B), HEPES and HBSS (Hank's balanced salt solution); wherein the final concentration of the fetal bovine serum 1-5% (such as 2%,% means volume percentage); the final concentration of penicillin in the antibacterial and antifungal agent (penicillin-streptomycin-amphotericin B) is 100-200U/mL (E.g.
  • the final concentration of streptomycin in the third antibacterial and antifungal agent is 100-200 ⁇ g/mL (such as 100 ⁇ g/mL);
  • the final concentration of amphotericin B in the antibacterial and antifungal third antibody is 250-500ng/mL (such as 250ng/mL);
  • the final concentration of HEPES is 8-12mM (Such as 10mM); the balance is HBSS.
  • composition of the third antibacterial and antifungal agent is as follows: each milliliter contains 10,000 units of penicillin (base), 10,000 ⁇ g of streptomycin (base) and 25 ⁇ g of amphotericin B.
  • the third antibacterial and antifungal agent is "Antibiotic-Antimycotic, 100X” (such as Gibco#15240062, or other products with the same composition).
  • the "Antibiotic-Antimycotic, 100X” contains 10000 units penicillin (alkali), 10000 ⁇ g streptomycin (alkali) and 25 ⁇ g amphotericin B per milliliter, using penicillin G (sodium salt) and streptomyces sulfate in the form of 0.85% saline solution And amphotericin B as Antifungal agent.
  • the brand product number of the fetal bovine serum is Gibco#16000-044; the brand product number of the third antibacterial and antifungal agent (penicillin-streptomycin-amphotericin B) is Gibco# 15240062; the brand item number of the HEPES is Gibco#15630080; the brand item number of the HBSS is Gibco#14170161.
  • the sample cleaning solution is composed of a third antibacterial and antifungal agent (penicillin-streptomycin-amphotericin B) and PBS; wherein, the third antibacterial and antifungal agent (penicillin-streptomycin-amphotericin B)
  • penicillin-streptomycin-amphotericin B The final concentration of penicillin is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the antibacterial and antifungal agent (penicillin-streptomycin-amphotericin B) is 100- 200 ⁇ g/mL (such as 100 ⁇ g/mL); the final concentration of amphotericin B in the antibacterial and antifungal agent (penicillin-streptomycin-amphotericin B) is 250-500ng/mL (such as 250ng/mL ); The balance is PBS.
  • composition of the third antibacterial and antifungal agent is as follows: each milliliter contains 10,000 units of penicillin (base), 10,000 ⁇ g of streptomycin (base) and 25 ⁇ g of amphotericin B.
  • the third antibacterial and antifungal agent is "Antibiotic-Antimycotic, 100X” (such as Gibco#15240062, or other products with the same composition).
  • the "Antibiotic-Antimycotic, 100X” contains 10000 units penicillin (alkali), 10000 ⁇ g streptomycin (alkali) and 25 ⁇ g amphotericin B per milliliter, using penicillin G (sodium salt) and streptomyces sulfate in the form of 0.85% saline solution And amphotericin B as Antifungal agent.
  • the brand product number of the third antibacterial and antifungal agent is Gibco#15240062; the brand product number of the PBS is Gibco#21-040- CVR.
  • the cell separation buffer is composed of double antibodies P/S (penicillin-streptomycin), heparin sodium and PBS; wherein the final concentration of penicillin in the double antibodies P/S (penicillin-streptomycin) is 100 -200U/mL (such as 100U/mL); the final concentration of streptomycin in the double antibody P/S (penicillin-streptomycin) is 100-200 ⁇ g/mL (such as 100 ⁇ g/mL); the heparin sodium The final concentration is 10IU/mL; the rest is PBS.
  • the brand product number of the double antibody P/S penicillin-streptomycin
  • the brand product number of the heparin sodium is Solarbio#H8270
  • the brand product number of the PBS is Gibco#21-040-CVR.
  • each 10mL of the cell digestion solution contains 4-6mL (such as 5mL) Accutase, a final concentration of 5mM EDTA (ie 10 ⁇ L 0.5M EDTA), 1.5-2.5mL (such as 2mL) TrypLE Express ,
  • the balance is PBS.
  • the Accutase is "StemPro TM Accutase TM Cell Dissociation Reagent” (such as Gibco#A11105-01, or other products with the same composition).
  • the Accutase is a single-component enzyme, dissolved in D-PBS, 0.5mM EDTA solution.
  • the TrypLE Express is "TrypLE TM Express Enzyme (1X), no phenol red” (such as Gibco#12604013, or other products with the same composition).
  • the "TrypLE TM Express Enzyme (1X), no phenol red” contains 200mg/L KCl, 200mg/L KH 2 PO 4 , 8000mg/L NaCl, 2160mg/L Na 2 HPO 4 ⁇ 7H 2 O , 457.6mg/L EDTA; also contains recombinant protease.
  • the brand product number of Accutase is Gibco#A11105-01; the brand product number of the 0.5M EDTA is Invitrogen#AM9261; the brand product number of the TrypLE Express is Gibco#12604013; the PBS The brand item number is Gibco#21-040-CVR.
  • the digestion termination solution is composed of fetal bovine serum, antibacterial and antifungal agents (penicillin-streptomycin-amphotericin B) and DMEM medium; wherein the final concentration of the fetal bovine serum is 8-12% ( For example, 10%,% means volume percentage content); the final concentration of penicillin in the third antibacterial and antifungal agent (penicillin-streptomycin-amphotericin B) is 100-200U/mL (such as 100U/mL) The final concentration of streptomycin in the third antibacterial and antifungal agent (penicillin-streptomycin-amphotericin B) is 100-200 ⁇ g/mL (such as 100 ⁇ g/mL); the third antibacterial and antifungal agent The final concentration of amphotericin B in (penicillin-streptomycin-amphotericin B) is 250-500ng/mL (such as 250ng/mL); the balance is all DMEM medium.
  • composition of the third antibacterial and antifungal agent is as follows: each milliliter contains 10,000 units of penicillin (base), 10,000 ⁇ g of streptomycin (base) and 25 ⁇ g of amphotericin B.
  • the third antibacterial and antifungal agent is "Antibiotic-Antimycotic, 100X” (such as Gibco#15240062, or other products with the same composition).
  • the "Antibiotic-Antimycotic, 100X” contains 10000 units penicillin (alkali), 10000 ⁇ g streptomycin (alkali) and 25 ⁇ g amphotericin B per milliliter, using penicillin G (sodium salt) and streptomyces sulfate in the form of 0.85% saline solution And amphotericin B as Antifungal agent.
  • the brand product number of the fetal bovine serum is Gibco#16000-044; the brand product number of the third antibacterial and antifungal agent (penicillin-streptomycin-amphotericin B) is Gibco# 15240062; The brand product number of the DMEM medium is Gibco#11965-092.
  • the cell cryopreservation solution is composed of Advanced DMEM/F12 medium, DMSO and 1% methylcellulose solution; wherein, the Advanced DMEM/F12 medium, the DMSO and the 1% methylcellulose solution are The volume ratio is 20:2:(0.8-1.2), such as 20:2:1; the 1% methylcellulose solution is a methylcellulose aqueous solution with a concentration of 1g/100ml.
  • the brand product number of the Advanced DMEM/F12 medium is Gibco#12634010; the brand product number of the DMSO is Sigma#D2438; and the brand product number of the methylcellulose is Sigma#M7027.
  • the sample preservation solution can be used for the temporary preservation of the sample after in vitro, and can maintain the activity of the cells in the sample in a short time after the sample is in vitro.
  • the sample storage solution can be stored for 1 month at 4°C after preparation.
  • the sample cleaning solution can be used for cleaning and disinfecting samples.
  • the sample cleaning solution needs to be prepared and used.
  • the sample dissociation solution can be used for the dissociation of the sample, and can dissociate the primary cells of the solid tumor of gastric cancer and/or gallbladder cholangiocarcinoma in the sample from the tissue.
  • the sample dissociation solution needs to be prepared for immediate use.
  • the collagenase I, collagenase II, and collagenase IV can be stored as a stock solution (mother solution) at -20°C for long-term storage, specifically 10 or 20 times the stock solution (mother solution) .
  • the 10 ⁇ collagenase I stock solution is composed of the collagenase I and PBS; wherein the final concentration of the collagenase I is 2000 U/mL; the 10 ⁇ collagenase II stock solution is composed of the collagenase II and PBS; The final concentration of the collagenase II is 2000 U/mL; the remainder is PBS; the 20 ⁇ collagenase IV stock solution is composed of the collagenase IV and PBS; wherein the final concentration of the collagenase IV is 2000 U/mL; The amount is PBS.
  • the enzymatic activities of the collagenase I, collagenase II and collagenase IV are defined in the foregoing.
  • the cell separation buffer is used to suspend cells in a bile sample. After the cell separation buffer is prepared, it can be stored at 4°C for 1 month.
  • the cell digestion solution can be used for the digestion and passage of cell masses, and can digest gastric cancer and/or gallbladder cholangiocarcinoma tumor masses into single cells.
  • the cell digestion solution needs to be prepared and used.
  • the digestion termination solution can be used to terminate the process of sample dissociation or cell digestion.
  • the digestion termination solution can be stored at 4°C for one month after preparation.
  • the gastric cancer and/or gallbladder cholangiocarcinoma primary cell culture medium can be used for the culture of gastric cancer and/or gallbladder cholangiocarcinoma primary cells.
  • the cell cryopreservation solution needs to be prepared and used.
  • the 1% methylcellulose solution can be stored for a long time at 4°C.
  • the present invention claims a method for culturing primary cells of gastric cancer and/or gallbladder cholangiocarcinoma.
  • the method for culturing primary cells of gastric cancer and/or gallbladder cholangiocarcinoma claimed in the present invention can be method A or method B:
  • Method A A method for culturing primary cells from solid tumors of gastric cancer and/or gallbladder cholangiocarcinoma, including the following steps:
  • Method B A method for culturing primary tumor cells from a bile sample of gallbladder cholangiocarcinoma, including the following steps:
  • the sample dissociation solution can be used to dissociate the solid tumor tissue of gastric cancer and/or gallbladder cholangiocarcinoma according to a method including the following steps: press 0.1-0.3mL (such as 0.1mL) The amount of the sample dissociation solution per mg of tissue is used to preheat the gastric cancer and/or gallbladder cholangiocarcinoma solid tumor tissue (for example, cut into small pieces of 0.8-1.2 mm 3 ) at 37°C. The sample dissociation solution is processed, and the sample is dissociated at 37° C., and the dissociation time is 15 minutes to 3 hours. Observe the dissociation of the sample under the microscope every 15 minutes until a large number of single cells are observed.
  • step (b1) the primary tumor cells of the gallbladder cholangiocarcinoma bile sample are separated from the gallbladder cholangiocarcinoma bile sample according to the method including the following steps: the gallbladder is suspended in the cell separation buffer described in the second aspect above The cells in the bile sample of cholangiocarcinoma are then subjected to density gradient centrifugation (using Ficoll lymphocyte separation solution) to obtain primary tumor cells in the bile sample of cholangiocarcinoma.
  • the primary gastric cancer and/or gallbladder cholangiocarcinoma solid tumor primary cells can be suspended and cultured in the medium according to the method including the following steps: use the cell culture container M to suspend the medium Cultivate the primary cells of the gastric cancer and/or gallbladder cholangiocarcinoma solid tumor at 37°C and 5% CO 2 and change the medium every 2-4 days (such as 3 days) until the cells form a diameter of 50-80 ⁇ m (Such as 80 ⁇ m) clumps.
  • the primary tumor cells of the gallbladder cholangiocarcinoma bile sample can be suspended and cultured in the medium according to the method including the following steps: using the cell culture container M, the medium is used to suspend the culture of the The primary tumor cells from the bile sample of gallbladder cholangiocarcinoma were cultured at 37°C and 5% CO 2 and the medium was changed every 2-4 days (such as 3 days) until the cells formed a cluster with a diameter of 50-80 ⁇ m (such as 80 ⁇ m) Piece.
  • the initial seeding density of 10 5 / cm 2 container bottom area an example in six-well plates, at a density of 106 per well were plated cells.
  • the cell culture container M can be any of the following: (1) a cell culture container made of polystyrene, a cell culture container made of polycarbonate, a cell culture container made of polymethyl methacrylate, a COC resin material A cell culture container, a cell culture container made of cycloolefin polymer, or a cell culture container with a low adsorption surface; (II) The cell culture container in (I) is modified by CYTOP.
  • the cell culture container is a cell culture dish, a cell culture well plate or a microplate chip for cell culture.
  • the cell culture container in (I) can be modified by CYTOP according to the method including the following steps: the cell culture container in (I) is etched with pure oxygen, and the etching conditions are The power is 20W and the etching time is 3 minutes; then the surface of the cell culture container is covered with 1% CYTOP solution, and the 1% CYTOP solution is dried to complete the CYTOP modification.
  • composition of the 1% CYTOP solution is as follows: every 100 mL of the 1% CYTOP solution contains 1 mL of CYTOP, and the balance is fluorine oil.
  • step (a1) the following step of dissociating the solid tumor tissue of the gastric cancer and/or gallbladder cholangiocarcinoma may also be included: washing the gastric cancer and/or the gastric cancer with 70-75% ethanol by volume / Or gallbladder cholangiocarcinoma solid tumor tissue sample surface; clean the gastric cancer and/or gallbladder cholangiocarcinoma solid tumor tissue sample 10-20 times (such as 10 times) with the sample cleaning solution described in the second aspect of the previous article, with sterile PBS solution washes the gastric cancer and/or gallbladder cholangiocarcinoma solid tumor tissue sample 5-10 times (such as 5 times); then removes impurities, connective tissue, and adipose tissue in the gastric cancer and/or gallbladder cholangiocarcinoma solid tumor tissue sample , Necrotic tissue and other components that affect primary cell culture.
  • gastric cancer and/or gallbladder cholangiocarcinoma solid tumor tissue sample subjected to the pre-dissociation treatment is isolated within 2 hours, and it has been stored in the second aspect of the foregoing before the pre-dissociation treatment In the sample preservation solution described in.
  • step (a1) after dissociating the gastric cancer and/or gallbladder cholangiocarcinoma solid tumor tissue with the sample dissociation solution, the method further includes the following steps: using 8-15 times (such as 10 times)
  • the volume of the digestion stop solution described in the second aspect of the preceding paragraph terminates the dissociation reaction, and the cell suspension is collected; the cell suspension is filtered with a 100 ⁇ m or 40 ⁇ m sterile cell strainer to remove tissue debris and adherent cells; 800-1000g (such as 800g) centrifuge at room temperature for 10-15 minutes (such as 10 minutes), discard the supernatant; then resuspend the cells with 3-5mL (such as 5mL) sterile PBS; then centrifuge at 800-1000g (such as 800g) at room temperature for 10-15 minutes ( For example, 10 minutes), discard the supernatant; then resuspend the cell pellet with the medium described in the first aspect above, observe the cell status under a microscope, and count the cells.
  • step (b1) it also includes a step of pre-processing the bile sample of gallbladder cholangiocarcinoma: removing impurities, clots and other components that affect cell density gradient separation in the bile sample of gallbladder cholangiocarcinoma.
  • step (a2) the following step may be further included: when the primary cells of the gastric cancer and/or gallbladder cholangiocarcinoma solid tumor form a mass with a diameter of 50-80 ⁇ m (such as 80 ⁇ m), the gastric cancer and / Or the primary cells of solid tumor of gallbladder cholangiocarcinoma are passaged.
  • step (b2) the following step may be further included: when the primary tumor cells of the gallbladder cholangiocarcinoma bile sample form a mass with a diameter of 50-80 ⁇ m (such as 80 ⁇ m), the gallbladder cholangiocarcinoma bile sample The primary tumor cells are passaged.
  • the cell digestion solution used in the passage is the cell digestion solution described in the second aspect above.
  • the digestion termination solution used in the passage is the digestion termination solution described in the second aspect above.
  • the digestion temperature used during the passage is 37°C.
  • the step of passage collect the cell clumps to be passaged, wash the cell clumps with sterile PBS solution after centrifugation, centrifuge again, and then resuspend the cell clumps with the cell digestion solution at 37 Perform digestion at °C until the cell clumps are digested into single cells, stop the digestion reaction with the digestion stop solution (the amount can be 5-10 times, such as 10 times the volume), collect the cell suspension; use after centrifugation in the foregoing first aspect of the media cell pellet was resuspended, counted, and then use the previously described cell culture vessel M cell suspension culture (initial seeding density of 10 5 / cm 2 area of the bottom of the container to an example six-well plates , Plated at a density of 10 6 cells per well), culture conditions are 37°C, 5% CO 2 . All centrifugation in the above passage steps can specifically be 800-1000g (such as 800g) centrifugation at room temperature for 10-20 minutes (such as 10 minutes).
  • the method may further include freezing and storing the primary cells of the gastric cancer and/or gallbladder cholangiocarcinoma solid tumor or the primary tumor cells of the gallbladder cholangiocarcinoma bile sample after 2-3 passages and expansion. / Or steps of resuscitation.
  • the cell cryopreservation solution used when performing the cryopreservation is the cell cryopreservation solution described in the second aspect above.
  • the specific steps of the freezing storage are performed: collecting the cell clumps to be frozen, washing the cell clumps with sterile PBS solution after centrifugation, and then centrifuging, and then resuspending the cell clumps with the cell digestion solution , Perform digestion at 37°C until the cell clumps are digested into single cells, stop the digestion reaction with the digestion stop solution (the amount can be 5-10 times, such as 10 times the volume), and collect the cell suspension; After centrifugation, use the cell cryopreservation solution to resuspend the cell pellet at a density of 0.5-2 ⁇ 10 6 /mL (for example, 10 6 /mL), and transfer to liquid nitrogen for long-term storage after freezing in a gradient cooling box overnight. All centrifugation in the above freezing step can specifically be 800-1000g (such as 800g) centrifugation at room temperature for 10-20 minutes (such as 10 minutes).
  • the cryotube containing the cells to be resuscitated from liquid nitrogen quickly thaw the cells in sterile water at 37-39°C (such as 37°C); centrifuge (such as 800-1000g) , Such as 800g room temperature centrifugation for 5-10 minutes, such as 10 minutes), then resuspend the cell pellet with the medium described in the first aspect above, and then use the cell culture vessel M described above to suspend the cells (the initial seeding density can be 10 5 Cells/cm 2 container bottom area), each tube of cells (10 6 cells) was recovered to a 3.5 cm petri dish), and the culture condition was 37°C, 5% CO 2 .
  • the initial seeding density can be 10 5 Cells/cm 2 container bottom area
  • each tube of cells (10 6 cells) was recovered to a 3.5 cm petri dish
  • the culture condition was 37°C, 5% CO 2 .
  • the present invention claims any of the following reagents:
  • C2 A tissue sample preservation solution for solid tumors of gastric cancer and/or gallbladder cholangiocarcinoma, which is the sample preservation solution described in the second aspect above;
  • the present invention claims any of the following methods:
  • (E1) A method for dissociating gastric cancer and/or gallbladder cholangiocarcinoma solid tumor primary cells from gastric cancer and/or gallbladder cholangiocarcinoma solid tumor tissue, including step (a1) in the method described in the fourth aspect above.
  • (E2) A method for preserving gastric cancer and/or gallbladder cholangiocarcinoma solid tumor tissue, comprising the steps of: placing the newly isolated gastric cancer and/or gallbladder cholangiocarcinoma solid tumor tissue in the sample preservation solution described in the second aspect above Stored in medium, the preservation time is within 2 hours.
  • (E3) A method for isolating primary tumor cells from a gallbladder cholangiocarcinoma bile sample from a gallbladder cholangiocarcinoma bile sample, including step (b1) in the method described in the fourth aspect above.
  • the gastric cancer may be primary gastric cancer;
  • the gallbladder cholangiocarcinoma may be primary gallbladder cholangiocarcinoma.
  • the gastric cancer may be a metastatic lesion of gastric cancer
  • the cholangiocarcinoma of the gallbladder may be a metastatic lesion of cholangiocarcinoma of the gallbladder.
  • the primary gastric cancer cell may be a primary gastric cancer solid tumor cell;
  • the gallbladder cholangiocarcinoma primary cell may be a primary cell of a solid tumor of the gallbladder cholangiocarcinoma or a primary tumor cell of a bile sample of a gallbladder cholangiocarcinoma.
  • the primary gastric cancer cells can be isolated from surgical samples of patients with gastric cancer; the primary cells of gallbladder cholangiocarcinoma can be isolated from surgical samples (solid tumor samples) and puncture samples (for Solid tumor samples) or bile samples.
  • the clinical staging of gastric cancer is stage II, stage III or stage IV (according to TNM staging).
  • surgical specimens weigh more than 20mg.
  • the clinical staging of the gallbladder cholangiocarcinoma is stage II, stage III or stage IV (according to TNM staging).
  • the weight of solid tumor tissue specimens of gallbladder and cholangiocarcinoma obtained from surgical samples should preferably exceed 20 mg.
  • the best bile sample is not less than 10mL. There are no less than 4 puncture samples.
  • PBSs can be 1 ⁇ PBS, pH 7.3-7.5.
  • the specific composition is as follows: the solvent is water, the solute and the concentration are: KH 2 PO 4 144 mg/L, NaCl 9000 mg/L, and Na 2 HPO 4 ⁇ 7H 2 O 795 mg/L.
  • Figure 1 shows the single cells obtained after treatment of gastric cancer tissue.
  • the scale is 100 ⁇ m, 100 times magnification.
  • Figure 2 shows the cell mass obtained after primary culture of gastric cancer tissue.
  • the scale is 100 ⁇ m, 100 times magnification.
  • Figure 3 is a HE staining image of a section of gastric cancer cell mass obtained after primary culture of gastric cancer tissue.
  • the scale is 100 ⁇ m, 200 times magnification.
  • Figure 4 is an immunofluorescence staining image of cancer cell masses obtained after primary culture of gastric cancer tissue.
  • the scale is 50 ⁇ m, 200 times magnification
  • Figure 5 shows that copy number variation analysis (CNV) based on the sequencing results shows that the copy number variation of primary gastric cancer cell cultures (P1, P2, P3, P4, P5) and primary gastric cancer tumor tissue (Tumor) are highly consistent .
  • CNV copy number variation analysis
  • Figure 6 shows the in vitro drug sensitivity test results of primary gastric cancer cells cultured in the present invention.
  • Figure 7 shows single cells obtained after treatment of cholangiocarcinoma tissue.
  • the scale is 100 ⁇ m, 100 times magnification.
  • Figure 8 shows cell masses obtained after primary culture of cholangiocarcinoma tissue.
  • the scale is 100 ⁇ m, 100 times magnification.
  • Figure 9 is an HE staining image of a cholangiocarcinoma cell mass section obtained after primary culture of cholangiocarcinoma tissue.
  • the scale is 100 ⁇ m, 200 times magnification.
  • Figure 10 is an immunohistochemical staining image of a paraffin section of cancer cell mass obtained after primary culture of cholangiocarcinoma tissue.
  • the scale is 100 ⁇ m, 200 times magnification.
  • Figure 11 shows the single cells obtained after processing a bile sample from cholangiocarcinoma.
  • the scale is 100 ⁇ m, 100 times magnification.
  • Figure 12 shows cell clumps obtained after primary culture of bile samples from cholangiocarcinoma.
  • the scale is 100 ⁇ m, 100 times magnification.
  • Figure 13 is an HE staining image of a section of a cholangiocarcinoma cell mass obtained after primary culture of a cholangiocarcinoma bile sample.
  • the scale is 100 ⁇ m, 100 times magnification.
  • Figure 14 is an immunohistochemical staining image of a paraffin section of cancer cell mass obtained after primary culture of a bile sample of cholangiocarcinoma.
  • the scale is 50 ⁇ m, 100 times magnification.
  • Figure 15 is a design diagram of the microplate chip of the present invention.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.
  • the quantitative tests in the following examples are all set to three repeated experiments, and the results are averaged.
  • Example 1 Preparation of reagents for culturing gastric cancer primary cells
  • sample preservation solution After the sample preservation solution is prepared, it is divided into 15 mL centrifuge tubes, 5 mL per tube. After aliquoting, it can be stored at 4°C for 1 month.
  • the sample cleaning solution needs to be prepared for immediate use.
  • sample dissociation solution is prepared for immediate use.
  • the unit U of collagenase (the collagenase I or the collagenase IV) is defined by the enzyme activity of the protease: under the conditions of 37°C and pH 7.5, treatment with 1 U protease Collagenase (the collagenase I or the collagenase IV) can release 1 ⁇ mol of L-leucine in 5 hours.
  • the cell digestion solution is prepared for immediate use.
  • the digestion stop solution After the digestion stop solution is prepared, it can be stored at 4°C for one month.
  • the primary cell culture medium for gastric cancer solid tumors After the preparation of the primary cell culture medium for gastric cancer solid tumors is completed, it is filtered and sterilized with a 0.22 ⁇ M syringe filter (Millipore SLGP033RS), and can be stored at 4°C for two weeks.
  • a 0.22 ⁇ M syringe filter (Millipore SLGP033RS)
  • Table 9 the preparation of human recombinant protein stock solution is shown in Table 11-16, the preparation of SB202190 stock solution is shown in Table 17, and the preparation of A83-01 stock solution is shown in Table 18.
  • N-acetyl-L- The preparation of cysteine stock solution is shown in Table 19, the preparation of Nicotinamide stock solution is shown in Table 20, the preparation of Cortisol stock solution is shown in Table 21, and the preparation of gastrin stock solution is shown in Table 22, Y-27632
  • the preparation of the stock solution is shown in Table 23.
  • the preparation of the 100 ⁇ BSA solution required when preparing these stock solutions is shown in Table 10.
  • the 100 ⁇ BSA solution is prepared for immediate use.
  • the 1% methylcellulose solution can be stored for a long time at 4°C after preparation.
  • the 1% CYTOP solution After the 1% CYTOP solution is prepared, it can be stored at room temperature for a long time.
  • the attending physician selects patients to be enrolled in accordance with the clinical indications specified in the medical guidelines, and selects appropriate samples for in vitro culture according to the clinical indications during the operation.
  • the selection criteria for samples are: primary gastric cancer, and the pathological stage is stage II , Stage III or Stage IV, various pathological types of gastric cancer or gastric cancer metastases, gastric cancer surgical specimens weighing more than 20mg samples.
  • the attending doctor provides basic clinical information such as the patient's gender, age, medical history, family history, smoking history, pathological staging, clinical diagnosis, etc.
  • the patient’s name, ID number and other information related to the patient’s privacy are concealed and replaced with a uniform experiment number.
  • the naming principle of the experiment number is the eight-digit date of the sample collection + the last four digits of the patient’s hospitalization number. For example, if the sample provided on January 1, 2018, the hospitalization number of the patient is T001512765, the sample experiment number is 201801012765.
  • the surgical equipment used in the following operations must be sterilized in advance by high temperature and high pressure, and can be used after drying.
  • the surgical instruments used in the following embodiments need to be sterilized by high temperature and high pressure in advance, and can be used after drying.
  • sample dissociation solution see Example 1
  • the medium used is the gastric cancer solid tumor primary cell culture medium in Example 1 (wherein, the final concentration of human recombinant protein EGF is 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
  • the concentration is 250ng/mL; the final concentration of human recombinant protein Noggin is 100ng/mL; the final concentration of SB202190 is 10 ⁇ M; the final concentration of A83-01 is 0.5 ⁇ M; the final concentration of N-acetyl-L-cysteine is 1mM; the final concentration of Nicotinamide
  • the final concentration is 10mM; the final concentration of cortisol is 25ng/mL; the final concentration of Y-27632 is 10 ⁇ M), take
  • cancer cells expanded to form cell clumps with a diameter of 80 ⁇ m.
  • the total number of tumor cells could exceed 10 7 , and the number of other types of cells was significantly reduced or even disappeared.
  • the method has been tested on a large number of samples, and the success rate of in vitro culture of primary gastric cancer cells can reach 80%.
  • the medium used is the gastric cancer solid tumor primary cell culture medium in Example 1. Take a six-well plate as an example, press 10 per well. The density of 6 cells is plated and cultured in a cell incubator at 37°C and 5% CO 2 .
  • the primary gastric cancer cells in suspension culture can be cryopreserved after 2-3 passages and expansion:
  • the cell freezing medium (see Example 1), according to 10 6 / mL density cell pellet was resuspended, 2 mL cryotubes 1mL of cell suspension per tube, the gradient was transferred to a liquid nitrogen cooling box overnight after long-term cryopreservation save.
  • the primary gastric cancer cells stored in liquid nitrogen can be recovered:
  • the gastric cancer solid tumor primary cell culture medium in Example 1 is collected by 800g centrifugation (where the final concentration of human recombinant protein EGF is 20ng/mL; the final concentration of human recombinant protein bFGF is 20ng/mL; human recombinant protein HGF
  • 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
  • the final concentration is 5 ⁇ M; the final concentration of A83-01 is 1 ⁇ 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; the final concentration of Y-27632 It is 10 ⁇ M) cultured primary gastric cancer solid tumor cell masses, fixed with 4% paraform
  • Figure 3 shows the HE staining effect of primary gastric cancer cells cultured in vitro. It can be seen that these cells generally have the characteristics of high nucleus to cytoplasmic ratio, deep staining of nuclei, intranuclear chromatin agglutination, multinucleation, and uneven cell size. , Dozens to hundreds of tumor cells gather to form tumor cell clusters with a certain three-dimensional structure.
  • Paraformaldehyde (Beijing Chemical Reagent Company, Analytical Pure), dissolve the paraformaldehyde powder in ultrapure water to prepare a 4% (4g/100mL) paraformaldehyde solution;
  • Methanol, dimethyl sulfoxide, and 35% hydrogen peroxide are mixed in a ratio of 4:4:1 (volume ratio) to make Dan's rinse solution;
  • Bovine serum albumin (Sigma, #A1933), dissolve bovine serum albumin with PBS solution to prepare a 3% (3g/100mL) BSA solution;
  • Example 1 where 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 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 The concentration is 10 ⁇ M; the final concentration of A83-01 is 0.5 ⁇ 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; the final concentration of Y-27632 Immunofluorescence staining was performed on the gastric cancer cell mass obtained by culture at 10 ⁇ M.
  • the concentration of human recombinant protein EGF is 50ng/
  • Figure 4 shows the effect of immunofluorescence staining of gastric cancer primary tumor cell clumps cultured in vitro. It can be seen that the cells that make up the cell clumps are all CK8/CK18 positive and are of epithelial origin, which confirms that the culture obtained by this method is Tumor cells with higher purity.
  • the primary cultures of 20 gastric cancer samples were identified by immunofluorescence staining. The statistical results showed that the proportion of tumor cells in the primary gastric cancer cells obtained by this method reached 70%-93% (Table 27).
  • Example 11 Primary gastric cancer cell culture and primary tumor tissue
  • the DNA extraction process mentioned in the following examples is performed using Tiangen Blood/Tissue/Cell Genome Extraction Kit (DP304).
  • the library construction process mentioned in the following examples is performed using NEB DNA sequencing library construction kit (E7645).
  • the high-throughput sequencing mentioned in the following examples refers to the Illumina Hiseq X-ten sequencing platform.
  • Example 2 After gastric cancer tissue is treated, use the gastric cancer solid tumor primary cell culture medium 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; human recombinant protein The final concentration of 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; SB202190 The final concentration of A83-01 is 10 ⁇ M; the final concentration of A83-01 is 1 ⁇ M; the final concentration of N-acetyl-L-cysteine is 1mM; the final concentration of Nicotinamide is 8mM; the final concentration of cortisol is 25ng/mL; the final concentration of Y-27632 (Concentration of 8 ⁇ M) After a period
  • Example 12 Comparison of the success rate of culturing gastric cancer primary cells with different primary cell culture media
  • Table 28 Formulation of primary cell culture medium for testing (100mL)
  • Each of the four primary cell culture media plans processed 20 samples.
  • the samples were processed and cultured according to the methods described in Examples 3, 4, and 5. After 10 days of culture, the success rate of primary cell culture of gastric cancer solid tumors was calculated as shown in Table 29 Show:
  • the primary cell culture medium has a great influence on the success rate of gastric cancer primary cells.
  • the gastric cancer solid tumor primary cell culture medium (Table 9) used in the present invention can stimulate the cancer in the gastric cancer solid tumor tissue sample to the greatest extent. Cell proliferation improves the success rate of primary cell culture of gastric cancer solid tumors.
  • Example 13 Comparison of the success rate of culturing gastric cancer primary cells with different sample preservation solutions
  • Each of the five sample preservation solutions processes 20 samples. After the samples are separated, they are temporarily stored in the sample preservation solution at 4°C. After being separated for 2 hours, the samples are processed and cultured according to the methods described in Examples 3, 4, and 5 After 10 days of culture, the success rate of primary cell culture of gastric cancer solid tumors is calculated as shown in Table 31:
  • sample preservation solution formula has a greater impact on the success rate of gastric cancer solid tumor primary cell culture.
  • the sample preservation solution (Table 1) used in the present invention can protect the cancer cells in the gastric cancer solid tumor tissue sample to the greatest extent. Activity, improve the success rate of cultivation.
  • Example 14 Comparison of the success rate of culturing gastric cancer primary cells with different sample dissociation solutions
  • the operation method and flow of the primary culture of all samples are completely the same (refer to the above), and only the sample dissociation solution formula is different.
  • the various sample dissociation fluids tested are shown in Table 32.
  • the scheme D is the formula used in the present invention, see Table 3 for details.
  • the sample dissociation solution is prepared for immediate use.
  • sample dissociation solution formula has a great influence on the success rate of gastric cancer solid tumor primary cell culture.
  • the sample dissociation solution used in the present invention (Table 3) can isolate cancer cells in the gastric cancer solid tumor tissue to the greatest extent , Improve the success rate of primary cell culture of gastric solid tumors.
  • Example 15 Comparison of the success rate of passage of primary gastric cancer cells in digestive juices of different cells
  • the cell digestion solution is prepared for immediate use.
  • the cell digestion solution (Table 7) used in the present invention can gently dissociate the cancer cells in the cell mass and make the sample It can be serially passaged to maintain the primary cell viability of gastric cancer solid tumors.
  • Example 16 Using cell culture consumables of different materials for gastric cancer primary tumor cell culture
  • PS Polystyrene
  • PC polycarbonate
  • PMMA poly-methyl methacrylate
  • COC resin Cyclo Olefin Polymer
  • Abbreviation COP Abbreviation COP
  • LAS low-attachment-surface
  • Example 17 Using CYTOP modified cell culture consumables for gastric cancer primary tumor cell culture
  • the method of CYTOP modification is as follows: firstly, the cell culture container is etched with pure oxygen, the etching condition is 20W power, and the etching time is 3 minutes. Then cover the surface of the petri dish or the culture plate with an appropriate amount (in a 96-well plate, 20 ⁇ L per well, an appropriate amount means completely covering the bottom of the petri dish) 1% CYTOP solution, and use it after the CYTOP solution is completely dried.
  • Example 18 Drug sensitivity test with gastric cancer primary tumor cells
  • the chemotherapy drugs Irinotecan, 5-Fluorouracil, and Oxaliplatin used in this example are all Selleck products.
  • the Celltiter-Glo cell viability detection kit mentioned in this example is a Promega product.
  • Example 20 Obtainment of postoperative specimens/biopsy puncture specimens for gallbladder cancer and cholangiocarcinoma
  • the attending doctor selects patients to be enrolled in accordance with the clinical indications specified in the medical guidelines, and selects appropriate samples for in vitro culture according to the intraoperative clinical indications.
  • the selection criteria for surgical samples are: primary gallbladder cancer or cholangiocarcinoma, the pathological stage is stage II, stage III, or stage IV, various pathological types of gallbladder cancer or cholangiocarcinoma metastasis, and the surgical specimen weight exceeds 20 mg.
  • the selection criteria for biopsy puncture samples are: primary gallbladder cancer or cholangiocarcinoma, the pathological stage is stage II, III or IV, various pathological types of gallbladder cancer or cholangiocarcinoma metastasis, and more than 4 puncture specimens .
  • the attending doctor provides basic clinical information such as the patient's gender, age, medical history, family history, smoking history, pathological staging, clinical diagnosis, etc.
  • the patient’s name, ID number and other information related to the patient’s privacy are concealed and replaced with a uniform experiment number.
  • the naming principle of the experiment number is the eight-digit date of the sample collection + the last four digits of the patient’s hospitalization number. For example, if the sample provided on January 1, 2018, the hospitalization number of the patient is T001512765, the sample experiment number is 201801012765.
  • the puncture surgeon collects fresh puncture specimens in a sterile environment in the puncture operating room, and places them in the pre-prepared sample preservation solution (see Example 19). After the sample is separated, it is temporarily stored on ice and transported to the laboratory within two hours for the next operation.
  • Example 21 Pretreatment of gallbladder cancer and cholangiocarcinoma samples before dissociation
  • the surgical equipment used in the following operations must be sterilized in advance by high temperature and high pressure, and can be used after drying.
  • Example 22 Dissociation of gallbladder cancer and cholangiocarcinoma tissue samples
  • the surgical instruments used in the following embodiments need to be sterilized by high temperature and high pressure in advance, and can be used after drying.
  • sample dissociation solution (see Example 19) per mg of tissue, treat the trimmed tissue sample with the preheated sample dissociation solution at 37°C, and perform the sample dissociation at 37°C. 15 minutes to 3 hours away. Observe the dissociation of the sample under the microscope every 15 minutes until a large number of single cells are observed.
  • Example 23 Primary cell culture of gallbladder cancer and cholangiocarcinoma
  • the medium used is the primary cell culture medium for gallbladder cancer and cholangiocarcinoma in Example 19 (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 ⁇ M; the final concentration of A83-01 is 0.5 ⁇ M; N-acetyl-L-cysteine The final concentration of Nicotinamide is 1mM; the final concentration of Nicotinamide is 10mM; the
  • cancer cells expanded to form cell masses with a diameter of 80 ⁇ m.
  • the total number of tumor cells could exceed 10 7 , and the number of other types of cells was significantly reduced or even disappeared.
  • This method has been tested on a large number of samples, and the success rate of in vitro culture of primary tumor cells of gallbladder cancer and cholangiocarcinoma can reach 80%.
  • Example 24 Passage of primary cells of gallbladder carcinoma and cholangiocarcinoma solid tumors
  • the medium used is the primary cell culture medium for gallbladder cancer and cholangiocarcinoma solid tumors in Example 19. Take the plate as an example. The plate is plated at a density of 10 6 cells per well and cultured in a cell incubator at 37°C and 5% CO 2 .
  • Example 25 Cryopreservation of primary cells from solid tumors of gallbladder cancer and cholangiocarcinoma
  • the primary cells of gallbladder cancer and cholangiocarcinoma solid tumors in suspension culture can be cryopreserved after 2-3 passages and expansion:
  • Example 26 Recovery of primary cells from solid tumors of gallbladder cancer and cholangiocarcinoma
  • the primary cells of gallbladder cancer and cholangiocarcinoma solid tumors stored in liquid nitrogen can be recovered:
  • Example 27 HE staining identification of primary cells of gallbladder carcinoma and cholangiocarcinoma solid tumors
  • Figure 9 shows the HE staining effect of primary cholangiocarcinoma cells cultured in vitro. It can be seen that these cells generally have high nuclear to cytoplasmic ratio, deep staining of nuclei, intranuclear chromatin agglutination, multinucleus, and uneven cell size. Characteristically, dozens to hundreds of tumor cells aggregate to form tumor cell clusters with a certain three-dimensional structure.
  • Example 28 Immunohistochemical staining identification of primary cells from solid tumors of gallbladder cancer and cholangiocarcinoma
  • Paraformaldehyde (Beijing Chemical Reagent Company, Analytical Pure), dissolve the paraformaldehyde powder in ultrapure water to prepare a 4% (4g/100mL) paraformaldehyde solution;
  • DAB color developing fluid ( DAB Substrate Kit, 8059S)
  • Example 19 Collect the primary cell culture media for gallbladder cancer and cholangiocarcinoma solid tumors in Example 19 (where 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 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 The concentration is 10 ⁇ M; the final concentration of A83-01 is 0.5 ⁇ 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; the final concentration of Y-27632 10 ⁇ M) cultured gallbladder cancer and chol
  • the dried sections can be observed or photographed under a microscope.
  • Figure 10 shows the results of immunohistochemical staining of primary tumor cell masses of cholangiocarcinoma cultured in vitro. It can be seen that the cells that make up the cell masses are pan-CK positive and are of epithelial origin, which confirms that this method is cultured. The ones are higher purity tumor cells.
  • the primary cultures of 5 gallbladder cancer and cholangiocarcinoma samples were identified by immunohistochemical staining. The statistical results showed that the proportion of tumor cells in the primary cells of gallbladder cancer and cholangiocarcinoma obtained by this method reached 84%-95% (Table 38).
  • Example 29 Using CYTOP modified cell culture consumables for primary tumor cell culture of gallbladder cancer and cholangiocarcinoma solid tumors
  • the method of CYTOP modification is as follows: firstly, the cell culture container is etched with pure oxygen, the etching condition is 20W power, and the etching time is 3 minutes. Then cover the surface of the petri dish or the culture plate with an appropriate amount (in a 96-well plate, 20 ⁇ L per well, an appropriate amount means completely covering the bottom of the petri dish) 1% CYTOP solution, and use it after the CYTOP solution is completely dried.
  • Table 39 The effect of CYTOP modified consumables on primary tumor cell culture of gallbladder cancer and cholangiocarcinoma solid tumors
  • Polystyrene Polystyrene, abbreviated PS.
  • Example 30 Preparation of reagents for culturing primary cells of gallbladder cancer and cholangiocarcinoma bile samples
  • the cell separation buffer After the cell separation buffer is prepared, it can be stored at 4°C for 1 month.
  • Example 31 Obtaining bile samples for gallbladder cancer and cholangiocarcinoma
  • the attending doctor selects patients to be enrolled in accordance with the clinical indications specified in the medical guidelines, and selects appropriate samples for in vitro culture according to the intraoperative clinical indications.
  • the selection criteria for surgical samples are: primary gallbladder cancer or cholangiocarcinoma, the pathological stage is stage II, stage III, or stage IV, and the volume of the bile sample exceeds 20 mL.
  • the attending doctor provides basic clinical information such as the patient's gender, age, medical history, family history, smoking history, pathological staging, clinical diagnosis, etc.
  • the patient’s name, ID number and other information related to the patient’s privacy are concealed and replaced with a uniform experiment number.
  • the naming principle of the experiment number is the eight-digit date of the sample collection + the last four digits of the patient’s hospitalization number. For example, if the sample provided on January 1, 2018, the hospitalization number of the patient is T001512765, the sample experiment number is 201801012765.
  • the patient's doctor in charge uses sterile equipment to collect more than 10 mL of fresh bile specimens.
  • the samples are temporarily stored on ice and transported to the laboratory for the next step within 48 hours.
  • Example 32 Pretreatment of bile samples for gallbladder cancer and cholangiocarcinoma
  • Example 33 Density gradient centrifugation of bile samples for gallbladder cancer and cholangiocarcinoma
  • Example 34 Primary cell culture of bile samples from gallbladder cancer and cholangiocarcinoma
  • the medium used is the primary cell culture medium for gallbladder cancer and cholangiocarcinoma bile samples in Example 30 ( Among them, 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 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 ⁇ M; the final concentration of A83-01 is 0.5 ⁇ M; N-acetyl-L -The final concentration of cysteine is 1 mM; the final concentration of Nicotinamide
  • cancer cells expanded to form cell masses with a diameter of 80 ⁇ m.
  • the total number of tumor cells could exceed 10 7 , and the number of other types of cells was significantly reduced or even disappeared.
  • This method has been tested on a large number of samples, and the success rate of in vitro culture of primary tumor cells from gallbladder cancer and cholangiocarcinoma bile samples can reach 70%.
  • Example 35 Passaging of primary cells from gallbladder cancer and cholangiocarcinoma bile samples
  • the medium used is the primary cell culture medium for gallbladder cancer and cholangiocarcinoma bile samples in Example 30. Take a six-well plate as an example. The plate is plated at a density of 10 6 cells per well and cultured in a cell incubator at 37°C and 5% CO 2 .
  • Example 36 Cryopreservation of primary cells from gallbladder cancer and cholangiocarcinoma bile samples
  • the primary cells of gallbladder cancer and cholangiocarcinoma bile samples in suspension culture can be cryopreserved after 2-3 passages and expansion:
  • Example 37 Recovery of primary cells from gallbladder cancer and cholangiocarcinoma bile samples
  • the primary cells of gallbladder cancer and cholangiocarcinoma bile samples stored in liquid nitrogen can be recovered:
  • Example 38 HE staining identification of primary cells from gallbladder carcinoma and cholangiocarcinoma bile samples
  • the primary cell culture medium for the gallbladder cancer and cholangiocarcinoma bile samples in Example 30 was collected by 800g centrifugation (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 mL; The final concentration of SB202190 is 5 ⁇ M; the final concentration of A83-01 is 1 ⁇ 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; Y- The final concentration of 27632 is 10
  • Figure 13 shows the HE staining effect of primary tumor cells from cholangiocarcinoma bile samples cultured in vitro. It can be seen that these cells generally have high nuclear to cytoplasmic ratio, deep nuclear staining, intranuclear chromatin agglutination, multinucleation, and uneven cell size. The characteristics of cancer cells, dozens to hundreds of tumor cells aggregate to form tumor cell clusters with a certain three-dimensional structure.
  • Example 39 Immunohistochemical staining identification of primary cells from gallbladder cancer and cholangiocarcinoma bile samples
  • Paraformaldehyde (Beijing Chemical Reagent Company, Analytical Pure), dissolve the paraformaldehyde powder in ultrapure water to prepare a 4% (4g/100mL) paraformaldehyde solution;
  • DAB color developing fluid ( DAB Substrate Kit, 8059S)
  • Example 30 Collect the primary cell culture medium of the gallbladder cancer and cholangiocarcinoma bile samples in Example 30 (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 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 The concentration is 10 ⁇ M; the final concentration of A83-01 is 0.5 ⁇ 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; the final concentration of Y-27632 The primary cell clumps of the gallbladder cancer
  • the dried sections can be observed or photographed under a microscope.
  • Figure 14 shows the effect of immunohistochemical staining of primary tumor cell masses of cholangiocarcinoma cultured in vitro. It can be seen that the cells that make up the cell masses are pan-CK positive and are of epithelial origin, which confirms that this method is cultured. The ones are higher purity tumor cells. Five primary cultures of gallbladder cancer and cholangiocarcinoma were identified by immunohistochemical staining. The statistical results showed that the proportion of tumor cells in the primary cells of gallbladder cancer and cholangiocarcinoma bile samples obtained by this method reached 64%-80% (Table 42).
  • Embodiment 40 microplate chip processing
  • microplate chips for culturing primary cells of gastric cancer and cholangiocarcinoma of the present invention.
  • the chip can be used for primary gastric cancer, gallbladder cholangiocarcinoma cell culture and in vitro drug sensitivity detection experiments.
  • the microplate chip design drawing is shown in Figure 15.
  • the PMMA material (or PS, PC, COC, COP, LAS and other materials) is used to prepare the design drawing of the microplate chip structure as shown in Figure 15, and then the above-mentioned CYTOP modification method (see implementation Example 29) The surface was modified with CYTOP to obtain the microplate chip that can be used for primary cell culture of gastric cancer and gallbladder cholangiocarcinoma.
  • the invention provides a method for extracting and culturing primary tumor cells of gastric cancer and cholangiocarcinoma of the gallbladder from fresh gastric cancer surgery samples, gallbladder cholangiocarcinoma surgery samples or biopsy puncture tissue samples or gallbladder cancer and cholangiocarcinoma bile samples and methods and supporting reagents. It has the following advantages: the amount of tissue samples is small, only about 20mg of surgical samples or about 10-20mL of bile samples; it can be used for the culture of primary tumor cells of gastric cancer and gallbladder cholangiocarcinoma, and it can also be used for gastric cancer and gallbladder bile duct.
  • the culture cycle is short, and primary tumor cells of the order of 10 6 -10 7 can be obtained in only 3-10 days; the culture stability is high, and qualified samples are cultured in vitro by this method
  • the success rate is as high as 70%; the cell purity is high.
  • the gastric cancer and gallbladder cholangiocarcinoma primary cell cultures obtained by the method of the present invention can be used for in vitro experiments at various cellular levels, second-generation sequencing, animal models, 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 gastric cancer and gallbladder cholangiocarcinoma.

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Abstract

一种胃癌和胆囊胆管癌原代细胞培养方法及配套试剂，该技术的核心是：(1)用温和细胞解离试剂处理胃癌和胆囊胆管癌实体瘤组织，用温和方法分离胆汁样本中的胆囊胆管癌原代肿瘤细胞，最大程度保证癌细胞的活力；(2)配制特殊无血清培养基，利用悬浮培养体系对胃癌和胆囊胆管癌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排除正常细胞的干扰。利用所述方法得到的胃癌和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，这种培养方法在胃癌和胆囊胆管癌的研究和临床诊断治疗领域具广泛应用前景。

Description

胃癌和胆囊胆管癌原代细胞培养方法及配套试剂 技术领域
本发明涉及生物技术领域，具体涉及一种胃癌和胆囊胆管癌原代细胞培养方法及配套试剂。
背景技术
胃癌是最常见的严重威胁人类的健康恶性肿瘤之一。我国是胃癌高发国家，胃癌发病数和死亡数分别占全球发病数和死亡数的42.6％和45％。在我国胃癌的发病率为11.8％，在所有恶性肿瘤中占第四位。而胃癌的死亡率为22.0％，在全部恶性肿瘤中占第五位。随着经济的发展、生活水平的提高和生活方式的改变，胃癌的发病率还将呈不断上升的趋势。另外，胃癌复发转移风险高，超过50％的胃癌患者会在根治性治疗后数月到数年内出现不同程度的复发转移。
胆囊胆管癌是发生在胆囊、胆管、肝内胆管部位的常见消化系统恶性肿瘤，包括胆囊癌、胆管细胞癌等。胆囊胆管相关恶性肿瘤在我国总发病率约3％，其中胆管癌占2％，在我国消化道恶性肿瘤中居第5位。虽其发病率不高，但胆囊胆管相关癌症均极为恶性，胆管癌甚至被称为“肝癌之王”、“癌中之王”。对于不可切除的胆囊癌，中位生存期仅8个月。
尽管世界各国的科研和医疗机构对胃癌和胆囊胆管癌的病因以及发生发展过程的研究都有很大力度的投入，但是人类对这种疾病仍然知之甚少。胃癌和胆囊胆管癌均属于复杂疾病，其发生、发展是一个动态的过程，涉及到诸多信号分子相互作用，形成了一个复杂的分子调控网络，同时还受到外界环境因素的影响。胃癌和胆囊胆管癌的病因和发生发展过程有很强的个体差异性，不能一概而论。因此将胃癌和胆囊胆管癌原代细胞培养物作为模型进行个体化精准研究是胃癌和胆囊胆管癌研究领域乃至胃癌诊断治疗领域的趋势。
现有的原代肿瘤细胞培养技术主要有2D培养，3D培养，重编程培养等几类，这些方法都不同程度的面临培养周期极长，培养成功率低，杂细胞难以去除等问题。
发明公开
为了有效解决上述技术问题，本发明提供了一种新的胃癌和胆囊胆管癌原代细胞培养方法及配套试剂，该技术的核心是：(1)用温和细胞解离试剂处理胃癌和胆囊胆管癌实体瘤组织，最大程度的保证了组织中癌细胞的活力；用温和方法分离胆汁样本中的胆囊胆管癌原代肿瘤细胞，最大程度保证癌细胞的活力；(2)配制特殊的无血清培养基，利用悬浮培养体系对胃癌和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。
第一方面，本发明要求保护一种用于培养胃癌和/或胆囊胆管癌原代细胞的培养基。
本发明所要求保护的用于培养胃癌和/或胆囊胆管癌原代细胞的培养基，由抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)、HEPES、GlutaMax、非必需氨基酸(Non-essential amino acids)、人重组蛋白EGF、人重组蛋白bFGF、人重组蛋白HGF、人重组蛋白FGF-10、人重组蛋白Wnt-3a、人重组蛋白Noggin、SB202190(4-(4-氟苯基)-2-(4-羟基苯基)-5-(4-吡啶基)-1H-咪唑)、A83-01(3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioami de)、Primocin TM、N-乙酰-L-半胱氨酸(N-acetyl-L-cysteine)、烟碱(Nicotinamide)、N-2 Supplement、皮质醇、B27、ITS-X(Insulin,Transferrin,Selenium,Ethanolamine Solution)、胃泌素(Gastin 1)、Y-27632和Advanced DMEM/F12培养基组成。其中，所述抗菌抗真菌剂三抗中的青霉素的终浓度为100-200U/mL(如100U/mL)；所述抗菌抗真菌剂三抗中的链霉素的终浓度为100-200μg/mL(如100μg/mL)；所述抗菌抗真菌剂三抗中的两性霉素B的终浓度为250-250ng/mL(如250ng/mL)；所述HEPES的终浓度为8-12mM(如10mM)；所述GlutaMax的终浓度为0.8-1.2％(如1％，％表示体积百分含量)；所述非必需氨基酸中的甘氨酸的浓度为80-120μM；所述非必需氨基酸中的L-丙氨酸的浓度为80-120μM(如100μM)；所述非必需氨基酸中的L-天冬酰胺的浓度为80-120μM(如100μM)；所述非必需氨基酸中的L-天冬氨酸的浓度为80-120μM(如100μM)；所述非必需氨基酸中的L-谷氨酸的浓度为80-120μM(如100μM)；所述非必需氨基酸中的L-脯氨酸的浓度为80-120μM(如100μM)；所述非必需氨基酸中的L-丝氨酸的浓度为80-120μM(如100μM)；所述人重组蛋白EGF的终浓度为10-100ng/mL；所述人重组蛋白bFGF的终浓度为10-50ng/mL；所述人重组蛋白HGF的终浓度为5-25ng/mL；所述人重组蛋白FGF-10的终浓度为5-25ng/mL；所述人重组蛋白Wnt-3a的终浓度为200-300ng/mL；所述人重组蛋白Noggin的终浓度为100-200ng/mL；所述SB202190的终浓度为5-10μM；所述A83-01的终浓度为0.25-1.25μM；所述Primocin的终浓度为1％(体积百分含量)；所述N-乙酰-L-半胱氨酸(N-acetyl-L-cysteine)的终浓度为0.5-2mM；所述烟碱(Nicotinamide)的终浓度为5-10mM；所述N-2 Supplement的终浓度为1％(体积百分含量)；所述皮质醇的终浓度为20-50ng/mL；所述B27的终浓度为1.5-2.5％(如2％，％表示体积百分含量)；所述ITS-X的终浓度为0.8-1.2％(如1％，％表示体积百分含量)；所述胃泌素(Gastrin 1)的终浓度为8-12nM(如10nM)；所述Y-27632的终浓度为5-20μM；余量均为Advanced DMEM/F12培养基。
进一步地，所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)组成如下：每毫升包含10000单位青霉素(碱)、10000μg链霉素(碱)和25μg两性霉素B。所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)为 “Antibiotic-Antimycotic,100X”(如Gibco#15240062，或与其组成相同的其他产品)。所述“Antibiotic-Antimycotic,100X”每毫升包含10000单位青霉素(碱)、10000μg链霉素(碱)和25μg两性霉素B，利用0.85％盐液形式的青霉素G(钠盐)、硫酸链霉素和两性霉素B作为
Figure PCTCN2019115306-appb-000001
抗真菌剂。所述GlutaMAX为“GlutaMAX TM Supplement”(如Gibco#35050061，或与其组成相同的其他产品)。所述“GlutaMAX TM Supplement”的成分为L-alanyl-L-glutamine，是L-glutamine的替代物，浓度为200nM，溶剂为0.85％NaCl溶液。所述非必需氨基酸组成如下：每毫升所述非必需氨基酸包含750μg甘氨酸、890μg L-丙氨酸、1320μg L-天冬酰胺、1330μg L-天冬氨酸、1470μg L-谷氨酸、1150μg L-脯氨酸、1050μg L-丝氨酸，溶剂为水(每毫升所述非必需氨基酸中上述涉及到的各种氨基酸的浓度均为10mM)。所述Primocin为原代细胞用抗菌剂(如Invivogene#ant-pm-1，或与其组成相同的其他产品)，用于保护原代细胞免受微生物污染的抗生素，对革兰氏阳性菌、革兰氏阴性菌、支原体和真菌均有杀伤作用。所述N-2 Supplement为“N-2 Supplement(100X)”(如Gibco#17502001，或与其组成相同的其他产品)。所述“N-2 Supplement(100X)”中含有终浓度为1mM的人全铁转铁蛋白(Human Transferrin(Holo))、500mg/L的重组胰岛素全链(Insulin Recombinant Full Chain)、0.63mg/L的孕酮(Progesterone)、10mM的腐胺(Putrescine)、0.52mg/L的亚硒酸盐(Selenite)。所述B27为“B-27 TM Supplement(50X),minus vitamin A”(如Gibco#12587010，或与其组成相同的其他产品)。所述“B-27 TM Supplement(50X),minus vitamin A”中含有生物素(Biotin)、DL-α-生育酚乙酸酯(DL Alpha Tocopherol Acetate)、DL-α-生育酚(DL Alpha-Tocopherol)、BSA(fatty acid free Fraction V)、过氧化氢酶(Catalase)、人重组胰岛素(Human Recombinant Insulin)、人转铁蛋白(Human Transferrin)、超氧化物歧化酶(Superoxide Dismutase)、皮质酮(Corticosterone)、D-半乳糖(D-Galactose)、乙醇胺盐酸(Ethanolamine HCl)、还原型谷胱甘肽(Glutathione(reduced))、左旋肉碱盐酸(L-Carnitine HCl)、亚油酸(Linoleic Acid)、亚麻酸(Linolenic Acid)、孕酮(Progesterone)、腐胺(Putrescine 2HCl)、亚硒酸钠(Sodium Selenite)、三碘甲状腺原氨酸(T3(triodo-I-thyronine))。所述ITS-X的溶剂为EBSS溶液(Earle's平衡盐溶液)，溶质及浓度如下：胰岛素1g/L；转铁蛋白0.55g/L；亚硒酸钠0.00067g/L；乙醇胺0.2g/L。所述GlutaMAX是一种高级细胞培养添加剂，可直接替代细胞培养基中的L-谷氨酰胺。所述GlutaMAX为“GlutaMAX TM Supplement”(如Gibco#35050061，或与其组成相同的其他产品)。所述Y-27632为“Y-27632dihydrochloride(一种ATP竞争性的ROCK-I和ROCK-II抑制剂，Ki分别为220nM和300nM)”(如MCE#129830-38-2，或与其组成相同的其他产品)。
在本发明的具体实施例中，所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)的品牌货号为Gibco#15240062；所述HEPES的品牌货号为 Gibco#15630080；所述GlutaMAX的品牌货号为Gibco#35050061；所述非必需氨基酸的品牌货号为Gibco#11140050；所述人重组蛋白EGF的品牌货号为Peprotech AF-100-15-100；所述人重组蛋白bFGF的品牌货号为Peprotech AF-100-18B-50；所述人重组蛋白HGF的品牌货号为Peprotech AF-100-39-100；所述人重组蛋白FGF-10的品牌货号为Peprotech AF-100-26-100；所述人重组蛋白Wnt-3a的品牌货号为R&D 5036-WN-500；所述人重组蛋白Noggin的品牌货号为上海近岸#C018；所述SB202190的品牌货号为Sigma#S7067；所述A83-01的品牌货号为Tocris#2939；所述Primocin TM的品牌货号为Invivogene#ant-pm-1；所述N-acetyl-L-cysteine的品牌货号为Sigma#A9165；所述Nicotinamide的品牌货号为Sigma#N0636；所述N-2 Supplement的品牌货号为Gibco#17502001；所述皮质醇的品牌货号为Sigma#H0888；所述B27的品牌货号为Gibco#12587010；所述ITS-X的品牌货号为Gibco#51500056；所述胃泌素的品牌货号为NJPeptide#Pep12307；所述Y-27632的品牌货号为MCE#129830-38-2；所述Advanced DMEM/F12培养基的品牌货号为Gibco#12634010。
进一步地，所述用于培养胃癌和/或胆囊胆管癌原代细胞的培养基的存在形式可为两种：
其一，所述用于培养胃癌和/或胆囊胆管癌原代细胞的培养基为含有所述抗菌抗真菌剂三抗、所述HEPES、所述GlutaMax、所述非必需氨基酸、所述人重组蛋白EGF、所述人重组蛋白bFGF、所述人重组蛋白HGF、所述人重组蛋白FGF-10、所述人重组蛋白Wnt-3a、所述人重组蛋白Noggin、所述SB202190、所述A83-01、所述Primocin TM、所述N-乙酰-L-半胱氨酸、所述烟碱、所述N-2 Supplement、所述皮质醇、所述B27、所述ITS-X、所述胃泌素、所述Y-27632和所述Advanced DMEM/F12培养基的溶液。
所述培养基配制好后需用0.22μM针头式滤器(Millipore SLGP033RS)过滤除菌，在4℃可以保存两周。
其二，所述用于培养胃癌和/或胆囊胆管癌原代细胞的培养基中的各组分单独存在，使用时按照配方进行配制。
更进一步地，其中的人重组蛋白EGF、人重组蛋白bFGF、人重组蛋白HGF、人重组蛋白FGF-10、人重组蛋白Wnt-3a、人重组蛋白Noggin可以储液(母液)形式存在(-80℃长期保存)，具体可为1000倍储液(母液)。SB202190、N-acetyl-L-cysteine、Nicotinamide、皮质醇、胃泌素和Y-27632可以储液(母液)形式存在(-20℃长期保存)，具体可为1000倍储液(母液)。A83-01可以储液(母液)形式存在(-20℃长期保存)，具体可为100000倍储液(母液)。
1000×人重组蛋白EGF储液由人重组蛋白EGF、BSA和PBS组成，其中所述人重组蛋白EGF的终浓度为20μg/mL，所述BSA的终浓度为0.01g/mL，余量均为PBS。
1000×人重组蛋白bFGF储液由人重组蛋白bFGF、BSA和PBS组成，其中 所述人重组蛋白bFGF的终浓度为20μg/mL，所述BSA的终浓度为0.01g/mL，余量均为PBS。
1000×人重组蛋白HGF储液由人重组蛋白HGF、BSA和PBS组成，其中所述人重组蛋白HGF的终浓度为20μg/mL，所述BSA的终浓度为0.01g/mL，余量均为PBS。
1000×人重组蛋白FGF-10储液由人重组蛋白FGF-10、BSA和PBS组成，其中所述人重组蛋白FGF-10的终浓度为20μg/mL，所述BSA的终浓度为0.01g/mL，余量均为PBS。
1000×人重组蛋白Wnt-3a储液由人重组蛋白Wnt-3a、BSA和PBS组成，其中所述人重组蛋白Wnt-3a的终浓度为200μg/mL，所述BSA的终浓度为0.01g/mL，余量均为PBS。
1000×人重组蛋白Noggin储液由人重组蛋白Noggin、BSA和PBS组成，其中所述人重组蛋白Noggin的终浓度为100μg/mL，所述BSA的终浓度为0.01g/mL，余量均为PBS。
上述六种1000倍储液中，所述BSA是可以100倍储液(母液)形式存在(现配现用)，具体由BSA和PBS组成，其中BSA(Sigma#A1933)的终浓度为0.1g/mL，余量均为PBS。
另外，1000×SB202190储液由SB202190和DMSO组成，其中所述SB202190的终浓度为10mM，余量均为DMSO。
100000×A83-01储液由A83-01和DMSO组成，其中所述A83-01的浓度为25mM，余量均为DMSO。
1000×N-acetyl-L-cysteine储液由N-acetyl-L-cysteine和超纯水组成，其中所述N-acetyl-L-cysteine的浓度为0.5M，余量均为超纯水。
1000×Nicotinamide储液由Nicotinamide和超纯水组成，其中所述Nicotinamide的浓度为5M，余量均为超纯水。
1000×皮质醇储液由皮质醇、无水乙醇和超纯水组成，其中所述皮质醇的终浓度为25μg/mL，所述无水乙醇的终浓度为5％(体积百分含量)，余量均为超纯水。
1000×胃泌素储液由胃泌素和超纯水组成，其中胃泌素的浓度为10μM，余量均为超纯水。
1000×Y-27632储液由Y-27632和超纯水组成，其中Y-27632的终浓度为10mM，余量均为超纯水。
第二方面，本发明要求保护一种用于培养胃癌和/或胆囊胆管癌原代细胞的成套试剂。
本发明所要求保护的成套试剂可为如下任一：
(A1)由前文第一方面中所述培养基如下中的全部或部分组成：样本解离液、样本保存液和样本清洗液。
(A2)由前文第一方面中所述培养基和细胞分离缓冲液组成。
(A3)由(A1)和如下试剂中的全部或部分组成：细胞消化液、消化终止液和细胞冻存液。
(A4)由(A2)和如下试剂中的全部或部分组成：细胞消化液、消化终止液和细胞冻存液。
所述样本解离液由胶原酶I、胶原酶II、胶原酶IV和PBS组成；其中，所述胶原酶I的终浓度为150-250U/mL(如200U/mL)；所述胶原酶II的终浓度为150-250U/mL(如200U/mL)；所述胶原酶IV的终浓度为50-150U/mL(如100U/mL)；余量均为PBS。
其中，用蛋白酶的酶活来定义胶原酶(所述胶原酶I、所述胶原酶II或所述胶原酶IV)的单位U：在37℃，pH 7.5的条件下，用1U蛋白酶处理胶原酶(所述胶原酶I、所述胶原酶II或所述胶原酶IV)5小时，可以释放L-亮氨酸1μmol。
在本发明的具体实施例中，所述胶原酶I的品牌货号为Gibco#17100-017。所述胶原酶II的品牌货号为Gibco#17101-015；所述胶原酶IV的品牌货号为Gibco#17104-019；所述PBS的品牌货号为Gibco#21-040-CVR。
所述样本保存液由胎牛血清、抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)、HEPES和HBSS(Hank's平衡盐溶液)组成；其中，所述胎牛血清的终浓度为1-5％(如2％，％表示体积百分含量)；所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)中的青霉素的终浓度为100-200U/mL(如100U/mL)；所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)中的链霉素的终浓度为100-200μg/mL(如100μg/mL)；所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)中的两性霉素B的终浓度为250-500ng/mL(如250ng/mL)；所述HEPES的终浓度为8-12mM(如10mM)；余量均为HBSS。
进一步地，所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)组成如下：每毫升包含10000单位青霉素(碱)、10000μg链霉素(碱)和25μg两性霉素B。所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)为“Antibiotic-Antimycotic,100X”(如Gibco#15240062，或与其组成相同的其他产品)。所述“Antibiotic-Antimycotic,100X”每毫升包含10000单位青霉素(碱)、10000μg链霉素(碱)和25μg两性霉素B，利用0.85％盐液形式的青霉素G(钠盐)、硫酸链霉素和两性霉素B作为
Figure PCTCN2019115306-appb-000002
抗真菌剂。
在本发明的具体实施例中，所述胎牛血清的品牌货号为Gibco#16000-044；所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)的品牌货号为Gibco#15240062；所述HEPES的品牌货号为Gibco#15630080；所述HBSS的品牌货号为Gibco#14170161。
所述样本清洗液由抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)和PBS组成；其中，所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)中的青霉素的终浓度为100-200U/mL(如100U/mL)；所述抗菌抗真菌剂三抗(青霉素- 链霉素-两性霉素B)中的链霉素的终浓度为100-200μg/mL(如100μg/mL)；所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)中的两性霉素B的终浓度为250-500ng/mL(如250ng/mL)；余量均为PBS。
进一步地，所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)组成如下：每毫升包含10000单位青霉素(碱)、10000μg链霉素(碱)和25μg两性霉素B。所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)为“Antibiotic-Antimycotic,100X”(如Gibco#15240062，或与其组成相同的其他产品)。所述“Antibiotic-Antimycotic,100X”每毫升包含10000单位青霉素(碱)、10000μg链霉素(碱)和25μg两性霉素B，利用0.85％盐液形式的青霉素G(钠盐)、硫酸链霉素和两性霉素B作为
Figure PCTCN2019115306-appb-000003
抗真菌剂。
在本发明的具体实施例中，所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)的品牌货号为Gibco#15240062；所述PBS的品牌货号为Gibco#21-040-CVR。
所述细胞分离缓冲液由双抗P/S(青霉素-链霉素)、肝素钠和PBS组成；其中，所述双抗P/S(青霉素-链霉素)中的青霉素的终浓度为100-200U/mL(如100U/mL)；所述双抗P/S(青霉素-链霉素)中的链霉素的终浓度为100-200μg/mL(如100μg/mL)；所述肝素钠的终浓度为10IU/mL；余量均为PBS。
在本发明的具体实施例中，所述双抗P/S(青霉素-链霉素)的品牌货号为Gibco#15140122；所述肝素钠的品牌货号为Solarbio#H8270；所述PBS的品牌货号为Gibco#21-040-CVR。
所述细胞消化液组成如下：每10mL所述细胞消化液中含有4-6mL(如5mL)Accutase，终浓度为5mM的EDTA(即10μL 0.5M EDTA)，1.5-2.5mL(如2mL)TrypLE Express，余量为PBS。
进一步地，所述Accutase为“StemPro TM Accutase TM Cell Dissociation Reagent”(如Gibco#A11105-01，或与其组成相同的其他产品)。所述Accutase是一种单一成分的酶，在D-PBS，0.5mM EDTA溶液中溶解。所述TrypLE Express为“TrypLE TM Express Enzyme(1X),no phenol red”(如Gibco#12604013，或与其组成相同的其他产品)。所述“TrypLE TM Express Enzyme(1X),no phenol red”中含有200mg/L的KCl、200mg/L的KH 2PO 4、8000mg/L的NaCl、2160mg/L的Na 2HPO 4·7H 2O、457.6mg/L的EDTA；还含有重组蛋白酶。
在本发明的具体实施例中，所述Accutase的品牌货号为Gibco#A11105-01；所述0.5M EDTA的品牌货号为Invitrogen#AM9261；所述TrypLE Express的品牌货号为Gibco#12604013；所述PBS的品牌货号为Gibco#21-040-CVR。
所述消化终止液由胎牛血清、抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)和DMEM培养基组成；其中，所述胎牛血清的终浓度为8-12％(如10％，％表示体积百分含量)；所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)中的青霉素的终浓度为100-200U/mL(如100U/mL)；所述抗菌抗真菌剂三抗 (青霉素-链霉素-两性霉素B)中的链霉素的终浓度为100-200μg/mL(如100μg/mL)；所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)中的两性霉素B的终浓度为250-500ng/mL(如250ng/mL)；余量均为DMEM培养基。
进一步地，所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)组成如下：每毫升包含10000单位青霉素(碱)、10000μg链霉素(碱)和25μg两性霉素B。所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)为“Antibiotic-Antimycotic,100X”(如Gibco#15240062，或与其组成相同的其他产品)。所述“Antibiotic-Antimycotic,100X”每毫升包含10000单位青霉素(碱)、10000μg链霉素(碱)和25μg两性霉素B，利用0.85％盐液形式的青霉素G(钠盐)、硫酸链霉素和两性霉素B作为
Figure PCTCN2019115306-appb-000004
抗真菌剂。
在本发明的具体实施例中，所述胎牛血清的品牌货号为Gibco#16000-044；所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)的品牌货号为Gibco#15240062；所述DMEM培养基的品牌货号为Gibco#11965-092。
所述细胞冻存液由Advanced DMEM/F12培养基、DMSO和1％甲基纤维素溶液组成；其中，所述Advanced DMEM/F12培养基、所述DMSO和所述1％甲基纤维素溶液的体积配比为20:2:(0.8-1.2)，如20:2:1；所述1％甲基纤维素溶液是浓度为1g/100ml的甲基纤维素水溶液。
在本发明的具体实施例中，所述Advanced DMEM/F12培养基的品牌货号为Gibco#12634010；所述DMSO的品牌货号为Sigma#D2438；所述甲基纤维素的品牌货号为Sigma#M7027。
所述样本保存液可用于样本离体后的暂时保存，可以在有样本离体后，短时间内维持样本中细胞的活性。所述样本保存液配制好后4℃可保存1个月。
所述样本清洗液可用于样本的清洗和消毒。所述样本清洗液需现配现用。
所述样本解离液可用于样本的解离，可以将样本中的胃癌和/或胆囊胆管癌实体瘤原代细胞从组织中解离出来。所述样本解离液需现配现用，其中的胶原酶I、胶原酶II和胶原酶IV可以储液(母液)形式-20℃长期保存，具体可为10或20倍储液(母液)。10×胶原酶I储液由所述胶原酶I和PBS组成；其中所述胶原酶I的终浓度为2000U/mL；10×胶原酶II储液由所述胶原酶II和PBS组成；其中所述胶原酶II的终浓度为2000U/mL；余量均为PBS；20×胶原酶IV储液由所述胶原酶IV和PBS组成；其中所述胶原酶IV的终浓度为2000U/mL；余量均为PBS。所述胶原酶I、胶原酶II和所述胶原酶IV的酶活定义见前文。
所述细胞分离缓冲液，用于悬浮胆汁样本中的细胞。所述细胞分离缓冲液配制完成后，可于4℃保存1个月。
所述细胞消化液可用于细胞团块的消化和传代，可以将胃癌和/或胆囊胆管癌肿瘤团块消化成单个细胞。所述细胞消化液需现配现用。
所述消化终止液可用于终止样本解离或细胞消化过程。所述消化终止液配制好后可在4℃保存一个月。
所述胃癌和/或胆囊胆管癌原代细胞培养基可用于胃癌和/或胆囊胆管癌原代细胞的培养。
所述细胞冻存液需现配现用。其中，所述1％甲基纤维素溶液可在4℃长期保存。
第三方面，本发明要求保护如下任一应用：
(B1)前文第一方面中所述的培养基在培养胃癌和/或胆囊胆管癌原代细胞中的应用；
(B2)前文第二方面中(A1)或(A3)所述的成套试剂在培养胃癌和/或胆囊胆管癌实体瘤原代细胞中的应用；
(B3)前文第二方面中(A2)或(A4)所述的成套试剂在培养胆囊胆管癌胆汁样本原代肿瘤细胞中的应用。
第四方面，本发明要求保护一种培养胃癌和/或胆囊胆管癌原代细胞的方法。
本发明所要求保护的培养胃癌和/或胆囊胆管癌原代细胞的方法，可为方法A或方法B：
方法A：一种培养胃癌和/或胆囊胆管癌实体瘤原代细胞的方法，包括如下步骤：
(a1)用前文第二方面中所述的样本解离液对胃癌和/或胆囊胆管癌实体瘤组织进行解离处理，获得胃癌和/或胆囊胆管癌实体瘤原代细胞；
(a2)利用前文第一方面所述培养基悬浮培养步骤(a1)解离出来的胃癌和/或胆囊胆管癌实体瘤原代细胞。
方法B：一种培养胆囊胆管癌胆汁样本原代肿瘤细胞的方法，包括如下步骤：
(b1)从胆囊胆管癌胆汁样本中分离获得胆囊胆管癌胆汁样本原代肿瘤细胞；
(b2)利用前文第一方面中所述培养基悬浮培养步骤(b1)分离出来的胆囊胆管癌胆汁样本原代肿瘤细胞。
进一步地，步骤(a1)中，可按照包括如下步骤的方法用所述样本解离液对所述胃癌和/或胆囊胆管癌实体瘤组织进行解离：按0.1-0.3mL(如0.1mL)所述样本解离液每mg组织的用量，将剪碎后的所述胃癌和/或胆囊胆管癌实体瘤组织(如剪成0.8-1.2mm 3的小块)用事先37℃预热的所述样本解离液进行处理，在37℃条件下进行样本解离，解离时间15分钟至3小时。每15分钟在显微镜下观察样本的解离情况，直到观察到大量的单个细胞。
进一步地，步骤(b1)中，可是按照包括如下步骤的方法从胆囊胆管癌胆汁样本中分离获得胆囊胆管癌胆汁样本原代肿瘤细胞：用前文第二方面中所述的细胞分离缓冲液悬浮胆囊胆管癌胆汁样本中的细胞，然后通过密度梯度离心(利用Ficoll淋巴细胞分离液)获得胆囊胆管癌胆汁样本原代肿瘤细胞。
进一步地，步骤(a2)中，可按照包括如下步骤的方法用所述培养基悬浮培养所述胃癌和/或胆囊胆管癌实体瘤原代细胞：使用细胞培养容器M，利用所述 培养基悬浮培养所述胃癌和/或胆囊胆管癌实体瘤原代细胞，37℃，5％CO 2条件下进行培养，每2-4天(如3天)更换一次培养基，直至细胞形成直径50-80μm(如80μm)的团块。
进一步地，步骤(b2)中，可按照包括如下步骤的方法用所述培养基悬浮培养所述胆囊胆管癌胆汁样本原代肿瘤细胞：使用细胞培养容器M，利用所述培养基悬浮培养所述胆囊胆管癌胆汁样本原代肿瘤细胞，37℃，5％CO 2条件下进行培养，每2-4天(如3天)更换一次培养基，直至细胞形成直径50-80μm(如80μm)的团块。
其中，初始接种密度可为10 5个/cm 2容器底面积，以六孔板为例，按每孔10 6个细胞的密度铺板。
其中，所述细胞培养容器M可为如下任一：(I)聚苯乙烯材质的细胞培养容器、聚碳酸酯材质的细胞培养容器、聚甲基丙烯酸甲酯材质的细胞培养容器、COC树脂材质的细胞培养容器、环烯烃聚合物材质的细胞培养容器或低吸附表面的细胞培养容器；(II)对(I)中的细胞培养容器进行CYTOP修饰后的细胞培养容器。
进一步地，所述细胞培养容器为细胞培养皿、细胞培养孔板或用于细胞培养的微孔板芯片。
所述(II)中，可按照包括如下步骤的方法对所述(I)中的细胞培养容器进行CYTOP修饰：对所述(I)中的细胞培养容器进行纯氧刻蚀，刻蚀条件为功率20W，刻蚀时间为3分钟；然后用1％CYTOP溶液覆盖所述细胞培养容器表面，晾干所述1％CYTOP溶液即完成CYTOP修饰。
其中，所述1％CYTOP溶液的组成如下：每100mL所述1％CYTOP溶液中含有1mL CYTOP，余量为氟油。
进一步地，在步骤(a1)之前，还可包括如下对所述胃癌和/或胆囊胆管癌实体瘤组织进行解离前处理的步骤：用体积百分含量为70-75％的乙醇清洗胃癌和/或胆囊胆管癌实体瘤组织样本表面；用前文第二方面中所述样本清洗液清洗所述胃癌和/或胆囊胆管癌实体瘤组织样本10-20次(如10次)，用无菌的PBS溶液清洗所述胃癌和/或胆囊胆管癌实体瘤组织样本5-10次(如5次)；然后除去所述胃癌和/或胆囊胆管癌实体瘤组织样本中的杂质、结缔组织、脂肪组织、坏死组织等影响原代细胞培养的成分。
对所述胃癌和/或胆囊胆管癌实体瘤组织进行解离前处理的步骤需要在冰上操作，整个操作步骤需要在10分钟内完成。
进一步地，进行所述解离前处理的所述胃癌和/或胆囊胆管癌实体瘤组织样本的离体时间为2小时以内，且在进行所述解离前处理之前一直保存于前文第二方面中所述样本保存液中。
进一步地，在步骤(a1)中，用所述样本解离液对所述胃癌和/或胆囊胆管癌实体瘤组织进行解离处理后还包括如下步骤：用8-15倍(如10倍)体积的前文 第二方面中所述消化终止液终止解离反应，收集细胞悬液；用100μm或40μm无菌细胞滤网过滤所述细胞悬液，去除组织残片和粘连细胞；800-1000g(如800g)室温离心10-15分钟(如10分钟)，弃去上清；后用3-5mL(如5mL)无菌PBS重悬细胞；再800-1000g(如800g)室温离心10-15分钟(如10分钟)，弃去上清；然后用前文第一方面中所述培养基重悬细胞沉淀，在显微镜下观察细胞状态，进行细胞计数。
进一步地，在步骤(b1)之前还包括对胆囊胆管癌胆汁样本进行分离前处理的步骤：去除胆囊胆管癌胆汁样本中的杂质、凝血块等影响细胞密度梯度分离的成分。
进一步地，在步骤(a2)中，还可包括如下步骤：待所述胃癌和/或胆囊胆管癌实体瘤原代细胞形成直径50-80μm(如80μm)的团块时，对所述胃癌和/或胆囊胆管癌实体瘤原代细胞进行传代。
进一步地，在步骤(b2)中，还可包括如下步骤：待所述胆囊胆管癌胆汁样本原代肿瘤细胞形成直径50-80μm(如80μm)的团块时，对所述胆囊胆管癌胆汁样本原代肿瘤细胞进行传代。
其中，进行所述传代时采用的细胞消化液为前文第二方面中所述的细胞消化液。
其中，进行所述传代时采用的消化终止液为前文第二方面中所述的消化终止液。
更进一步地，进行所述传代时采用的消化温度为37℃。
更加具体地，进行所述传代的步骤：收集待传代的细胞团块，离心后用无菌的PBS溶液清洗细胞团块，再离心，然后用所述细胞消化液重悬细胞团块，在37℃条件下进行消化，直到细胞团块都被消化为单个细胞，用所述消化终止液(其用量可为5-10倍，如10倍体积)终止消化反应，收集细胞悬液；离心后用前文第一方面中所述培养基重悬细胞沉淀，计数，然后使用前文所述细胞培养容器M悬浮培养细胞(初始接种密度可为10 5个/cm 2容器底面积，以六孔板为例，按每孔10 6个细胞的密度铺板)，培养条件为37℃，5％CO 2。上述传代步骤中的所有离心均具体可为800-1000g(如800g)室温离心10-20分钟(如10分钟)。
进一步地，所述方法还可包括对经过2-3次传代扩增后的所述胃癌和/或胆囊胆管癌实体瘤原代细胞或所述胆囊胆管癌胆汁样本原代肿瘤细胞进行冻存和/或复苏的步骤。
进行所述冻存时采用的细胞冻存液为前文第二方面中所述的细胞冻存液。
更进一步地，进行所述冻存的具体步骤：收集待冻存的细胞团块，离心后用无菌的PBS溶液清洗细胞团块，再离心，然后用所述细胞消化液重悬细胞团块，在37℃条件下进行消化，直到细胞团块都被消化为单个细胞，用所述消化终止液(其用量可为5-10倍，如10倍体积)终止消化反应，收集细胞悬液；离 心后用所述细胞冻存液，按0.5-2×10 6/mL(如10 6/mL)的密度重悬细胞沉淀，梯度降温盒过夜冻存后转移至液氮中长期保存。上述冻存步骤中的所有离心均具体可为800-1000g(如800g)室温离心10-20分钟(如10分钟)。
更进一步地，进行所述复苏的具体步骤：将装有待复苏细胞的冻存管从液氮中取出，在37-39℃(如37℃)无菌水中迅速融化细胞；离心(如800-1000g，如800g室温离心5-10分钟，如10分钟)后用前文第一方面中所述培养基重悬细胞沉淀，然后使用前文所述细胞培养容器M悬浮培养细胞(初始接种密度可为10 5个/cm 2容器底面积)，每管细胞(10 6个)复苏至3.5cm培养皿)，培养条件为37℃，5％CO 2。
第五方面，本发明要求保护如下任一试剂：
(C1)胃癌和/或胆囊胆管癌实体瘤组织样本解离液，为前文第二方面中所述的样本解离液；
(C2)胃癌和/或胆囊胆管癌实体瘤组织样本保存液，为前文第二方面中所述的样本保存液；
(C3)胆囊胆管癌胆汁样本分离缓冲液，为前文第二方面中所述的细胞分离缓冲液。
第六方面，本发明要求保护如下任一应用：
(D1)前文第五方面中(C1)所述样本解离液在从胃癌和/或胆囊胆管癌实体瘤组织中解离出胃癌和/或胆囊胆管癌实体瘤原代细胞中的应用。
(D2)前文第五方面中(C2)所述样本保存液在保存胃癌和/或胆囊胆管癌实体瘤组织中的应用。
(D3前文第五方面中(C3)所述细胞分离缓冲液在从胆囊胆管癌胆汁样本中分离出胆囊胆管癌胆汁样本原代肿瘤细胞中的应用。
第七方面，本发明要求保护如下任一方法：
(E1)一种从胃癌和/或胆囊胆管癌实体瘤组织中解离出胃癌和/或胆囊胆管癌实体瘤原代细胞的方法，包括前文第四方面所述方法中的步骤(a1)。
(E2)一种保存胃癌和/或胆囊胆管癌实体瘤组织的方法，包括如下步骤：将刚刚离体的胃癌和/或胆囊胆管癌实体瘤组织置于前文第二方面中所述样本保存液中保存，保存时间为2小时以内。
(E3)一种从胆囊胆管癌胆汁样本中分离出胆囊胆管癌胆汁样本原代肿瘤细胞的方法，包括前文第四方面所述方法中的步骤(b1)。
在上述各方面中，所述胃癌可为原发性胃癌；所述胆囊胆管癌可为原发性胆囊胆管癌。
在上述各方面中，所述胃癌可为胃癌转移病灶；所述胆囊胆管癌可为胆囊胆管癌转移病灶。
在上述各方面中，所述胃癌原代细胞可为胃癌实体瘤原代细胞；所述胆囊胆管癌原代细胞可为胆囊胆管癌实体瘤原代细胞或胆囊胆管癌胆汁样本原代肿 瘤细胞。
在上述各方面中，所述胃癌原代细胞可分离自胃癌患者的手术样本；所述胆囊胆管癌原代细胞可分离自胆囊胆管癌患者的手术样本(为实体瘤样本)、穿刺样本(为实体瘤样本)或者胆汁样本。
在上述各方面中，所述胃癌的临床分期为II期、III期或IV期(按TNM分期)。其中，手术标本重量超过20mg的样本。
在上述各方面中，所述胆囊胆管癌的临床分期为II期、III期或IV期(按TNM分期)。其中，手术样本获得的胆囊胆管癌实体瘤组织标本最好重量超过20mg。胆汁样本最好不少于10mL。穿刺样本不少于4条。
在本发明中，以上所有的所述PBS均可为1×PBS，pH7.3-7.5。其具体组成如下：溶剂为水，溶质及浓度为：KH 2PO 4 144mg/L，NaCl 9000mg/L，Na 2HPO 4·7H 2O 795mg/L。
附图说明
图1为胃癌组织经过处理后得到的单细胞。标尺为100μm，100倍放大。
图2为胃癌组织原代培养后得到的细胞团块。标尺为100μm，100倍放大。
图3为胃癌组织原代培养后得到的胃癌细胞团块切片HE染色图。标尺为100μm，200倍放大。
图4为胃癌组织原代培养后得到的癌细胞团块免疫荧光染色图。标尺为50μm，200倍放大
图5为根据测序结果进行拷贝数变异分析(CNV)显示各代胃癌原代细胞培养物(P1、P2、P3、P4、P5)与原发胃癌肿瘤组织(Tumor)的拷贝数变异情况高度一致。
图6为用本发明培养得到的原代胃癌细胞进行体外药物敏感性测试结果。
图7为胆管癌组织经过处理后得到的单细胞。标尺为100μm，100倍放大。
图8为胆管癌组织原代培养后得到的细胞团块。标尺为100μm，100倍放大。
图9为胆管癌组织原代培养后得到的胆管癌细胞团块切片HE染色图。标尺为100μm，200倍放大。
图10为胆管癌组织原代培养后得到的癌细胞团块石蜡切片免疫组化染色图。标尺为100μm，200倍放大。
图11为胆管癌胆汁样本经过处理后得到的单细胞。标尺为100μm，100倍放大。
图12为胆管癌胆汁样本原代培养后得到的细胞团块。标尺为100μm，100倍放大。
图13为胆管癌胆汁样本原代培养后得到的胆管癌细胞团块切片HE染色图。标尺为100μm，100倍放大。
图14为胆管癌胆汁样本原代培养后得到的癌细胞团块石蜡切片免疫组化染 色图。标尺为50μm，100倍放大。
图15为本发明微孔板芯片设计图。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
实施例1、配制用于培养胃癌原代细胞的试剂
1、样本保存液(100mL)
样本保存液(100mL)的具体配方如表1所示。
表1样本保存液(100mL)
Figure PCTCN2019115306-appb-000005
样本保存液配制完成后，用15mL离心管进行分装，每管5mL。分装后可于4℃保存1个月。
2、样本清洗液(100mL)
样本清洗液(100mL)的具体配方如表2所示。
表2样本清洗液(100mL)
Figure PCTCN2019115306-appb-000006
样本清洗液需现配现用。
3、样本解离液(10mL)
样本解离液(10mL)的具体配方如表3所示。
表3样本解离液(10mL)
Figure PCTCN2019115306-appb-000007
注：样本解离液现配现用。
表3中，胶原酶储液的配制如表4-6所示。
表4 10×胶原酶I储液(100mL)
Figure PCTCN2019115306-appb-000008
10×胶原酶I储液配制后，用1.5mL无菌离心管分装，每管1mL。该储液可在-20℃长期保存。
表5 10×胶原酶II储液(100mL)
Figure PCTCN2019115306-appb-000009
10×胶原酶II储液配制后，用1.5mL无菌离心管分装，每管1mL。该储液可在-20℃长期保存。
表6 20×胶原酶IV储液(100mL)
Figure PCTCN2019115306-appb-000010
20×胶原酶IV储液配制后，用1.5mL无菌离心管分装，每管1mL。该储液可在-20℃长期保存。
表4、表5和表6中，用蛋白酶的酶活来定义胶原酶(所述胶原酶I或所述胶原酶IV)的单位U：在37℃，pH 7.5的条件下，用1U蛋白酶处理胶原酶(所述胶原酶I或所述胶原酶IV)5小时，可以释放L-亮氨酸1μmol。
4、细胞消化液(10mL)
细胞消化液(10mL)的具体配方如表7所示。
表7细胞消化液(10mL)
Figure PCTCN2019115306-appb-000011
细胞消化液现配现用。
5、消化终止液(100mL)
消化终止液(100mL)的具体配方如表8所示。
表8消化终止液(100mL)
Figure PCTCN2019115306-appb-000012
消化终止液配制后，可在4℃保存一个月。
6、胃癌实体瘤原代细胞培养基(100mL)
胃癌实体瘤原代细胞培养基(100mL)的具体配方如表9所示。
表9胃癌实体瘤原代细胞培养基(100mL)
Figure PCTCN2019115306-appb-000013
胃癌实体瘤原代细胞培养基配制完成后，用0.22μM针头式滤器(Millipore SLGP033RS)过滤除菌，在4℃可以保存两周。
表9中，人重组蛋白储液的配制如表11-表16所示，SB202190储液的配制如表17所示，A83-01储液的配制如表18所示，N-acetyl-L-cysteine储液的配制如表19所示，Nicotinamide储液的配制如表20所示，皮质醇储液的配制如表21所示，胃泌素储液的配制如表22所示，Y-27632储液的配制如表23所示。配制这些储液时所需的100×BSA溶液配制如表10所示。
表10 100×BSA溶液(1mL)
Figure PCTCN2019115306-appb-000014
100×BSA溶液现配现用。
表11 1000×人重组蛋白EGF储液(5mL)
Figure PCTCN2019115306-appb-000015
1000×人重组蛋白EGF储液配制后，用1.5mL无菌离心管分装，该储液可在-80℃长期保存。
表12 1000×人重组蛋白bFGF储液(2.5mL)
Figure PCTCN2019115306-appb-000016
1000×人重组蛋白bFGF储液配制后，用1.5mL无菌离心管分装，该储液可在-80℃长期保存。
表13 1000×人重组蛋白HGF储液(5mL)
Figure PCTCN2019115306-appb-000017
1000×人重组蛋白HGF储液配制后，用1.5mL无菌离心管分装，该储液可在-80℃长期保存。
表14 1000×人重组蛋白FGF-10储液(5mL)
Figure PCTCN2019115306-appb-000018
1000×人重组蛋白FGF-10储液配制后，用1.5mL无菌离心管分装，该储液可在-80℃长期保存。
表15 1000×人重组蛋白Wnt-3a储液(2.5mL)
Figure PCTCN2019115306-appb-000019
Figure PCTCN2019115306-appb-000020
1000×人重组蛋白Wnt-3a储液配制后，用1.5mL无菌离心管分装，该储液可在-80℃长期保存。
表16 1000×人重组蛋白Noggin储液(5mL)
Figure PCTCN2019115306-appb-000021
1000×人重组蛋白Noggin储液配制后，用1.5mL无菌离心管分装，该储液可在-80℃长期保存。
表17 1000×SB202190储液(1.51mL)
Figure PCTCN2019115306-appb-000022
1000×SB202190储液配制后，用0.5mL无菌离心管分装，该储液可在-20℃长期保存。
表18 100000×A83-01储液(1.05mL)
Figure PCTCN2019115306-appb-000023
1000×A83-01储液配制后，用0.5mL无菌离心管分装，该储液可在-20℃长期保存。
表19 1000×N-acetyl-L-cysteine储液(5mL)
Figure PCTCN2019115306-appb-000024
1000×N-acetyl-L-cysteine储液配制后，用0.5mL无菌离心管分装，该储液可在-20℃长期保存。
表20 1000×Nicotinamide储液(4mL)
Figure PCTCN2019115306-appb-000025
1000×Nicotinamide储液配制后，用0.5mL无菌离心管分装，该储液可在-20℃长期保存。
表21 1000×皮质醇储液(100mL)
Figure PCTCN2019115306-appb-000026
1000×皮质醇储液配制后，用1.5mL无菌离心管分装，该储液可在-20℃长期保存。
表22 1000×胃泌素储液(48mL)
Figure PCTCN2019115306-appb-000027
1000×胃泌素储液配制后，用0.5mL无菌离心管分装，该储液可在-20℃长期保存。
表23 1000×Y-27632储液(3.125mL)
Figure PCTCN2019115306-appb-000028
1000×Y-27632储液配制后，用0.5mL无菌离心管分装，该储液可在-20℃长期保存。
7、细胞冻存液
细胞冻存液的具体配方如表24所示。
表24细胞冻存液
Figure PCTCN2019115306-appb-000029
细胞冻存液现配现用。
表24中，1％甲基纤维素溶液的配制如表25所示。
表25 1％甲基纤维素溶液(10mL)
Figure PCTCN2019115306-appb-000030
1％甲基纤维素溶液配制后可在4℃长期保存。
8、1％CYTOP溶液
表26 1％CYTOP溶液(100mL)
Figure PCTCN2019115306-appb-000031
Figure PCTCN2019115306-appb-000032
1％CYTOP溶液配制完成后，可于常温长期保存。
实施例2、胃癌术后标本的获取
1、与三甲医院合作，合作的开展通过了正规的医学伦理审查。
2、主治医生医生按照医学指南规定的临床指征选择入组患者，并根据术中临床指征选择合适的样本用于体外培养，样本的选取标准为：原发性胃癌，病理分期为II期、III期或IV期，各种病理分型的胃癌或胃癌转移病灶，胃癌手术标本重量超过20mg的样本。
3、主治医生提供患者的性别、年龄、病史、家族史、吸烟史、病理分期分型、临床诊断等基本临床信息。隐去患者的姓名、身份证号等与病人隐私相关的信息，用统一的实验编号代替，实验编号的命名原则为采集样本的八位数字日期+患者住院号后四位。例如2018年1月1日提供的样本，患者住院号为T001512765，则样本实验编号为201801012765。
4、术中由外科医生，在手术室无菌环境中采集新鲜标本，置于事先准备好的样本保存液(见实施例1)中。样本离体后在冰上暂存，两小时内运输到实验室进行下一步操作。
实施例3、胃癌组织样本解离前处理
下述操作需要在冰上操作，整个操作步骤需要在10分钟内完成。
下述操作中用到的手术器材，均需事先高温高压灭菌，烘干后才能使用。
1、样本称重。
2、用75％(体积百分含量)乙醇清洗样本表面10到30秒。
3、用样本清洗液清洗样本10次，用无菌的PBS溶液清洗样本5次。
4、用眼科剪、眼科镊、手术刀等器材，小心将样本中的脂肪组织、结缔组织、坏死组织剥离。
实施例4、胃癌组织样本解离
下述实施例中用到的手术器材，均需事先高温高压灭菌，烘干后才能使用。
1、用眼科剪将组织剪碎成1mm 3左右的小块。
2、按0.1mL样本解离液(见实施例1)每mg组织的用量，用事先37℃预热的样本解离液处理剪碎的组织样本，在37℃条件下进行样本解离，解离时间15分钟至3小时。每15分钟在显微镜下观察样本的解离情况，直到观察到大量的单个细胞。
3、用10倍体积的消化终止液(见实施例1)终止解离反应，收集细胞悬液。
4、用40μm无菌细胞滤网过滤细胞悬液，去除组织残片和粘连细胞。
5、800g室温离心10分钟，弃去上清。
6、用5mL无菌PBS重悬细胞，800g室温离心10分钟，弃去上清。
7、用胃癌实体瘤原代细胞培养基(见实施例1)重悬细胞沉淀，在显微镜下观察细胞状态，进行细胞计数。
如图1所示，解离得到的单细胞悬液中，除了肿瘤细胞以外还混杂着大量各种类型的其他细胞，如红细胞，淋巴细胞，纤维细胞等等。本方法的优势之一就是在后续的培养过程中，只有癌细胞可以进行大量扩增，而其他细胞的比例逐渐减少甚至消失，最终获得纯度较高的胃癌原代肿瘤细胞。
实施例5、胃癌原代细胞培养
1、使用低吸附表面(low-attachment-surface)进行胃癌原代细胞悬浮培养，所用培养基即为实施例1中的胃癌实体瘤原代细胞培养基(其中，人重组蛋白EGF的终浓度为50ng/mL；人重组蛋白bFGF的终浓度为20ng/mL；人重组蛋白HGF的终浓度为20ng/mL；人重组蛋白FGF-10的终浓度为20ng/mL；人重组蛋白Wnt-3a的终浓度为250ng/mL；人重组蛋白Noggin的终浓度为100ng/mL；SB202190的终浓度为10μM；A83-01的终浓度为0.5μM；N-acetyl-L-cysteine的终浓度为1mM；Nicotinamide的终浓度为10mM；皮质醇的终浓度为25ng/mL；Y-27632的终浓度为10μM)，以六孔板为例，按每孔10 6个细胞的密度铺板，37℃，5％CO 2条件下在细胞培养箱中进行培养。
2、每天观察细胞状态，每3天更换一次培养基，直至细胞形成直径80μm左右的团块。
如图2所示，经过3-10天的培养，癌细胞大量扩增形成直径80μm大小的细胞团块，肿瘤细胞总数量可以超过10 7，其他类型的细胞数量明显减少甚至消失。本方法经过大量样本测试，胃癌原代肿瘤细胞体外培养成功率可以达到80％。
实施例6、胃癌原代细胞传代
1、收集培养皿中的细胞团块，800g室温离心10分钟，弃去上清。
2、用无菌的PBS溶液清洗细胞团块，800g室温离心10分钟，弃去上清。
3、用细胞消化液(见实施例1)重悬细胞团块，在37℃条件下进行消化。每5分钟在显微镜下观察细胞团块消化的情况，直到细胞团块都被消化为单个细胞。
4、用10倍体积的消化终止液(见实施例1)终止解离反应，收集细胞悬液。
5、800g室温离心10分钟，弃去上清。
6、用胃癌实体瘤原代细胞培养基重悬细胞沉淀，细胞计数。
7、使用低吸附表面(low-attachment-surface)进行胃癌原代细胞培养，所用培养基即为实施例1中的胃癌实体瘤原代细胞培养基，以六孔板为例，按每孔10 6个细胞的密度铺板，37℃，5％CO 2条件下在细胞培养箱中进行培养。
实施例7、胃癌原代细胞的冻存
悬浮培养的胃癌原代细胞经过2-3次传代扩增后，可以进行冻存：
1、收集培养皿中的细胞团块，800g室温离心10分钟，弃去上清。
2、用无菌的PBS溶液清洗细胞团块，800g室温离心10分钟，弃去上清。
3、用细胞消化液(见实施例1)重悬细胞团块，在37℃条件下进行消化。每15分钟在显微镜下观察细胞团块消化的情况，直到细胞团块都被消化为单个细胞。
4、用10倍体积的消化终止液(见实施例1)终止解离反应，收集细胞悬液，细胞计数。
5、800g室温离心10分钟，弃去上清。
6、用细胞冻存液(见实施例1)，按10 6/mL的密度重悬细胞沉淀，2mL冻存管每管1mL细胞悬液，梯度降温盒过夜冻存后转移至液氮中长期保存。
实施例8、胃癌原代细胞的复苏
液氮中保存的胃癌原代细胞可以进行复苏：
1、提前五分钟准备37℃无菌水。
2、将冻存管从液氮中取出，在37℃无菌水中迅速融化细胞。
3、800g室温离心10分钟，弃去上清。
4、用胃癌实体瘤原代细胞培养基(见实施例1)重悬细胞沉淀，使用低吸附表面进行胃癌原代细胞培养，每管细胞复苏至3.5cm培养皿中，37℃，5％CO 2条件下在细胞培养箱中进行培养。
实施例9、胃癌原代细胞的HE染色鉴定
下述实施例中用到的试剂耗材说明：
HE染色试剂盒(北京索莱宝生物科技有限公司，#G1120)；
阳离子防脱玻片(北京中杉金桥生物科技有限公司)；
二甲苯、甲醇、丙酮(北京化学试剂公司，分析纯)；
中性树脂胶(北京益利精细化学品有限公司)。
1、800g离心收集用实施例1中的胃癌实体瘤原代细胞培养基(其中，人重组蛋白EGF的终浓度为20ng/mL；人重组蛋白bFGF的终浓度为20ng/mL；人重组蛋白HGF的终浓度为20ng/mL；人重组蛋白FGF-10的终浓度为20ng/mL；人重组蛋白Wnt-3a的终浓度为200ng/mL；人重组蛋白Noggin的终浓度为100ng/mL；SB202190的终浓度为5μM；A83-01的终浓度为1μM；N-acetyl-L-cysteine的终浓度为1mM；Nicotinamide的终浓度为10mM；皮质醇的终浓度为25ng/mL；Y-27632的终浓度为10μM)培养得到的胃癌实体瘤原代细胞团块，用4％多聚甲醛固定。细胞团块沉淀用石蜡包埋并进行切片，切片厚度为5μm。
2、石蜡切片浸泡在二甲苯溶液中室温孵育5分钟进行脱蜡，重复3次后，用去离子水冲洗切片2次。
3、将切片浸入无水乙醇中室温孵育10分钟，重复两次。
4、姜切片浸入95％乙醇中室温孵育10分钟，重复两次后，用去离子水冲洗切片给两次。
5、待玻片上水分微干时加入100μL苏木精染液染色1mins。
6、吸去苏木精染液，用自来水清洗玻片3次。
7、滴加100μL分化液分化1mins。
8、吸去分化液，依次用自来水清洗玻片2次，蒸馏水清洗玻片1次。
9、吸去玻片表面水分，滴加200μL伊红染液染色40s。
10、吸去伊红染液，依次用75％、80％、90％、100％乙醇漂洗脱水20s、20s、40s、40s。
11、等乙醇晾干后，滴加50μL二甲苯进行细胞通透。
12、等二甲苯晾干完全后，滴加一滴中性树脂胶，用盖玻片封片，在显微镜下观察并拍照。
图3展示了体外培养得到的胃癌原代肿瘤细胞HE染色效果图，可以看到这些细胞普遍具有核质比高、核深染、核内染色质凝集、多核、细胞大小不均一等癌细胞特征，几十到数百个肿瘤细胞聚集形成具有一定立体结构的肿瘤细胞团块。
实施例10、胃癌原代细胞的免疫荧光染色鉴定
下述实施例中用到的试剂说明：
多聚甲醛(北京化学试剂公司，分析纯)，用超纯水溶解多聚甲醛粉末，制成4％(4g/100mL)多聚甲醛溶液；
甲醇、二甲基亚砜(北京化学试剂公司，分析纯)；
双氧水(北京化学试剂公司，35％)；
甲醇、二甲基亚砜、35％双氧水按照4:4:1(体积比)的比例混合制成丹氏漂洗液；
牛血清白蛋白(Sigma，#A1933)，用PBS溶液溶解牛血清白蛋白，制成3％(3g/100mL)的BSA溶液；
免疫荧光一抗抗体(Abcam，#ab17139)；
免疫荧光二抗抗体(CST，#4408)；
Hoechst染液(北京索莱宝生物科技有限公司，#C0021)；
按以下步骤对用实施例1中的胃癌实体瘤原代细胞培养基(其中，人重组蛋白EGF的终浓度为50ng/mL；人重组蛋白bFGF的终浓度为25ng/mL；人重组蛋白HGF的终浓度为25ng/mL；人重组蛋白FGF-10的终浓度为25ng/mL；人重组蛋白Wnt-3a的终浓度为300ng/mL；人重组蛋白Noggin的终浓度为200ng/mL；SB202190的终浓度为10μM；A83-01的终浓度为0.5μM；N-acetyl-L-cysteine的终浓度为1mM；Nicotinamide的终浓度为10mM；皮质醇的终浓度为25ng/mL；Y-27632的终浓度为10μM)培养得到的胃癌细胞团块进行免疫荧光染色，一抗为CK8+CK18，表征上皮来源的细胞。
1、收集培养皿中的细胞团块，用PBS清洗一遍后，用4％多聚甲醛重悬细胞沉淀，4℃过夜固定。
2、800g离心弃去上清，用预冷的甲醇溶液重悬细胞沉淀，在冰上放置1小时。
3、800g离心弃去上清，丹氏漂洗液重悬细胞沉淀，室温放置2小时。
4、800g离心弃去上清，依次用75％、50％、25％(体积百分含量)用PBS稀释的甲醇溶液清洗细胞，每次10分钟。
5、800g离心弃去上清，用3％BSA溶液悬浮细胞沉淀，室温封闭2小时。
6、按1：500的比例，用3％BSA溶液稀释一抗，并用抗体稀释液(3％BSA溶液)重悬细胞沉淀，4℃一抗过夜。
7、800g离心弃去上清，用PBS溶液清洗细胞沉淀5次，每次20分钟。
8、按1：2000的比例，用3％BSA溶液稀释二抗，并用抗体稀释液(3％BSA溶液)重悬细胞沉淀，室温二抗2小时。
9、800g离心弃去上清，用PBS溶液清洗细胞沉淀5次，每次20分钟。
10、按1/100的体积比加入100×Hoechst染液，室温染色20分钟。
11、用PBS溶液清洗细胞沉淀2次，每次10分钟。使用激光共聚焦显微镜观察细胞团块的染色情况。
图4展示了体外培养的胃癌原代肿瘤细胞团块免疫荧光染色的效果图，可以看到组成细胞团块的细胞都是CK8/CK18阳性，是上皮来源的，证实了本方法培养得到的是纯度较高的肿瘤细胞。对20个胃癌样本原代培养物进行免疫荧光染色鉴定，统计结果显示经本方法得到的胃癌原代细胞中，肿瘤细胞的比例达到70％-93％(表27)。
表27胃癌样本原代培养物免疫荧光染色鉴定
Figure PCTCN2019115306-appb-000033
Figure PCTCN2019115306-appb-000034
实施例11、胃癌原代细胞培养物与原发肿瘤组织
下述实施例中提及的DNA提取流程采用天根血液/组织/细胞基因组提取试剂盒(DP304)进行。
下述实施例中提及的建库流程采用NEB DNA测序建库试剂盒(E7645)进行。
下述实施例中提及的高通量测序是指Illumina HiSeq X-ten测序平台。
1、取得胃癌实体瘤样本，进行体外培养操作之前，先取胃癌实体瘤样本10mg进行DNA提取，建库及全基因组高通量测序(WGS)，测序深度300×，剩余实体瘤样本用于胃癌原代细胞体外培养。
2、胃癌组织处理后用实施例1中的胃癌实体瘤原代细胞培养基(其中，人重组蛋白EGF的终浓度为50ng/mL；人重组蛋白bFGF的终浓度为20ng/mL；人重组蛋白HGF的终浓度为20ng/mL；人重组蛋白FGF-10的终浓度为20ng/mL；人重组蛋白Wnt-3a的终浓度为250ng/mL；人重组蛋白Noggin的终浓度为100ng/mL；SB202190的终浓度为10μM；A83-01的终浓度为1μM；N-acetyl-L-cysteine的终浓度为1mM；Nicotinamide的终浓度为8mM；皮质醇的终浓度为25ng/mL；Y-27632的终浓度为8μM)经过一段时间的培养，形成直径100μm以上的细胞团块记为P0代细胞，之后按传代的次数依次记为P1，P2，…，Pn。从P1、P2、P3、P4、P5代的胃癌原代肿瘤细胞培养物中各取10 6个细胞，进行DNA提取，建库及全基因组高通量测序(WGS)，测序深度300×。
3、各组测序结果分别进行拷贝数变异分析(CNV)，比较原发胃癌肿瘤组织与各代胃癌原代细胞培养物之间的拷贝数变异，如图5所示，各代胃癌原代细胞培养物(P1、P2、P3、P4、P5)与原发胃癌肿瘤组织(Tumor)的拷贝数变异情况高度一致，因此经本方法得到的胃癌原代细胞能够代表患者原发肿瘤的真实情况。
实施例12、不同原代细胞培养基培养胃癌原代细胞成功率比较
本实施例中所有样本原代培养的操作方法流程均完全一致(参照前文所述)，仅培养基配方有所区别。进行测试的各种原代细胞培养基见表28。其中方案D为本发明中采用的配方，具体见表9(其中，人重组蛋白EGF的终浓度为50ng/mL；人重组蛋白bFGF的终浓度为20ng/mL；人重组蛋白HGF的终浓度为20ng/mL；人重组蛋白FGF-10的终浓度为20ng/mL；人重组蛋白Wnt-3a的终浓度为250ng/mL；人重组蛋白Noggin的终浓度为100ng/mL；SB202190的终浓度为10μM；A83-01的终浓度为1μM；N-acetyl-L-cysteine的终浓度为1mM；Nicotinamide的终浓度为8mM；皮质醇的终浓度为25ng/mL；Y-27632的终浓度为8μM)。
表28测试用原代细胞培养基配方(100mL)
Figure PCTCN2019115306-appb-000035
原代细胞培养基配制完成后，用0.22μM针头式滤器(Millipore SLGP033RS)过滤除菌，在4℃可以保存两周。
四种原代细胞培养基方案各处理20例样本，按实施例3、4、5中所述方法进行样本处理和培养操作，培养10天后统计胃癌实体瘤原代细胞培养成功率如表29所示：
表29不同培养基培养情况
Figure PCTCN2019115306-appb-000036
可以看到，原代细胞培养基对胃癌原代细胞的培养成功率影响极大，本发明使用的胃癌实体瘤原代细胞培养基(表9)可以最大程度的刺激胃癌实体瘤组织样本中癌细胞增殖，提高胃癌实体瘤原代细胞培养的成功率。
实施例13、不同样本保存液培养胃癌原代细胞成功率比较
本实施例中所有样本原代培养的操作方法流程均完全一致(参照前文所述)，仅样本保存液配方有所区别。进行测试的各种样本保存液见表30。其中方案E为本发明中采用的配方，具体见表1。
表30测试用样本保存液配方(100mL)
Figure PCTCN2019115306-appb-000037
Figure PCTCN2019115306-appb-000038
上表中各种样本保存液配制完成后，用15mL离心管进行分装，每管5mL。分装后可于4℃保存1个月。
五种样本保存液方案各处理20例样本，样本离体后在样本保存液中4℃暂存，离体2小时后，按实施例3、4、5中所述方法进行样本处理和培养操作，培养10天后统计胃癌实体瘤原代细胞培养成功率如表31：
表31不同样本保存液培养情况
Figure PCTCN2019115306-appb-000039
可以看到，样本保存液配方对胃癌实体瘤原代细胞培养的成功率有较大的影响，本发明使用的样本保存液(表1)可以最大程度的保护胃癌实体瘤组织样本中癌细胞的活性，提高培养的成功率。
实施例14、不同样本解离液培养胃癌原代细胞成功率比较
本实施例中所有样本原代培养的操作方法流程均完全一致(参照前文所述)，仅样本解离液配方有所区别。进行测试的各种样本解离液见表32。其中方案D为本发明中采用的配方，具体见表3。
表32测试用样本解离液配方(10mL)
Figure PCTCN2019115306-appb-000040
Figure PCTCN2019115306-appb-000041
样本解离液现配现用。
选取20例胃癌实体瘤组织块重量超过100mg的样本，平均分成四份，分别用上述四种样本解离液，按实施例3、4、5中所述方法进行样本处理和培养操作。培养10天后统计胃癌实体瘤原代细胞培养成功率如下表33：
表33不同样本解离液培养情况
Figure PCTCN2019115306-appb-000042
可以看到，样本解离液配方对胃癌实体瘤原代细胞培养的成功率有很大的 影响，本发明使用的样本解离液(表3)可以最大程度分离胃癌实体瘤组织中的癌细胞，提高胃癌实体瘤原代细胞培养的成功率。
实施例15、不同细胞消化液传代胃癌原代细胞成功率比较
本实施例中所有样本原代细胞传代操作方法流程均完全一致(参照前文所述)，仅细胞消化液配方有所区别。进行测试的各种样本解离液见表34。其中方案D为本发明中采用的配方，具体见表7。
表34测试用细胞消化液配方(10mL)
Figure PCTCN2019115306-appb-000043
细胞消化液现配现用。
选取20例培养成功的胃癌样本，将培养得到的胃癌实体瘤原代细胞，分别用上述四种细胞消化液，按实施例6中所述方法进行连续传代操作。每当癌细胞扩增形成直径100μm大小的细胞团时就进行传代(不超过10次)，记录最大传代次数。统计结果如表35：
表35不同细胞消化液培养情况
Figure PCTCN2019115306-appb-000044
Figure PCTCN2019115306-appb-000045
可以看到，细胞消化液配方对胃癌实体瘤原代细胞传代的成功率有很大的影响，本发明使用的细胞消化液(表7)可以温和解离细胞团块中的癌细胞，使样本可以进行连续传代而保持胃癌实体瘤原代细胞活性。
实施例16、用不同材质的细胞培养耗材进行胃癌原代肿瘤细胞培养
本实施例中所有样本原代培养的操作方法流程均完全一致(参照前文所述)，仅细胞培养耗材材质(未经修饰)有所区别(表36)。
表36未经修饰的不同材质培养耗材对胃癌原代肿瘤细胞培养的影响
Figure PCTCN2019115306-appb-000046
注：聚苯乙烯(Polystyrene，缩写PS)、聚碳酸酯(Polycarbonate，缩写PC)、聚甲基丙烯酸甲酯(poly-methyl methacrylate，缩写PMMA)、COC树脂、环烯烃聚合物(Cyclo Olefin Polymer，缩写COP)、低吸附表面(low-attachment-surface，缩写LAS)。
由表36可见：不同材质培养耗材对胃癌实体瘤原代细胞的培养成功率有一定的影响，其中低吸附表面(LAS)培养成功率最高。
实施例17、用CYTOP修饰的细胞培养耗材进行胃癌原代肿瘤细胞培养
本实施例中所有样本原代培养的操作方法流程均完全一致(参照前文所述)，CYTOP的修饰方法均完全一致，仅细胞培养耗材材质有所区别(表37)。
其中CYTOP修饰的方法为：首先对细胞培养容器进行纯氧刻蚀，刻蚀条件为功率20W，刻蚀时间为3分钟。然后用适量(以96孔板为例，每孔20μL，适量是指完全覆盖培养皿底部)1％CYTOP溶液覆盖培养皿或培养平板表面，待CYTOP溶液完全晾干后即可使用。
表37 CYTOP修饰的不同材质CYTOP修饰培养耗材对胃癌原代肿瘤细胞培养的影响
Figure PCTCN2019115306-appb-000047
由表37可见：可以看到CYTOP修饰可以有效提高各种材料的培养成功率。
实施例18、用胃癌原代肿瘤细胞进行药物敏感性测试
本实施例中用到的化疗药物Irinotecan、5-Fluorouracil、Oxaliplatin均为Selleck产品。
本实施例中提到的Celltiter-Glo细胞活力检测试剂盒为Promega产品。
用本发明培养得到的原代胃癌细胞进行体外药物敏感性测试：用标准尺寸96孔低吸附细胞培养平板，按10 5/孔的密度进行原代细胞接种，每种药物设置5个药物浓度梯度，n＝3。加药后细胞在37℃，5％CO 2条件下孵育7天。药物作用结束后，用Celltiter-Glo细胞活力检测试剂盒检测各孔细胞活力。实验结果见图6。该结果说明：本方法获得的原代胃癌细胞可以用于体外药物敏感性检测。
实施例19、配制用于培养胆囊癌、胆管癌实体瘤原代细胞的试剂
1、样本保存液(100mL)
样本保存液(100mL)的具体配方(表1)以及配制方法见实施例1步骤1。
2、样本清洗液(100mL)
样本清洗液(100mL)的具体配方(表2)以及配制方法见实施例1步骤2。
3、样本解离液(10mL)
样本解离液(10mL)的具体配方(表3)以及配制方法见实施例1步骤3。
4、细胞消化液(10mL)
细胞消化液(10mL)的具体配方(表7)以及配制方法见实施例1步骤4。
5、消化终止液(100mL)
消化终止液(100mL)的具体配方(表8)以及配制方法见实施例1步骤5。
6、胆囊癌、胆管癌实体瘤原代细胞培养基(100mL)
胆囊癌、胆管癌实体瘤原代细胞培养基(100mL)的具体配方(表9)以及配制方法见实施例1步骤6。
7、细胞冻存液
细胞冻存液的具体配方(表24)以及配制方法见实施例1步骤7。
8、1％CYTOP溶液
1％CYTOP溶液的具体配方(表26)以及配制方法见实施例1步骤8。
实施例20、胆囊癌、胆管癌术后标本/活检穿刺标本的获取
1、与三甲医院合作，合作的开展通过了正规的医学伦理审查。
2、主治医生医生按照医学指南规定的临床指征选择入组患者，并根据术中临床指征选择合适的样本用于体外培养。手术样本的选取标准为：原发性胆囊癌或胆管癌，病理分期为II期、III期或IV期，各种病理分型的胆囊癌或胆管癌转移病灶，手术标本重量超过20mg的样本。活检穿刺样本的选取标准为：原发性胆囊癌或胆管癌，病理分期为II期、III期或IV期，各种病理分型的胆囊癌或胆管癌转移病灶，穿刺标本4条以上的样本。
3、主治医生提供患者的性别、年龄、病史、家族史、吸烟史、病理分期分型、临床诊断等基本临床信息。隐去患者的姓名、身份证号等与病人隐私相关的信息，用统一的实验编号代替，实验编号的命名原则为采集样本的八位数字日期+患者住院号后四位。例如2018年1月1日提供的样本，患者住院号为T001512765，则样本实验编号为201801012765。
4、术中由外科医生，在手术室无菌环境中采集新鲜手术标本，置于事先准备好的样本保存液(见实施例19)中。样本离体后在冰上暂存，两小时内运输到实验室进行下一步操作。
5、穿刺术医生，在穿刺手术室无菌环境中采集新鲜穿刺标本，置于事先准备好的样本保存液(见实施例19)中。样本离体后在冰上暂存，两小时内运输到实验室进行下一步操作。
实施例21、胆囊癌、胆管癌样本解离前处理
下述操作需要在冰上操作，整个操作步骤需要在10分钟内完成。
下述操作中用到的手术器材，均需事先高温高压灭菌，烘干后才能使用。
1、样本称重。
2、用75％(体积百分含量)乙醇清洗样本表面10到30秒。
3、用样本清洗液清洗样本10次，用无菌的PBS溶液清洗样本5次。
4、用眼科剪、眼科镊、手术刀等器材，小心将样本中的脂肪组织、结缔组织、坏死组织剥离。
实施例22、胆囊癌、胆管癌组织样本解离
下述实施例中用到的手术器材，均需事先高温高压灭菌，烘干后才能使用。
1、用眼科剪将组织剪碎成1mm 3左右的小块。
2、按0.1mL样本解离液(见实施例19)每mg组织的用量，用事先37℃预热的样本解离液处理剪碎的组织样本，在37℃条件下进行样本解离，解离时间15分钟至3小时。每15分钟在显微镜下观察样本的解离情况，直到观察到大量的单个细胞。
3、用10倍体积的消化终止液(见实施例19)终止解离反应，收集细胞悬液。
4、用40μm无菌细胞滤网过滤细胞悬液，去除组织残片和粘连细胞。
5、800g室温离心10分钟，弃去上清。
6、用5mL无菌PBS重悬细胞，800g室温离心10分钟，弃去上清。
7、用胆囊癌、胆管癌实体瘤原代细胞培养基(见实施例19)重悬细胞沉淀，在显微镜下观察细胞状态，进行细胞计数。
如图7所示，解离得到的单细胞悬液中，除了肿瘤细胞以外还混杂着大量各种类型的其他细胞，如红细胞，淋巴细胞，纤维细胞等等。本方法的优势之一就是在后续的培养过程中，只有癌细胞可以进行大量扩增，而其他细胞的比例逐渐减少甚至消失，最终获得纯度较高的胆囊癌、胆管癌原代肿瘤细胞。
实施例23、胆囊癌、胆管癌原代细胞培养
1、使用低吸附表面(low-attachment-surface)进行胆囊癌、胆管癌实体瘤原代细胞悬浮培养，所用培养基即为实施例19中的胆囊癌、胆管癌原代细胞培养基(其中，人重组蛋白EGF的终浓度为50ng/mL；人重组蛋白bFGF的终浓度为20ng/mL；人重组蛋白HGF的终浓度为20ng/mL；人重组蛋白FGF-10的终浓度为20ng/mL；人重组蛋白Wnt-3a的终浓度为250ng/mL；人重组蛋白Noggin的终浓度为100ng/mL；SB202190的终浓度为10μM；A83-01的终浓度为0.5μM；N-acetyl-L-cysteine的终浓度为1mM；Nicotinamide的终浓度为10mM；皮质醇的终浓度为25ng/mL；Y-27632的终浓度为10μM)，以六孔板为例，按每孔10 6个细胞的密度铺板，37℃，5％CO 2条件下在细胞培养箱中进行培养。
2、每天观察细胞状态，每3天更换一次培养基，直至细胞形成直径80μm左右的团块。
如图8所示，经过3-10天的培养，癌细胞大量扩增形成直径80μm大小的细胞团块，肿瘤细胞总数量可以超过10 7，其他类型的细胞数量明显减少甚至消失。本方法经过大量样本测试，胆囊癌、胆管癌原代肿瘤细胞体外培养成功率可以达到80％。
实施例24、胆囊癌、胆管癌实体瘤原代细胞传代
1、收集培养皿中的细胞团块，800g室温离心10分钟，弃去上清。
2、用无菌的PBS溶液清洗细胞团块，800g室温离心10分钟，弃去上清。
3、用细胞消化液(见实施例19)重悬细胞团块，在37℃条件下进行消化。每5分钟在显微镜下观察细胞团块消化的情况，直到细胞团块都被消化为单个细胞。
4、用10倍体积的消化终止液(见实施例19)终止解离反应，收集细胞悬液。
5、800g室温离心10分钟，弃去上清。
6、用胆囊癌、胆管癌原代细胞培养基重悬细胞沉淀，细胞计数。
7、使用低吸附表面(low-attachment-surface)进行胆囊癌、胆管癌原代细胞培养，所用培养基即为实施例19中的胆囊癌、胆管癌实体瘤原代细胞培养基，以六孔板为例，按每孔10 6个细胞的密度铺板，37℃，5％CO 2条件下在细胞培养箱中进行培养。
实施例25、胆囊癌、胆管癌实体瘤原代细胞的冻存
悬浮培养的胆囊癌、胆管癌实体瘤原代细胞经过2-3次传代扩增后，可以进行冻存：
1、收集培养皿中的细胞团块，800g室温离心10分钟，弃去上清。
2、用无菌的PBS溶液清洗细胞团块，800g室温离心10分钟，弃去上清。
3、用细胞消化液(见实施例19)重悬细胞团块，在37℃条件下进行消化。每15分钟在显微镜下观察细胞团块消化的情况，直到细胞团块都被消化为单个细胞。
4、用10倍体积的消化终止液(见实施例19)终止解离反应，收集细胞悬液，细胞计数。
5、800g室温离心10分钟，弃去上清。
6、用细胞冻存液(见实施例19)，按10 6/mL的密度重悬细胞沉淀，2mL冻存管每管1mL细胞悬液，梯度降温盒过夜冻存后转移至液氮中长期保存。
实施例26、胆囊癌、胆管癌实体瘤原代细胞的复苏
液氮中保存的胆囊癌、胆管癌实体瘤原代细胞可以进行复苏：
1、提前五分钟准备37℃无菌水。
2、将冻存管从液氮中取出，在37℃无菌水中迅速融化细胞。
3、800g室温离心10分钟，弃去上清。
4、用胆囊癌、胆管癌实体瘤原代细胞培养基(见实施例19)重悬细胞沉淀，使用低吸附表面进行胆囊癌、胆管癌实体瘤原代细胞培养，每管细胞复苏至3.5cm培养皿中，37℃，5％CO 2条件下在细胞培养箱中进行培养。
实施例27、胆囊癌、胆管癌实体瘤原代细胞的HE染色鉴定
下述实施例中用到的试剂耗材说明：
HE染色试剂盒(北京索莱宝生物科技有限公司，#G1120)；
阳离子防脱玻片(北京中杉金桥生物科技有限公司)；
二甲苯、甲醇、丙酮(北京化学试剂公司，分析纯)；
中性树脂胶(北京益利精细化学品有限公司)。
1、800g离心收集用实施例19中的胆囊癌、胆管癌实体瘤原代细胞培养基(其中，人重组蛋白EGF的终浓度为20ng/mL；人重组蛋白bFGF的终浓度为20ng/mL；人重组蛋白HGF的终浓度为20ng/mL；人重组蛋白FGF-10的终浓度为20ng/mL；人重组蛋白Wnt-3a的终浓度为200ng/mL；人重组蛋白Noggin的终浓度为100ng/mL；SB202190的终浓度为5μM；A83-01的终浓度为1μM；N-acetyl-L-cysteine的终浓度为1mM；Nicotinamide的终浓度为10mM；皮质醇的终浓度为25ng/mL；Y-27632的终浓度为10μM)培养得到的胆囊癌、胆管癌实体瘤原代细胞团块，用4％多聚甲醛固定。细胞团块沉淀用石蜡包埋并进行切片，切片厚度为5μm。
2、石蜡切片浸泡在二甲苯溶液中室温孵育5分钟进行脱蜡，重复3次后，用去离子水冲洗切片2次。
3、将切片浸入无水乙醇中室温孵育10分钟，重复两次。
4、姜切片浸入95％乙醇中室温孵育10分钟，重复两次后，用去离子水冲洗切片给两次。
5、待玻片上水分微干时加入100μL苏木精染液染色1mins。
6、吸去苏木精染液，用自来水清洗玻片3次。
7、滴加100μL分化液分化1mins。
8、吸去分化液，依次用自来水清洗玻片2次，蒸馏水清洗玻片1次。
9、吸去玻片表面水分，滴加200μL伊红染液染色40s。
10、吸去伊红染液，依次用75％、80％、90％、100％乙醇漂洗脱水20s、20s、40s、40s。
11、等乙醇晾干后，滴加50μL二甲苯进行细胞通透。
12、等二甲苯晾干完全后，滴加一滴中性树脂胶，用盖玻片封片，在显微镜下观察并拍照。
图9展示了体外培养得到的胆管癌原代肿瘤细胞HE染色效果图，可以看到这些细胞普遍具有核质比高、核深染、核内染色质凝集、多核、细胞大小不均一等癌细胞特征，几十到数百个肿瘤细胞聚集形成具有一定立体结构的肿瘤细胞团块。
实施例28、胆囊癌、胆管癌实体瘤原代细胞的免疫组化染色鉴定
下述实施例中用到的试剂说明：
多聚甲醛(北京化学试剂公司，分析纯)，用超纯水溶解多聚甲醛粉末，制成4％(4g/100mL)多聚甲醛溶液；
双氧水(北京化学试剂公司，35％)；
封闭用正常山羊血清(Solarbio，SL038)；
免疫组化一抗抗体(Abcam，ab215838)；
免疫组化二抗抗体(Abcam，ab205719)；
EDTA修复液(Abcam，ab93684)；
DAB显色液(
Figure PCTCN2019115306-appb-000048
DAB Substrate Kit，8059S)
收集实施例19中的胆囊癌、胆管癌实体瘤原代细胞培养基(其中，人重组蛋白EGF的终浓度为50ng/mL；人重组蛋白bFGF的终浓度为25ng/mL；人重组蛋白HGF的终浓度为25ng/mL；人重组蛋白FGF-10的终浓度为25ng/mL；人重组蛋白Wnt-3a的终浓度为300ng/mL；人重组蛋白Noggin的终浓度为200ng/mL；SB202190的终浓度为10μM；A83-01的终浓度为0.5μM；N-acetyl-L-cysteine的终浓度为1mM；Nicotinamide的终浓度为10mM；皮质醇的终浓度为25ng/mL；Y-27632的终浓度为10μM)培养得到的胆囊癌、胆管癌细胞团块进行石蜡切片，按以下步骤对用pan-CK抗体，表征上皮来源的细胞。
1.将切片依次浸入二甲苯Ⅰ10min，二甲苯Ⅱ(10min)。
2.再次浸入无水乙醇Ⅰ(5min)-无水乙醇Ⅱ(5min)-95％酒精(5min)-80％酒精(5min)-70％酒精(5min)，然后去离子水冲洗2次，每次2min。
3.将组织切片放入修复盒，然后加入适量稀释好的EDTA修复液(pH 9.0)，液面要浸没组织。
4.微波中档修复10min(液体沸腾时开始计时)，此过程中勿使组织干片。
5.将修复盒从微波炉中拿出，自然冷却降温，当修复液降至室温后取出玻片，PBS(pH 7.4)冲洗3遍，每次3min(冲洗过程中切勿对着组织冲洗，以免弄破组织)。
6.将配制好的3％的过氧化氢(去离子水稀释30％过氧化氢)滴加于切片组织上以阻断内源性过氧化物酶，室温孵育15min，PBS冲洗3次，每次3min。
7.吸水纸吸干PBS，在玻片上滴加10％的山羊血清(与二抗种属来源一致或相似)，37℃封闭60min。
8.用吸水纸擦干玻片组织周围的液体，用油性笔在组织周围画圈，然后滴加稀释好的一抗，于湿盒中4℃孵育过夜。
9.PBS冲洗切片3次，每次3min，吸水纸擦干切片后滴加辣根过氧化物酶标记二抗，室温孵育60min。
10.PBS冲洗切片3次，每次3min，甩去PBS液体后吸水纸擦干切片，每张切片滴加新鲜配制的DAB显色液，显微镜下观察，阳性信号后自来水冲洗切片终止显色。
11.苏木素复染，1min，水洗后使用酸性乙醇分化液分化，再用自来水冲洗返蓝。
12.将切片置于水中冲洗后，将切片依次放入：70％酒精-80％酒精-90％酒精-95％酒精-无水乙醇Ⅰ-无水乙醇Ⅱ-二甲苯Ⅰ-二甲苯Ⅱ中脱水透明，每 个试剂中放置2min，最后在通风橱中风干切片。
13.使用中性树胶封片，用盖玻片盖上。置于通风橱中晾干。
14.晾干的切片可以在显微镜下观察或拍照。
图10展示了体外培养的胆管癌原代肿瘤细胞团块免疫组化染色的效果图，可以看到组成细胞团块的细胞都是pan-CK阳性，是上皮来源的，证实了本方法培养得到的是纯度较高的肿瘤细胞。对5个胆囊癌、胆管癌样本原代培养物进行免疫组化染色鉴定，统计结果显示经本方法得到的胆囊癌、胆管癌原代细胞中，肿瘤细胞的比例达到84％-95％(表38)。
表38胆囊癌、胆管癌原代培养物免疫组化染色鉴定
Figure PCTCN2019115306-appb-000049
实施例29、用CYTOP修饰的细胞培养耗材进行胆囊癌、胆管癌实体瘤原代肿瘤细胞培养
本实施例中所有样本原代培养的操作方法流程均完全一致(参照前文所述)，CYTOP的修饰方法均完全一致，仅细胞培养耗材材质有所区别(表39)。
其中CYTOP修饰的方法为：首先对细胞培养容器进行纯氧刻蚀，刻蚀条件为功率20W，刻蚀时间为3分钟。然后用适量(以96孔板为例，每孔20μL，适量是指完全覆盖培养皿底部)1％CYTOP溶液覆盖培养皿或培养平板表面，待CYTOP溶液完全晾干后即可使用。
表39 CYTOP修饰耗材对胆囊癌、胆管癌实体瘤原代肿瘤细胞培养的影响
Figure PCTCN2019115306-appb-000050
注：聚苯乙烯(Polystyrene，缩写PS)。
由表39可见：可以看到CYTOP修饰后可以大幅度提高样本培养成功率。
实施例30、配制用于培养胆囊癌胆管癌胆汁样本原代细胞的试剂
1、细胞分离缓冲液(100mL)
细胞分离缓冲液(100mL)的具体配方如表40所示：
表40细胞分离缓冲液(100mL)
Figure PCTCN2019115306-appb-000051
细胞分离缓冲液配制完成后，可于4℃保存1个月。
表40中，肝素钠溶液的配制如表41所示。
表41 1000×肝素钠(1mL)
Figure PCTCN2019115306-appb-000052
1000×肝素钠溶液现配现用。
2、细胞消化液(10mL)
细胞消化液(10mL)的具体配方(表7)以及配制方法见实施例1步骤4。
3、消化终止液(100mL)
消化终止液(100mL)的具体配方(表8)以及配制方法见实施例1步骤5。
4、胆囊癌、胆管癌胆汁样本原代细胞培养基(100mL)
胆囊癌、胆管癌胆汁样本原代细胞培养基(100mL)的具体配方(表9)以及配制方法见实施例1步骤6。
5、细胞冻存液
细胞冻存液的具体配方(表24)以及配制方法见实施例1步骤7。
实施例31、胆囊癌、胆管癌胆汁样本的获取
1、与三甲医院合作，合作的开展通过了正规的医学伦理审查。
2、主治医生医生按照医学指南规定的临床指征选择入组患者，并根据术中临床指征选择合适的样本用于体外培养。手术样本的选取标准为：原发性胆囊癌或胆管癌，病理分期为II期、III期或IV期，胆汁样本的体积超过20mL的样本。
3、主治医生提供患者的性别、年龄、病史、家族史、吸烟史、病理分期分型、临床诊断等基本临床信息。隐去患者的姓名、身份证号等与病人隐私相关的信息，用统一的实验编号代替，实验编号的命名原则为采集样本的八位数字日期+患者住院号后四位。例如2018年1月1日提供的样本，患者住院号为T001512765，则样本实验编号为201801012765。
4、由患者主管医生，使用无菌器材采集新鲜胆汁标本10mL以上。样本在冰上暂存，48小时内运输到实验室进行下一步操作。
实施例32、胆囊癌、胆管癌胆汁样本预处理
下述操作需要在冰上操作，整个操作步骤需要在10分钟内完成。
1、胆汁样本冰上静置30分钟左右，使样本中的凝血块和大的不溶固体沉降至样本管底部；
2、小心将上清液转移至50mL无菌离心管中，加入一倍体积预冷的PBS混匀；
3、2000g，4℃离心5分钟，弃去上清；
4、用细胞分离缓冲液(见实施例30)重悬细胞沉淀，2000g，4℃离心5分钟，弃去上清；
5、用细胞分离缓冲液(见实施例30)重悬细胞沉淀，调整细胞浓度至10 7/mL。
实施例33、胆囊癌、胆管癌胆汁样本密度梯度离心
1、用50mL无菌离心管取与细胞悬液等体积的Ficoll细胞分离液(MP#50494)。
2、小心将细胞悬液加在细胞分离液上层，使两者之间形成一个清晰的界面。
3、2000g水平式常温离心20分钟。
4、吸取中间层白膜，至新管中。
5、用20mL无菌的PBS重悬细胞沉淀，1500g常温离心10分钟，弃去上清。
6、用胆囊癌、胆管癌胆汁样本原代细胞培养基(见实施例30)重悬细胞沉淀，在显微镜下观察细胞状态，进行细胞计数。
如图11所示，分离得到的单细胞悬液中，除了肿瘤细胞以外还混杂着大量各种类型的其他细胞，如红细胞，淋巴细胞，纤维细胞等等。本方法的优势之一就是在后续的培养过程中，只有癌细胞可以进行大量扩增，而其他细胞的比例逐渐减少甚至消失，最终获得纯度较高的胆囊癌、胆管癌原代肿瘤细胞。
实施例34、胆囊癌、胆管癌胆汁样本原代细胞培养
1、使用低吸附表面(low-attachment-surface)进行胆囊癌、胆管癌胆汁样本原代细胞悬浮培养，所用培养基即为实施例30中的胆囊癌、胆管癌胆汁样本原代细胞培养基(其中，人重组蛋白EGF的终浓度为50ng/mL；人重组蛋白bFGF的终浓度为20ng/mL；人重组蛋白HGF的终浓度为20ng/mL；人重组蛋白FGF-10的终浓度为20ng/mL；人重组蛋白Wnt-3a的终浓度为250ng/mL；人重组蛋白Noggin的终浓度为100ng/mL；SB202190的终浓度为10μM；A83-01的终浓度为0.5μM；N-acetyl-L-cysteine的终浓度为1mM；Nicotinamide的终浓度为10mM；皮质醇的终浓度为25ng/mL；Y-27632的终浓度为10μM)，以六孔板为例，按每孔10 6个细胞的密度铺板，37℃，5％CO 2条件下在细胞培养箱中进行培养。
2、每天观察细胞状态，每3天更换一次培养基，直至细胞形成直径80μm左右的团块。
如图12所示，经过3-10天的培养，癌细胞大量扩增形成直径80μm大小的细胞团块，肿瘤细胞总数量可以超过10 7，其他类型的细胞数量明显减少甚至消 失。本方法经过大量样本测试，胆囊癌、胆管癌胆汁样本原代肿瘤细胞体外培养成功率可以达到70％。
实施例35、胆囊癌、胆管癌胆汁样本原代细胞传代
1、收集培养皿中的细胞团块，800g室温离心10分钟，弃去上清。
2、用无菌的PBS溶液清洗细胞团块，800g室温离心10分钟，弃去上清。
3、用细胞消化液(见实施例30)重悬细胞团块，在37℃条件下进行消化。每5分钟在显微镜下观察细胞团块消化的情况，直到细胞团块都被消化为单个细胞。
4、用10倍体积的消化终止液(见实施例30)终止解离反应，收集细胞悬液。
5、800g室温离心10分钟，弃去上清。
6、用胆囊癌、胆管癌原代细胞培养基重悬细胞沉淀，细胞计数。
7、使用低吸附表面(low-attachment-surface)进行胆囊癌、胆管癌胆汁样本原代细胞培养，所用培养基即为实施例30中的胆囊癌、胆管癌胆汁样本原代细胞培养基，以六孔板为例，按每孔10 6个细胞的密度铺板，37℃，5％CO 2条件下在细胞培养箱中进行培养。
实施例36、胆囊癌、胆管癌胆汁样本原代细胞的冻存
悬浮培养的胆囊癌、胆管癌胆汁样本原代细胞经过2-3次传代扩增后，可以进行冻存：
1、收集培养皿中的细胞团块，800g室温离心10分钟，弃去上清。
2、用无菌的PBS溶液清洗细胞团块，800g室温离心10分钟，弃去上清。
3、用细胞消化液(见实施例30)重悬细胞团块，在37℃条件下进行消化。每15分钟在显微镜下观察细胞团块消化的情况，直到细胞团块都被消化为单个细胞。
4、用10倍体积的消化终止液(见实施例30)终止解离反应，收集细胞悬液，细胞计数。
5、800g室温离心10分钟，弃去上清。
6、用细胞冻存液(见实施例30)，按10 6/mL的密度重悬细胞沉淀，2mL冻存管每管1mL细胞悬液，梯度降温盒过夜冻存后转移至液氮中长期保存。
实施例37、胆囊癌、胆管癌胆汁样本原代细胞的复苏
液氮中保存的胆囊癌、胆管癌胆汁样本原代细胞可以进行复苏：
1、提前五分钟准备37℃无菌水。
2、将冻存管从液氮中取出，在37℃无菌水中迅速融化细胞。
3、800g室温离心10分钟，弃去上清。
4、用胆囊癌、胆管癌胆汁样本原代细胞培养基(见实施例30)重悬细胞沉淀，使用低吸附表面进行胆囊癌、胆管癌胆汁样本原代细胞培养，每管细胞复苏至3.5cm培养皿中，37℃，5％CO 2条件下在细胞培养箱中进行培养。
实施例38、胆囊癌、胆管癌胆汁样本原代细胞的HE染色鉴定
下述实施例中用到的试剂耗材说明：
HE染色试剂盒(北京索莱宝生物科技有限公司，#G1120)；
阳离子防脱玻片(北京中杉金桥生物科技有限公司)；
二甲苯、甲醇、丙酮(北京化学试剂公司，分析纯)；
中性树脂胶(北京益利精细化学品有限公司)。
1、800g离心收集用实施例30中的胆囊癌、胆管癌胆汁样本原代细胞培养基(其中，人重组蛋白EGF的终浓度为20ng/mL；人重组蛋白bFGF的终浓度为20ng/mL；人重组蛋白HGF的终浓度为20ng/mL；人重组蛋白FGF-10的终浓度为20ng/mL；人重组蛋白Wnt-3a的终浓度为200ng/mL；人重组蛋白Noggin的终浓度为100ng/mL；SB202190的终浓度为5μM；A83-01的终浓度为1μM；N-acetyl-L-cysteine的终浓度为1mM；Nicotinamide的终浓度为10mM；皮质醇的终浓度为25ng/mL；Y-27632的终浓度为10μM)培养得到的胆囊癌、胆管癌实体瘤原代细胞团块，用4％多聚甲醛固定。细胞团块沉淀用石蜡包埋并进行切片，切片厚度为5μm。
2、石蜡切片浸泡在二甲苯溶液中室温孵育5分钟进行脱蜡，重复3次后，用去离子水冲洗切片2次。
3、将切片浸入无水乙醇中室温孵育10分钟，重复两次。
4、姜切片浸入95％乙醇中室温孵育10分钟，重复两次后，用去离子水冲洗切片给两次。
5、待玻片上水分微干时加入100μL苏木精染液染色1mins。
6、吸去苏木精染液，用自来水清洗玻片3次。
7、滴加100μL分化液分化1mins。
8、吸去分化液，依次用自来水清洗玻片2次，蒸馏水清洗玻片1次。
9、吸去玻片表面水分，滴加200μL伊红染液染色40s。
10、吸去伊红染液，依次用75％、80％、90％、100％乙醇漂洗脱水20s、20s、40s、40s。
11、等乙醇晾干后，滴加50μL二甲苯进行细胞通透。
12、等二甲苯晾干完全后，滴加一滴中性树脂胶，用盖玻片封片，在显微镜下观察并拍照。
图13展示了体外培养得到的胆管癌胆汁样本原代肿瘤细胞HE染色效果图，可以看到这些细胞普遍具有核质比高、核深染、核内染色质凝集、多核、细胞大小不均一等癌细胞特征，几十到数百个肿瘤细胞聚集形成具有一定立体结构的肿瘤细胞团块。
实施例39、胆囊癌、胆管癌胆汁样本原代细胞的免疫组化染色鉴定
下述实施例中用到的试剂说明：
多聚甲醛(北京化学试剂公司，分析纯)，用超纯水溶解多聚甲醛粉末， 制成4％(4g/100mL)多聚甲醛溶液；
双氧水(北京化学试剂公司，35％)；
封闭用正常山羊血清(Solarbio，SL038)；
免疫组化一抗抗体(Abcam，ab215838)；
免疫组化二抗抗体(Abcam，ab205719)；
EDTA修复液(Abcam，ab93684)；
DAB显色液(
Figure PCTCN2019115306-appb-000053
DAB Substrate Kit，8059S)
收集实施例30中的胆囊癌、胆管癌胆汁样本原代细胞培养基(其中，人重组蛋白EGF的终浓度为50ng/mL；人重组蛋白bFGF的终浓度为25ng/mL；人重组蛋白HGF的终浓度为25ng/mL；人重组蛋白FGF-10的终浓度为25ng/mL；人重组蛋白Wnt-3a的终浓度为300ng/mL；人重组蛋白Noggin的终浓度为200ng/mL；SB202190的终浓度为10μM；A83-01的终浓度为0.5μM；N-acetyl-L-cysteine的终浓度为1mM；Nicotinamide的终浓度为10mM；皮质醇的终浓度为25ng/mL；Y-27632的终浓度为10μM)培养得到的胆囊癌、胆管癌胆汁样本原代细胞团块进行石蜡切片，按以下步骤对用pan-CK抗体，表征上皮来源的细胞。
1.将切片依次浸入二甲苯Ⅰ10min，二甲苯Ⅱ(10min)。
2.再次浸入无水乙醇Ⅰ(5min)-无水乙醇Ⅱ(5min)-95％酒精(5min)-80％酒精(5min)-70％酒精(5min)，然后去离子水冲洗2次，每次2min。
3.将组织切片放入修复盒，然后加入适量稀释好的EDTA修复液(pH 9.0)，液面要浸没组织。
4.微波中档修复10min(液体沸腾时开始计时)，此过程中勿使组织干片。
5.将修复盒从微波炉中拿出，自然冷却降温，当修复液降至室温后取出玻片，PBS(pH 7.4)冲洗3遍，每次3min(冲洗过程中切勿对着组织冲洗，以免弄破组织)。
6.将配制好的3％的过氧化氢(去离子水稀释30％过氧化氢)滴加于切片组织上以阻断内源性过氧化物酶，室温孵育15min，PBS冲洗3次，每次3min。
7.吸水纸吸干PBS，在玻片上滴加10％的山羊血清(与二抗种属来源一致或相似)，37℃封闭60min。
8.用吸水纸擦干玻片组织周围的液体，用油性笔在组织周围画圈，然后滴加稀释好的一抗，于湿盒中4℃孵育过夜。
9.PBS冲洗切片3次，每次3min，吸水纸擦干切片后滴加辣根过氧化物酶标记二抗，室温孵育60min。
10.PBS冲洗切片3次，每次3min，甩去PBS液体后吸水纸擦干切片，每张切片滴加新鲜配制的DAB显色液，显微镜下观察，阳性信号后自来水冲洗切片终止显色。
11.苏木素复染，1min，水洗后使用酸性乙醇分化液分化，再用自来水冲洗 返蓝。
12.将切片置于水中冲洗后，将切片依次放入：70％酒精-80％酒精-90％酒精-95％酒精-无水乙醇Ⅰ-无水乙醇Ⅱ-二甲苯Ⅰ-二甲苯Ⅱ中脱水透明，每个试剂中放置2min，最后在通风橱中风干切片。
13.使用中性树胶封片，用盖玻片盖上。置于通风橱中晾干。
14.晾干的切片可以在显微镜下观察或拍照。
图14展示了体外培养的胆管癌原代肿瘤细胞团块免疫组化染色的效果图，可以看到组成细胞团块的细胞都是pan-CK阳性，是上皮来源的，证实了本方法培养得到的是纯度较高的肿瘤细胞。对5个胆囊癌、胆管癌样本原代培养物进行免疫组化染色鉴定，统计结果显示经本方法得到的胆囊癌、胆管癌胆汁样本原代细胞中，肿瘤细胞的比例达到64％-80％(表42)。
表42胆囊癌、胆管癌胆汁样本原代培养物免疫组化染色鉴定
Figure PCTCN2019115306-appb-000054
实施例40、微孔板芯片加工
本实施例中使用注塑加工的方式，用PMMA材料(或者PS、PC、COC、COP、LAS等材料)加工得到用于培养本发明胃癌、胆囊胆管癌原代细胞所用的微孔板芯片。该芯片可以用于原代胃癌、胆囊胆管癌细胞培养以及体外药敏检测实验。微孔板芯片设计图纸见图15。
实际应用过程中，具体是采用PMMA材料(或者PS、PC、COC、COP、LAS等材料)制备得到设计图纸见图15的微孔板芯片的结构，然后再通过上述的CYTOP修饰方法(见实施例29)对其表面进行CYTOP修饰，就得到了这里所说的可用于胃癌、胆囊胆管癌原代细胞培养的微孔板芯片。
工业应用
本发明提供了一种从新鲜胃癌手术样本、胆囊胆管癌手术样本或活检穿刺组织样本或胆囊癌、胆管癌胆汁样本中提取培养胃癌、胆囊胆管癌原代肿瘤细胞的方法和配套试剂，该方法具有以下优点：组织样本用量少，仅需20mg左右的手术样本或者10-20mL左右的胆汁样本；可用于胃癌、胆囊胆管癌原发肿瘤原代肿瘤细胞的培养，也可用于胃癌、胆囊胆管癌转移病灶原代肿瘤细胞的培养；培养周期短，仅需3-10天即可获得10 6-10 7数量级的原代肿瘤细胞；培养稳定性高，用本方法对合格的样本进行体外培养的成功率高达70％；细胞纯度高， 利用本方法得到的胃癌、胆囊胆管癌原代细胞培养物中，癌细胞的比例可以达到60％-95％，杂细胞干扰少。利用本发明方法得到的胃癌、胆囊胆管癌原代细胞培养物可以用于多种细胞水平的体外实验、二代测序、构建动物模型、构建细胞系等等。可以预见，这种培养方法在胃癌、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。

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  1. 一种用于培养胃癌和/或胆囊胆管癌原代细胞的培养基，由抗菌抗真菌剂三抗、HEPES、GlutaMax、非必需氨基酸、人重组蛋白EGF、人重组蛋白bFGF、人重组蛋白HGF、人重组蛋白FGF-10、人重组蛋白Wnt-3a、人重组蛋白Noggin、SB202190、A83-01、Primocin、N-乙酰-L-半胱氨酸、烟碱、N-2Supplement、皮质醇、B27、ITS-X、胃泌素、Y-27632和Advanced DMEM/F12培养基组成；其中，所述抗菌抗真菌剂三抗中的青霉素的终浓度为100-200U/mL；所述抗菌抗真菌剂三抗中的链霉素的终浓度为100-200μg/mL；所述抗菌抗真菌剂三抗中的两性霉素B的终浓度为250-250ng/mL；所述HEPES的终浓度为8-12mM；所述GlutaMax的终浓度为0.8-1.2％(体积百分含量)；所述非必需氨基酸中的甘氨酸的浓度为80-120μM；所述非必需氨基酸中的L-丙氨酸的浓度为80-120μM；所述非必需氨基酸中的L-天冬酰胺的浓度为80-120μM；所述非必需氨基酸中的L-天冬氨酸的浓度为80-120μM；所述非必需氨基酸中的L-谷氨酸的浓度为80-120μM；所述非必需氨基酸中的L-脯氨酸的浓度为80-120μM；所述非必需氨基酸中的L-丝氨酸的浓度为80-120μM；所述人重组蛋白EGF的终浓度为10-100ng/mL；所述人重组蛋白bFGF的终浓度为10-50ng/mL；所述人重组蛋白HGF的终浓度为5-25ng/mL；所述人重组蛋白FGF-10的终浓度为5-25ng/mL；所述人重组蛋白Wnt-3a的终浓度为200-300ng/mL；所述人重组蛋白Noggin的终浓度为100-200ng/mL；所述SB202190的终浓度为5-10μM；所述A83-01的终浓度为0.25-1.25μM；所述Primocin的终浓度为1％(体积百分含量)；所述N-乙酰-L-半胱氨酸的终浓度为0.5-2mM；所述烟碱的终浓度为5-10mM；所述N-2Supplement的终浓度为1％(体积百分含量)；所述皮质醇的终浓度为20-50ng/mL；所述B27的终浓度为1.5-2.5％(体积百分含量)；所述ITS-X的终浓度为0.8-1.2％(体积百分含量)；所述胃泌素的终浓度为8-12nM；所述Y-27632的终浓度为5-20μM；余量均为Advanced DMEM/F12培养基。
  2. 一种用于培养胃癌和/或胆囊胆管癌原代细胞的成套试剂，为如下任一：
    (A1)由权利要求1所述培养基如下中的全部或部分组成：样本解离液、样本保存液和样本清洗液；
    所述样本解离液由胶原酶I、胶原酶II、胶原酶IV和PBS组成；其中，所述胶原酶I的终浓度为150-250U/mL；所述胶原酶II的终浓度为150-250U/mL；所述胶原酶IV的终浓度为50-150U/mL；余量均为PBS；
    所述样本保存液由胎牛血清、抗菌抗真菌剂三抗、HEPES和HBSS组成；其中，所述胎牛血清的终浓度为1-5％(体积百分含量)；所述抗菌抗真菌剂三抗中的青霉素的终浓度为100-200U/mL；所述抗菌抗真菌剂三抗中的链霉素的终浓度为100-200μg/mL；所述抗菌抗真菌剂三抗中的两性霉素B的终浓度为250-500ng/mL；所述HEPES的终浓度为8-12mM；余量均为HBSS；
    所述样本清洗液由抗菌抗真菌剂三抗和PBS组成；其中，所述抗菌抗真菌剂三抗中的青霉素的终浓度为100-200U/mL；所述抗菌抗真菌剂三抗中的链霉素的终浓度为100-200μg/mL；所述抗菌抗真菌剂三抗中的两性霉素B的终浓度为250-500ng/mL；余量均为PBS；
    (A2)由权利要求1所述培养基和细胞分离缓冲液组成；
    所述细胞分离缓冲液由双抗P/S、肝素钠和PBS组成；其中，所述双抗P/S中的青霉素的终浓度为100-200U/mL；所述双抗P/S中的链霉素的终浓度为100-200μg/mL；所述肝素钠的终浓度为10IU/mL；余量均为PBS；
    (A3)由(A1)和如下试剂中的全部或部分组成：细胞消化液、消化终止液和细胞冻存液；
    (A4)由(A2)和如下试剂中的全部或部分组成：细胞消化液、消化终止液和细胞冻存液；
    所述细胞消化液组成如下：每10mL所述细胞消化液中含有4-6mL Accutase，终浓度为5mM的EDTA，1.5-2.5mL TrypLE Express，余量为PBS；
    所述消化终止液由胎牛血清、抗菌抗真菌剂三抗和DMEM培养基组成；其中，所述胎牛血清的终浓度为8-12％(体积百分含量)；所述抗菌抗真菌剂三抗中的青霉素的终浓度为100-200U/mL；所述抗菌抗真菌剂三抗中的链霉素的终浓度为100-200μg/mL；所述抗菌抗真菌剂三抗中的两性霉素B的终浓度为250-500ng/mL；余量均为DMEM培养基；
    所述细胞冻存液由Advanced DMEM/F12培养基、DMSO和1％甲基纤维素溶液组成；其中，所述Advanced DMEM/F12培养基、所述DMSO和所述1％甲基纤维素溶液的体积配比为20:2:(0.8-1.2)；所述1％甲基纤维素溶液是浓度为1g/100ml的甲基纤维素水溶液。
  3. 如下任一应用：
    (B1)权利要求1所述的培养基在培养胃癌和/或胆囊胆管癌原代细胞中的应用；
    (B2)权利要求2中(A1)或(A3)所述的成套试剂在培养胃癌和/或胆囊胆管癌实体瘤原代细胞中的应用；
    (B3)权利要求2中(A2)或(A4)所述的成套试剂在培养胆囊胆管癌胆汁样本原代肿瘤细胞中的应用。
  4. 一种培养胃癌和/或胆囊胆管癌原代细胞的方法，为方法A或方法B：
    方法A：一种培养胃癌和/或胆囊胆管癌实体瘤原代细胞的方法，包括如下步骤：
    (a1)用权利要求2中所述的样本解离液对胃癌和/或胆囊胆管癌实体瘤组织进行解离处理，获得胃癌和/或胆囊胆管癌实体瘤原代细胞；
    (a2)利用权利要求1所述培养基悬浮培养步骤(a1)解离出来的胃癌和/或胆囊胆管癌实体瘤原代细胞；
    方法B：一种培养胆囊胆管癌胆汁样本原代肿瘤细胞的方法，包括如下步骤：
    (b1)从胆囊胆管癌胆汁样本中分离获得胆囊胆管癌胆汁样本原代肿瘤细胞；
    (b2)利用权利要求1所述培养基悬浮培养步骤(b1)分离出来的胆囊胆管癌胆汁样本原代肿瘤细胞。
  5. 根据权利要求4所述的方法，其特征在于：步骤(a1)中，是按照包括如下步骤的方法用所述样本解离液对所述胃癌和/或胆囊胆管癌实体瘤组织进行解离的：按0.1-0.3mL所述样本解离液每mg组织的用量，将剪碎后的所述胃癌和/或胆囊胆管癌癌实体瘤组织用事先37℃预热的所述样本解离液进行处理，在37℃条件下进行样本解离，解离时间15分钟至3小时；
    步骤(b1)中，是按照包括如下步骤的方法从胆囊胆管癌胆汁样本中分离获得胆囊胆管癌胆汁样本原代肿瘤细胞的：用权利要求2中所述的细胞分离缓冲液悬浮胆囊胆管癌胆汁样本中的细胞，然后通过密度梯度离心获得胆囊胆管癌胆汁样本原代肿瘤细胞。
  6. 根据权利要求4或5所述的方法，其特征在于：步骤(a2)中，是按照包括如下步骤的方法用所述培养基悬浮培养所述胃癌和/或胆囊胆管癌实体瘤原代细胞的：使用细胞培养容器M，利用所述培养基悬浮培养所述胃癌和/或胆囊胆管癌实体瘤原代细胞，37℃，5％CO 2条件下进行培养，每2-4天更换一次培养基；
    步骤(b2)中，是按照包括如下步骤的方法用所述培养基悬浮培养所述胆囊胆管癌胆汁样本原代肿瘤细胞的：使用细胞培养容器M，利用所述培养基悬浮培养所述胆囊胆管癌胆汁样本原代肿瘤细胞，37℃，5％CO 2条件下进行培养，每2-4天更换一次培养基；
    所述细胞培养容器M为如下任一：(I)聚苯乙烯材质的细胞培养容器、聚碳酸酯材质的细胞培养容器、聚甲基丙烯酸甲酯材质的细胞培养容器、COC树脂材质的细胞培养容器、环烯烃聚合物材质的细胞培养容器或低吸附表面的细胞培养容器；(II)对(I)中的细胞培养容器进行CYTOP修饰后的细胞培养容器。
  7. 根据权利要求6所述的方法，其特征在于：所述(II)中，是按照包括如下步骤的方法对所述(I)中的细胞培养容器进行CYTOP修饰的：对所述(I)中的细胞培养容器进行纯氧刻蚀，刻蚀条件为功率20W，刻蚀时间为3分钟；然后用1％CYTOP溶液覆盖所述细胞培养容器表面，晾干所述1％CYTOP溶液即完成CYTOP修饰；
    所述1％CYTOP溶液的组成如下：每100mL所述1％CYTOP溶液中含有1mL CYTOP，余量为氟油。
  8. 根据权利要求4-7中任一所述的方法，其特征在于：在步骤(a1)之前，还包括如下对所述胃癌和/或胆囊胆管癌实体瘤组织进行解离前处理的步骤：用 体积百分含量为70-75％的乙醇清洗胃癌和/或胆囊胆管癌实体瘤组织样本表面；用权利要求2中所述样本清洗液和无菌的PBS溶液先后清洗所述胃癌和/或胆囊胆管癌实体瘤组织样本。
  9. 根据权利要求8所述的方法，其特征在于：进行所述解离前处理的所述胃癌和/或胆囊胆管癌实体瘤组织样本的离体时间为2小时以内，且在进行所述解离前处理之前一直保存于权利要求2中所述样本保存液中。
  10. 根据权利要求4-9中任一所述的方法，其特征在于：在步骤(a1)中，用所述样本解离液对所述胃癌和/或胆囊胆管癌实体瘤组织进行解离处理后还包括如下步骤：用权利要求2中所述消化终止液终止解离反应，收集细胞悬液；过滤所述细胞悬液，去除组织残片和粘连细胞；离心后用无菌PBS重悬细胞；再离心，然后用权利要求1所述培养基重悬细胞沉淀。
  11. 根据权利要求4-10中任一所述的方法，其特征在于：在步骤(a2)中，还包括如下步骤：待所述胃癌和/或胆囊胆管癌实体瘤原代细胞形成直径50-80μm的团块时，对所述胃癌和/或胆囊胆管癌实体瘤原代细胞进行传代；
    在步骤(b2)中，还包括如下步骤：待所述胆囊胆管癌胆汁样本原代肿瘤细胞形成直径50-80μm的团块时，对所述胆囊胆管癌胆汁样本原代肿瘤细胞进行传代。
  12. 根据权利要求11所述的方法，其特征在于：进行所述传代时采用的细胞消化液为权利要求2中所述的细胞消化液。
  13. 根据权利要求11或12所述的方法，其特征在于：进行所述传代时采用的消化终止液为权利要求2中所述的消化终止液。
  14. 根据权利要求4-13中任一所述的方法，其特征在于：所述方法还包括对经过2-3次传代扩增后的所述胃癌和/或胆囊胆管癌实体瘤原代细胞或所述胆囊胆管癌胆汁样本原代肿瘤细胞进行冻存和/或复苏的步骤；
    进行所述冻存时采用的细胞冻存液为权利要求2中所述的细胞冻存液。
  15. 如下任一试剂：
    (C1)胃癌和/或胆囊胆管癌实体瘤组织样本解离液，为权利要求2中所述的样本解离液；
    (C2)胃癌和/或胆囊胆管癌实体瘤组织样本保存液，为权利要求2中所述的样本保存液；
    (C3)胆囊胆管癌胆汁样本分离缓冲液，为权利要求2中所述的细胞分离缓冲液。
  16. 如下任一应用：
    (D1)权利要求15中(C1)所述样本解离液在从胃癌和/或胆囊胆管癌实体瘤组织中解离出胃癌和/或胆囊胆管癌实体瘤原代细胞中的应用；
    (D2)权利要求15中(C2)所述样本保存液在保存胃癌和/或胆囊胆管癌实体瘤组织中的应用；
    (D3)权利要求15中(C3)所述细胞分离缓冲液在从胆囊胆管癌胆汁样本中分离出胆囊胆管癌胆汁样本原代肿瘤细胞中的应用。
  17. 如下任一方法：
    (E1)一种从胃癌和/或胆囊胆管癌实体瘤组织中解离出胃癌和/或胆囊胆管癌实体瘤原代细胞的方法，包括权利要求4-14任一中的步骤(a1)；
    (E2)一种保存胃癌和/或胆囊胆管癌实体瘤组织的方法，包括如下步骤：将刚刚离体的胃癌和/或胆囊胆管癌实体瘤组织置于权利要求2中所述样本保存液中保存，保存时间为2小时以内；
    (E3)一种从胆囊胆管癌胆汁样本中分离出胆囊胆管癌胆汁样本原代肿瘤细胞的方法，包括权利要求4-14任一中的步骤(b1)。
  18. 根据权利要求1-17中任一所述的培养基或成套试剂或应用或方法，其特征在于：所述胃癌为原发性胃癌；所述胆囊胆管癌为原发性胆囊胆管癌。
  19. 根据权利要求1-17中任一所述的培养基或成套试剂或应用或方法，其特征在于：所述胃癌为胃癌转移病灶；所述胆囊胆管癌为胆囊胆管癌转移病灶。
  20. 根据权利要求1-19中任一所述的培养基或成套试剂或应用或方法，其特征在于：所述胃癌原代细胞为胃癌实体瘤原代细胞；所述胆囊胆管癌原代细胞为胆囊胆管癌实体瘤原代细胞或胆囊胆管癌胆汁样本原代肿瘤细胞。
  21. 根据权利要求1-20中任一所述的培养基或成套试剂或应用或方法，其特征在于：所述胃癌原代细胞分离自胃癌患者的手术样本；所述胆囊胆管癌原代细胞分离自胆囊胆管癌患者的手术样本、穿刺样本或者胆汁样本。
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