WO2019238143A2 - 结直肠癌实体瘤原代细胞和结直肠癌腹水原代肿瘤细胞培养方法及配套试剂 - Google Patents

结直肠癌实体瘤原代细胞和结直肠癌腹水原代肿瘤细胞培养方法及配套试剂 Download PDF

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WO2019238143A2
WO2019238143A2 PCT/CN2019/099245 CN2019099245W WO2019238143A2 WO 2019238143 A2 WO2019238143 A2 WO 2019238143A2 CN 2019099245 W CN2019099245 W CN 2019099245W WO 2019238143 A2 WO2019238143 A2 WO 2019238143A2
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colorectal cancer
solution
cell
final concentration
primary
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PCT/CN2019/099245
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French (fr)
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WO2019238143A3 (zh
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张函槊
尹申意
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北京吉尚立德生物科技有限公司
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Priority claimed from CN201810607216.2A external-priority patent/CN110592018A/zh
Priority claimed from CN201810607714.7A external-priority patent/CN110583619B/zh
Priority claimed from CN201810607219.6A external-priority patent/CN110592019A/zh
Priority claimed from CN201810607728.9A external-priority patent/CN110592020A/zh
Application filed by 北京吉尚立德生物科技有限公司 filed Critical 北京吉尚立德生物科技有限公司
Priority to JP2021518844A priority Critical patent/JP7507751B2/ja
Priority to US17/251,537 priority patent/US20210284970A1/en
Priority to AU2019284405A priority patent/AU2019284405A1/en
Priority to EP19818946.6A priority patent/EP3896154A4/en
Publication of WO2019238143A2 publication Critical patent/WO2019238143A2/zh
Publication of WO2019238143A3 publication Critical patent/WO2019238143A3/zh

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Definitions

  • the invention relates to the field of biotechnology, in particular to a method for culturing primary tumor cells of solid tumors of colorectal cancer and primary tumor cells of ascites of colorectal cancer and supporting reagents.
  • Colorectal cancer is one of the most common malignant tumors that seriously threaten human health.
  • the incidence of colorectal cancer in China is 9.24%, accounting for the fourth place among all malignant tumors.
  • the mortality rate of colorectal cancer is 11.77%, which is the fifth highest among all malignant tumors.
  • the risk of colorectal cancer recurrence and metastasis is high, and more than 50% of patients with colorectal cancer will have different degrees of recurrence and metastasis within months to years after radical treatment.
  • Colorectal cancer is a complex disease.
  • the occurrence and development of colorectal cancer is a dynamic process that involves the interaction of many signaling molecules, forming a complex molecular regulatory network, and also affected by external environmental factors.
  • the cause and development of colorectal cancer have strong individual differences and cannot be generalized. Therefore, the use of primary cell cultures of solid tumors of colorectal cancer as a model for individualized and accurate research is a trend in the field of colorectal cancer research and even in the field of colorectal cancer diagnosis and treatment.
  • colorectal cancer cell lines as a model for colorectal cancer research. It is difficult to represent the true situation of cancer cells in thousands of different colorectal cancer patients, which has great limitations.
  • the PDX model which represents the concept of precision medicine, is difficult to overcome the weakness of the modeling cycle being too long to guide clinical treatment. Patients with intermediate and advanced colorectal cancer often have ascites and need to be excreted in time. Therefore, ascites is a very easy to obtain clinical sample, and shedding colorectal cancer cells can often be found in ascites.
  • the use of colorectal cancer solid tumor primary cell cultures and colorectal cancer ascites primary tumor cell cultures as models for individualized and precise research is a trend in the field of colorectal cancer research and even in the field of colorectal cancer diagnosis and treatment.
  • the existing primary tumor cell culture technologies mainly include 2D culture, 3D culture, reprogramming culture, etc. These methods are faced with problems such as extremely long culture cycles, low culture success rates, and difficult removal of heterogeneous cells.
  • the present invention provides a new culture technology and supporting reagents for primary colorectal cancer solid tumor primary cells and colorectal cancer ascites primary tumor cells.
  • Cell dissociation reagents treat solid tumor tissues of colorectal cancer to ensure the viability of cancer cells in the tissue to the greatest extent;
  • (2) Prepare a special serum-free medium and use suspension culture system to treat tumor cells derived from solid tumors of colorectal cancer and Colorectal cancer ascites primary tumor cells are cultured in vitro to ensure normal expansion of cancer cells while maximally eliminating interference from normal cells.
  • the invention claims a medium for culturing colorectal cancer primary cells.
  • the medium claimed in the present invention for culturing primary cells of colorectal cancer is composed of a third antibody (penicillin-streptomycin-amphoterin B), HEPES, GlutaMax, human recombinant protein EGF, and human recombinant protein.
  • bFGF human recombinant protein HGF, human recombinant protein Wnt-3a, human recombinant protein Noggin
  • SB202190 (4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl)- 1H-imidazole
  • A83-01 (3- (6-Methyl-2-pyridinyl) -N-phenyl-4- (4-quinolinyl) -1H-pyrazole-1-carbothioamide
  • Primocin TM N-acetyl-L -N-acetyl-L-cysteine
  • Nicotinamide N-2Supplement
  • Cortisol B27
  • ITS-X Insulin, Transferrin, Selenium, Ethanolamine Solution
  • Y-27632 Advanced DMEM / F12 medium composition.
  • the final concentration of penicillin in the third antibody of the antibacterial and antifungal agent is 100-200U / mL (such as 100U / mL); the final concentration of streptomycin in the third antibody of the antibacterial and antifungal agent is 100-200 ⁇ g / mL (such as 100 ⁇ g / mL); the final concentration of amphotericin B in the third antibody of the antibacterial and antifungal agent is 100-250ng / 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 content); the final concentration of human recombinant protein EGF is 10-100ng / mL (such as 20ng / mL or 40ng / mL mL); the final concentration of the human recombinant protein bFGF is 10-50ng / mL (such as 20ng / mL
  • the third antibody (penicillin-streptomycin-amphotericin B) of the antibacterial and antifungal agent is composed as follows: each ml contains 10,000 units of penicillin (base), 10,000 ⁇ g of streptomycin (base), and 25 ⁇ g of amphotericin B.
  • the third antibody (penicillin-streptomycin-amphoterin B) of the antibacterial and antifungal agent is "Antibiotic-Antimycotic, 100X" (such as Gibco # 15240062, or other products with the same composition).
  • the "Antibiotic-Antimycotic, 100X" contains 10,000 units of penicillin (base), 10,000 ⁇ g of streptomycin (base), and 25 ⁇ g of amphotericin B per milliliter.
  • the GlutaMAX is "GlutaMAX TM Supplement” (such as Gibco # 35050061, or other products with the same composition).
  • the component 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 a 0.85% NaCl solution.
  • the Primocin TM is an antibacterial agent for primary cells (such as Invivogene # ant-pm-1, or other products with the same composition), an antibiotic used to protect primary cells from microbial contamination, against Gram-positive bacteria, Gram-negative bacteria, mycoplasma and fungi all have killing effects.
  • the N-2Supplement is "N-2Supplement (100X)” (such as Gibco # 17502001, or other products with the same composition).
  • the "N-2 Supplement (100X)” contains human Human Transferrin (Holo) at a final concentration of 1 mM, 500 mg / L of Insulin Recombinant Full Chain, 0.63 mg / L Progesterone, 10 mM putrescine, 0.52 mg / L 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, DL- ⁇ -tocopherol Acetate, DL- ⁇ -tocopherol (DL Alpha- Tocopherol), BSA (fatty acid free Fraction V), catalase, human recombinant insulin (Human Recombinant Insulin), human transferrin, superoxide dismutase, corticosterone (Corticosterone), D-Galactose, Ethanolamine HCl, Reduced Glutathione (Reduced), L-Carnitine Hydrochloride (L-Carnitine HCl), Linoleic Acid), Linolenic Acid, Progesterone, Putrescine 2HCl, Sodium Selenite, Triodo-I-thyronine.
  • the solvent of the ITS-X is an EBSS solution (Earle's balanced salt solution).
  • the solute and concentration are as follows: insulin 1g / L; transferrin 0.55g / L; sodium selenite 0.0067g / L; ethanolamine 0.2g / L.
  • the GlutaMAX is an advanced cell culture additive, which can directly replace L-glutamine in a cell culture medium.
  • the GlutaMAX is "GlutaMAX TM Supplement" (such as Gibco # 35050061, or other products with the same composition).
  • the Y-27632 is "Y-27632 dihydrochloride (an ATP-competitive ROCK-I and ROCK-II inhibitor, Ki is 220nM and 300nM, respectively)" (such as MCE # 129830-38-2, or has the same composition Other products).
  • the brand name of the anti-fungal antifungal agent (penicillin-streptomycin-amphoterin B) is Gibco # 15240062; the brand name of the HEPES is Gibco # 15630080; The brand name of GlutaMAX is Gibco # 35050061; the brand name of the human recombinant protein EGF is Peprotech AF-100-15-100; the brand name of the human recombinant protein bFGF is Peprotech AF-100-18B-50; the person The brand name of the recombinant protein HGF is Peprotech AF-100-39-100; the brand name of the human recombinant protein Wnt-3a is R & D 5036-WN-500; the brand name of the human recombinant protein Noggin is Shanghai Jinan # C018
  • the brand number of the SB202190 is Sigma # S7067; the brand number of the A83-01 is Tocris # 2939; the brand number of the Primocin
  • the medium for culturing colorectal cancer primary cells can exist in two forms:
  • the medium for culturing colorectal cancer primary cells is composed of the antibacterial antifungal third antibody, the HEPES, the GlutaMax, the human recombinant protein EGF, the human recombinant protein bFGF, The human recombinant protein HGF, 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 Y-27632, and the Advanced DMEM / F12 medium.
  • the medium After the medium is prepared, it needs to be filtered and sterilized by a 0.22 ⁇ M needle filter (Millipore SLGP033RS), and it can be stored at 4 ° C. for two weeks.
  • a 0.22 ⁇ M needle filter (Millipore SLGP033RS)
  • each component in the medium for culturing colorectal cancer primary cells exists separately, and is formulated according to a formula when used.
  • human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein Wnt-3a, and human recombinant protein Noggin can exist in the form of a stock solution (mother liquor) (-80 ° C can be stored for a long time), specifically Can be 1000 times the stock solution (mother solution).
  • SB202190, N-acetyl-L-cysteine, Nicotinamide, Cortisol, and Y-27632 can exist in the form of stock solution (mother liquor) (long-term storage at -20 ° C), which can be 1,000 times the stock solution (mother liquor).
  • A83-01 can exist in the form of stock solution (mother liquor) (long-term storage at -20 ° C), which can be 100,000 times the stock solution (mother liquor).
  • human recombinant protein EGF stock solution is composed of human recombinant proteins EGF, BSA, and PBS.
  • the final concentration of the human recombinant protein EGF is 20 ⁇ g / mL
  • the final concentration of the BSA is 0.01 g / mL.
  • the balance is all PBS.
  • 1000 ⁇ human recombinant protein bFGF stock solution is composed of human recombinant proteins bFGF, BSA, and PBS.
  • the final concentration of the human recombinant protein bFGF is 20 ⁇ g / mL, and the final concentration of the BSA is 0.01 g / mL.
  • the balance is all PBS.
  • 1000 ⁇ human recombinant protein HGF stock solution is composed of human recombinant proteins HGF, BSA, and PBS.
  • the final concentration of the human recombinant protein HGF is 20 ⁇ g / mL, and the final concentration of the BSA is 0.01 g / mL.
  • the balance is PBS.
  • 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, the balance is PBS.
  • 1000 ⁇ human recombinant protein Noggin stock solution is composed of human recombinant proteins 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 as a 100-fold stock solution (mother solution) (now equipped with current use), and specifically consists of BSA and PBS, where the final concentration of BSA (Sigma # A1933) is 0.1 g / mL, the balance is PBS.
  • the 1000 ⁇ SB202190 stock solution was composed of SB202190 and DMSO.
  • the final concentration of the SB202190 was 10 mM, and the balance was DMSO.
  • the 100,000 ⁇ A83-01 stock solution was composed of A83-01 and DMSO.
  • the concentration of the A83-01 was 25 mM, and the balance was DMSO.
  • the 1000 ⁇ N-acetyl-L-cysteine stock solution is composed of N-acetyl-L-cysteine and ultrapure water.
  • the concentration of the N-acetyl-L-cysteine is 0.5M, and the balance is ultrapure water.
  • the 1000 ⁇ Nicotinamide stock solution is composed of Nicotinamide and ultrapure water.
  • the concentration of Nicotinamide is 5M, and the balance is ultrapure water.
  • 1000 ⁇ cortisol stock solution is composed of cortisol, absolute ethanol and ultrapure water, wherein the final concentration of cortisol is 25 ⁇ g / mL, and the final concentration of absolute ethanol is 5% (volume percentage content), The balance is ultra pure water.
  • 1000 ⁇ Y-27632 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 for culturing primary cells of colorectal cancer.
  • the set of reagents claimed in the present invention may be any of the following:
  • (A1) It consists of all or part of the culture medium described in the first aspect above: a sample dissociation solution, a sample preservation solution, and a sample washing solution.
  • (A2) consists of the medium and cell separation buffer described in the first aspect above.
  • (A3) is composed of (A1) and all or part of the following reagents: a cell digestion solution, a digestion termination solution, and a cell cryopreservation solution.
  • (A4) is composed of (A2) and all or part of the following reagents: a cell digestion solution, a digestion termination solution, and a cell cryopreservation solution.
  • the sample dissociation solution consists of collagenase I, collagenase II, collagenase IV, and PBS; wherein the final concentration of the collagenase I in the sample dissociation solution is 150-250 U / mL (such as 200 U / mL) mL); the final concentration of the collagenase II in the sample dissociation solution is 150-250U / mL (such as 200U / mL); the final concentration of the collagenase IV in the sample dissociation solution is 50 -150U / mL (such as 100U / mL); the balance is PBS.
  • the final concentration of the collagenase I in the sample dissociation solution is 150-250 U / mL (such as 200 U / mL) mL)
  • the final concentration of the collagenase II in the sample dissociation solution is 150-250U / mL (such as 200U / mL)
  • 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, pH 7.5, the collagenase is treated with 1U protease (The collagenase I, the collagenase II, or the collagenase IV) for 5 hours, 1 ⁇ mol of L-leucine can be released.
  • the brand name of the collagenase I is Gibco # 17100-017; the brand name of the collagenase II is Gibco # 17101-015; the brand name of the collagenase IV is Gibco # 17104-019; the brand name of the PBS is Gibco # 21-040-CVR.
  • the sample preservation solution is composed of fetal bovine serum, an antibacterial antifungal tertiary antibody (penicillin-streptomycin-amphoterin B), HEPES, and HBSS (Hank's balanced salt solution); wherein the fetal bovine serum is in the
  • the final concentration in the sample preservation solution is 1-5% (for example, 2%,% means volume percentage); the penicillin in the third antibody (penicillin-streptomycin-amphoterin B) of the antibacterial and antifungal agent is in all
  • the final concentration in the sample preservation solution is 100-200 U / mL (such as 100 U / mL); streptomycin in the third antibody (penicillin-streptomycin-amphoterin B) of the antibacterial and antifungal agent is in the sample
  • the final concentration in the preservation solution is 100-200 ⁇ g / mL (such as 100 ⁇ g / mL); the amphotericin B in the third antibody (penicillin-streptomycin-amphoter
  • the third antibody (penicillin-streptomycin-amphotericin B) of the antibacterial and antifungal agent is composed as follows: each ml contains 10,000 units of penicillin (base), 10,000 ⁇ g of streptomycin (base), and 25 ⁇ g of amphotericin B.
  • the third antibody (penicillin-streptomycin-amphoterin B) of the antibacterial and antifungal agent is "Antibiotic-Antimycotic, 100X" (such as Gibco # 15240062, or other products with the same composition).
  • the "Antibiotic-Antimycotic, 100X” contains 10,000 units of penicillin (alkali), 10,000 ⁇ g of streptomycin (base), and 25 ⁇ g of amphotericin B per milliliter, using penicillin G (sodium salt), streptomycin sulfate in the form of 0.85% salt solution. And amphotericin B as Antifungal.
  • the brand number of the fetal bovine serum is Gibco # 16000-044; the brand number of the anti-fungal antifungal agent (penicillin-streptomycin-amphoterin B) is Gibco # 15240062; the brand number of the HEPES is Gibco # 15630080; the brand number of the HBSS is Gibco # 14170161.
  • the sample cleaning solution is composed of a third antibody (penicillin-streptomycin-amphoterin B) and a PBS; wherein the third antibody (penicillin-streptomycin-amphoterin B) is an antifungal agent.
  • the final concentration of penicillin in the sample cleaning solution is 100-200 U / mL (such as 100 U / mL); streptomyces in the third antibacterial and antifungal agent (penicillin-streptomycin-amphoterin B)
  • the final concentration in the sample cleaning solution is 100-200 ⁇ g / mL (eg, 100 ⁇ g / mL); the amphotericin B in the anti-fungal antifungal agent third antibody (penicillin-streptomycin-amphotericin B)
  • the final concentration in the sample cleaning solution is 250-500 ng / mL (such as 250 ng / mL); the balance is all PBS.
  • the third antibody (penicillin-streptomycin-amphotericin B) of the antibacterial and antifungal agent is composed as follows: each ml contains 10,000 units of penicillin (base), 10,000 ⁇ g of streptomycin (base), and 25 ⁇ g of amphotericin B.
  • the third antibody (penicillin-streptomycin-amphoterin B) of the antibacterial and antifungal agent is "Antibiotic-Antimycotic, 100X" (such as Gibco # 15240062, or other products with the same composition).
  • the "Antibiotic-Antimycotic, 100X" contains 10,000 units of penicillin (base), 10,000 ⁇ g of streptomycin (base), and 25 ⁇ g of amphotericin B per milliliter. Penicillin G (sodium salt), Streptomyces sulfate And amphotericin B as Antifungal.
  • the brand name of the anti-fungal antifungal agent is Gibco # 15240062; the brand name of the PBS is Gibco # 21-040- CVR.
  • the cell isolation buffer is composed of a third antibody (penicillin-streptomycin-amphotericin B), heparin sodium, and PBS; wherein the third antibody (penicillin-streptomycin-amphoteric) is an antifungal agent
  • the final concentration of penicillin in B.mycin B) is 100-200 U / mL (such as 100 U / mL); the final concentration of streptomycin in the third antibody (penicillin-streptomycin-amphoterin B) of the antibacterial antifungal agent
  • the concentration is 100-200 ⁇ g / mL (for example, 100 ⁇ g / mL);
  • the final concentration of amphotericin B in the third antibody (penicillin-streptomycin-amphotericin B) of the antibacterial and antifungal agent is 250-500ng / mL ( Such as 250ng / mL); the final concentration of heparin sodium is 10IU / mL; the balance is all PBS.
  • the third antibody (penicillin-streptomycin-amphotericin B) of the antibacterial and antifungal agent is composed as follows: each ml contains 10,000 units of penicillin (base), 10,000 ⁇ g of streptomycin (base) and 25 ⁇ g of amphotericin B.
  • the third antibody (penicillin-streptomycin-amphoterin B) of the antibacterial and antifungal agent is "Antibiotic-Antimycotic, 100X" (such as Gibco # 15240062, or other products with the same composition).
  • the "Antibiotic-Antimycotic, 100X” contains 10,000 units of penicillin (alkali), 10,000 ⁇ g of streptomycin (base), and 25 ⁇ g of amphotericin B per milliliter, using penicillin G (sodium salt), streptomycin sulfate in the form of 0.85% salt solution. And amphotericin B as Antifungal.
  • the brand name of the antibacterial and antifungal agent third antibody is Gibco # 15240062; the brand name of the heparin sodium is Solarbio # H8270; The brand name of PBS is Gibco # 21-040-CVR.
  • the composition of the cell digestive fluid is as follows: every 10mL of the cell digestive fluid contains 4-6mL (such as 5mL) Accutase, the final concentration of 5mM EDTA (ie 10 ⁇ L 0.5M EDTA), 1.5-2.5mL (such as 2mL) , 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 that is dissolved in D-PBS, 0.5 mM 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 200 mg / L of KCl, 200 mg / L of KH 2 PO 4 , 8000 mg / L of NaCl, and 2160 mg / L of Na 2 HPO 4 ⁇ 7H 2 O EDTA, 457.6mg / L; also contains recombinant protease.
  • the brand name of Accutase is Gibco # A11105-01; the brand name of 0.5M EDTA is Invitrogen # AM9261; the brand name of TrypLE Express is Gibco # 12604013; the PBS Brand name is Gibco # 21-040-CVR.
  • the digestion termination liquid is composed of fetal calf serum, an antibacterial antifungal tertiary antibody (penicillin-streptomycin-amphoterin B), and DMEM culture medium; wherein the end of the fetal calf serum in the digestion termination liquid
  • concentration is 8-12% (for example, 10%,% means volume percentage); the penicillin in the third antibody (penicillin-streptomycin-amphoterin B) of the antibacterial and antifungal agent is in the digestion termination solution.
  • the final concentration is 100-200 U / mL (such as 100 U / mL); the final concentration of streptomycin in the antibacterial antifungal agent third antibody (penicillin-streptomycin-amphotericin B) in the digestion termination solution
  • the final concentration of amphotericin B in the tertiary antibody (penicillin-streptomycin-amphotericin B) of the antibacterial and antifungal agent (penicillin-streptomycin-amphotericin B) in the digestion termination solution is 250-500ng / mL (such as 250ng / mL); the rest are DMEM medium.
  • the third antibody (penicillin-streptomycin-amphotericin B) of the antibacterial and antifungal agent is composed as follows: each ml contains 10,000 units of penicillin (base), 10,000 ⁇ g of streptomycin (base) and 25 ⁇ g of amphotericin B.
  • the third antibody (penicillin-streptomycin-amphoterin B) of the antibacterial and antifungal agent is "Antibiotic-Antimycotic, 100X" (such as Gibco # 15240062, or other products with the same composition).
  • the "Antibiotic-Antimycotic, 100X” contains 10,000 units of penicillin (alkali), 10,000 ⁇ g of streptomycin (base), and 25 ⁇ g of amphotericin B per milliliter, using penicillin G (sodium salt), streptomycin sulfate in the form of 0.85% salt solution. And amphotericin B as Antifungal.
  • the brand number of the fetal bovine serum is Gibco # 16000-044; the brand number of the anti-fungal antifungal agent (penicillin-streptomycin-amphoterin B) is Gibco # 15240062; the brand name of the DMEM medium is Gibco # 11965-092.
  • the cell cryopreservation solution is composed of Advanced DMEM / F12 medium, DMSO and 1% methyl cellulose solution; wherein, the Advanced DMEM / F12 medium, the DMSO and the 1% methyl cellulose solution
  • the volume ratio is 20: 2: (0.8-1.2), such as 20: 2: 1; the 1% methyl cellulose solution is a methyl cellulose aqueous solution having a concentration of 1 g / 100 ml.
  • the brand name of the Advanced DMEM / F12 medium is Gibco # 12634010; the brand name of the DMSO is Sigma # D2438; and the brand name of the methyl cellulose is Sigma # M7027.
  • the sample storage solution can be used for temporary storage after the sample is isolated, and can maintain the cell activity in the sample for a short time after the sample is isolated. After the sample storage solution is prepared, it can be stored at 4 ° C for one month.
  • the sample cleaning solution can be used for cleaning and disinfecting samples.
  • the sample cleaning solution needs to be prepared immediately.
  • the sample dissociation liquid can be used for dissociation of the sample, and can dissociate primary cells of solid colorectal cancer in the sample from the tissue.
  • the sample dissociation solution needs to be prepared immediately.
  • the collagenase I, collagenase II, and collagenase IV can be stored in the form of a stock solution (mother solution) at -20 ° C for a long time, which can be 10 or 20 times the stock solution (mother solution). .
  • 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; 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 balance is PBS; a 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 amounts are all PBS.
  • the enzyme activities of the collagenase I, collagenase II and collagenase IV see the foregoing.
  • the cell separation buffer is used for suspending cells in ascites to prepare cells for density gradient separation. After the cell isolation buffer is prepared, it can be stored at 4 ° C for one month.
  • the cell digestive fluid can be used for digestion and passaging of cell masses, and can digest colorectal cancer tumor masses into single cells.
  • the cell digestive fluid should be prepared immediately.
  • the digestion termination solution can be used to terminate the sample dissociation or cell digestion process. After the digestion stop solution is prepared, it can be stored at 4 ° C for one month.
  • the cell cryopreservation solution should be prepared immediately.
  • the 1% methyl cellulose solution can be stored at 4 ° C for a long time.
  • the present invention claims a method for culturing primary cells of colorectal cancer.
  • the method for culturing colorectal cancer primary cells as claimed in the present invention is Method A or Method B:
  • Method A A method for culturing primary cells of solid tumors of colorectal cancer, which may include the following steps:
  • Method B A method for culturing primary tumor cells of colorectal cancer ascites, which may include the following steps:
  • the sample dissociation liquid may be used to dissociate the solid tumor tissue of colorectal cancer according to a method including the following steps: dissolve the sample according to 0.1-0.3mL (such as 0.1mL) Dosage per mg of tissue, and cut the colorectal cancer solid tumor tissue (for example, cut into small pieces of 0.8-1.2 mm 3 ) after cutting with the sample dissociation solution pre-heated at 37 ° C. Dissociate the sample at 37 ° C for 15 minutes to 3 hours. The dissociation of the sample was observed under a microscope every 15 minutes until a large number of individual cells were observed.
  • 0.1-0.3mL such as 0.1mL
  • colorectal cancer ascites primary tumor cells can be isolated from colorectal cancer ascites according to a method including the steps of: suspending colorectal cancer with the cell separation buffer described in the second aspect above. Cells in ascites were then subjected to density gradient centrifugation (using Ficoll lymphocyte separation fluid) to obtain primary tumor cells of colorectal cancer ascites.
  • the colorectal cancer solid tumor primary cell culture medium may be suspended and cultured in the colorectal cancer solid tumor primary cell culture medium according to a method including the following steps: using a cell culture container M, using The culture medium is used to suspend and culture the primary colorectal cancer solid tumor primary cells.
  • the culture is performed under the conditions of 37 ° C and 5% CO 2 , and the culture medium is replaced every 2-4 days (such as 3 days) until the cells form a diameter of 50-80 ⁇ m. (Such as 80 ⁇ m).
  • the colorectal cancer ascites primary tumor cells can be suspension-cultured with the medium according to a method including the following steps: using the cell culture container M, culturing the colony with the medium in suspension Primary tumor cells of rectal cancer ascites were cultured under the conditions of 37 ° C and 5% CO 2 , and the culture medium was changed every 2-4 days (for example, 3 days) until the cells formed clumps with a diameter of 50-80 ⁇ m (such as 80 ⁇ m).
  • 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 may be any of the following: (I) a cell culture container made of polystyrene, a cell culture container made of polycarbonate, a cell culture container made of polymethylmethacrylate, or a COC resin Cell culture container, cell culture container made of cycloolefin polymer, or cell culture container with low adsorption surface; (II) Cell culture container modified by CYTOP in the cell culture container in (I).
  • the cell culture container is a cell culture dish, a cell culture well plate, or a micro-well plate chip for cell culture.
  • the cell culture container in the step (I) may be subjected to CYTOP modification according to a method including the following steps: the cell culture container in the step (I) is subjected to pure oxygen etching, 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 a 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.
  • the method may further include the step of pre-dissociating the colorectal cancer solid tumor tissue as follows: washing the knot with 70-75% (eg, 75%) ethanol by volume. Colorectal cancer solid tumor tissue sample surface for 10 to 30 seconds; wash the colorectal cancer solid tumor tissue sample with a sample cleaning solution 10-20 times (such as 10 times), and wash the colorectal cancer solid tumor with a sterile PBS solution Tissue samples 5-10 times (such as 5 times); then impurities, connective tissue, adipose tissue, necrotic tissue and other components that affect primary cell culture are removed from the colorectal cancer solid tumor tissue samples.
  • the step of pre-dissociating the solid tumor tissue of colorectal cancer needs to be operated on ice, and the entire operation step needs to be completed within 10 minutes.
  • the ex vivo time of the colorectal cancer solid tumor tissue sample subjected to the pre-dissociation treatment must be within 2 hours, and it must be stored in the sample preservation solution before the pre-dissociation treatment.
  • the method may further include the following steps: using 8-15 times (such as 10 times) the volume of all Said digestion termination solution terminates the dissociation reaction and collects the cell suspension; 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 (such as 10 minutes), discard the supernatant Clear; then resuspend the cell pellet in the medium described in the first aspect above, observe the cell state under a microscope, and count the cells.
  • 8-15 times such as 10 times
  • step (b1) a step of pre-separating the colorectal cancer ascites sample is further included: removing impurities, blood clots and other components that affect cell density gradient separation in the colorectal cancer ascites sample.
  • the method may further include the following step: when the primary colorectal cancer solid tumor primary cells form a mass with a diameter of 50-80 ⁇ m (eg, 80 ⁇ m), the colorectal cancer solid tumor Passage cells for passage.
  • the method may further include the following step: when the colorectal cancer ascites primary tumor cells form a mass with a diameter of 50-80 ⁇ m (such as 80 ⁇ m), the colorectal cancer ascites primary Tumor cells were passaged.
  • the cell digestive solution used in the passage is the cell digestive solution described in the second aspect above.
  • the digestion terminating liquid used in the passage is the digestion terminating liquid described in the second aspect above.
  • the digestion temperature used in the passage was 37 ° C.
  • the step of passaging is performed: the cell mass to be passaged is collected, the cell mass is washed with a sterile PBS solution after centrifugation, and then centrifuged, and then the cell mass is resuspended with the cell digestion solution at 37 Digest at °C until the cell mass is digested into single cells.
  • the digestion termination solution (the amount can be 5-10 times, such as 10 times the volume) to terminate the digestion reaction, collect the cell suspension; 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), and the culture conditions were 37 ° C and 5% CO 2 . All centrifugation in the above-mentioned passaging step may specifically be performed at 800-1000g (for example, 800g) at room temperature for 10-20 minutes (for example, 10 minutes).
  • the method may further include a step of cryopreserving and / or resuscitating the primary colorectal cancer solid tumor primary cells or the colorectal cancer ascites primary tumor cells expanded after 2-3 passages.
  • the cell cryopreservation solution used in performing the cryopreservation is the cell cryopreservation solution described in the second aspect above.
  • the specific steps of performing the cryopreservation are performed: collecting the cell clumps to be cryopreserved, washing the cell clumps with a sterile PBS solution after centrifugation, centrifuging, and then resuspending the cell clumps with the cell digestion solution. Digestion is performed at 37 ° C until the cell mass is digested into single cells, and the digestion termination solution (the amount of which can be 5-10 times, such as 10 times the volume) is used to terminate the digestion reaction, and the cell suspension is collected; After centrifugation, the cell cryopreservation solution is used to resuspend the cell pellet at a density of 0.5-2 ⁇ 10 6 / mL (such as 10 6 / mL). The cells are stored in a gradient cooling box overnight and then transferred to liquid nitrogen for long-term storage. All the centrifugation in the freezing step can be specifically performed at 800-1000g (for example, 800g) at room temperature for 10-20 minutes (for example, 10 minutes).
  • the specific steps of performing the recovery are: removing the cryopreserved tube containing the cells to be recovered from liquid nitrogen, and rapidly thawing the cells in sterile water at 37-39 ° C (such as 37 ° C); centrifuging (such as 800-1000g) Centrifuge at 800g at room temperature for 5-10 minutes, such as 10 minutes. Then resuspend the cell pellet with the medium described in the first aspect, and then use the cell culture container M described above to suspend the cultured cells (the initial seeding density can be 10 5 cells / cm 2 area of the bottom of the container), each tube cells (10 6) to 3.5cm recovery dish), the culture conditions of 37 °C, 5% CO 2.
  • the present invention claims any of the following reagents:
  • (C1) a dissociation solution of a solid tumor tissue sample of colorectal cancer, which is the dissociation solution of the sample described in the second aspect;
  • (C3) Colorectal cancer ascites cell isolation buffer is the cell isolation buffer described in the second aspect above.
  • the present invention claims any of the following methods:
  • step (E1) A method for dissociating primary cells of solid colorectal cancer from colorectal cancer solid tumor tissue, comprising step (a1) in the method described in the fourth aspect above.
  • (E2) A method for preserving solid tumor tissue of colorectal cancer, comprising the steps of: storing freshly excised colorectal cancer solid tumor tissue in the sample preservation solution described in the second aspect, and the preservation time is 2 hours Within.
  • (E3) A method for isolating colorectal cancer ascites primary tumor cells from colorectal cancer ascites, comprising step (b1) in the fourth aspect above.
  • the colorectal cancer may be primary colorectal cancer.
  • the pathological classification was colorectal cancer or colorectal cancer metastatic lesions.
  • the pathological stage was stage II or III or IV.
  • sample used when isolating colorectal cancer primary cells from solid tumor tissue of colorectal cancer may be a colorectal cancer stage II or III or IV sample.
  • Colorectal cancer stage IV samples were used to isolate colorectal cancer primary cells from colorectal cancer ascites.
  • the colorectal cancer primary cell may be a solid colorectal cancer primary cell or a colorectal cancer ascites primary tumor cell.
  • the primary colorectal cancer cells can be isolated from a surgical sample (a solid tumor sample), a colonoscopy sample, or an ascites sample from a patient with colorectal cancer.
  • solid tumor tissue specimens of colorectal cancer obtained from surgical samples preferably weigh more than 20 mg.
  • the ascites sample is preferably not less than 50mL.
  • Enteroscopy samples are not less than 2 pieces.
  • all of the above PBSs can be 1 ⁇ PBS, pH 7.3-7.5. Its specific composition is as follows: the solvent is water, the solute and concentration are: 144 mg / L of KH 2 PO 4 , 9000 mg / L of NaCl, and 795 mg / L of Na 2 HPO 4 ⁇ 7H 2 O.
  • Figure 1 shows single cells obtained after colorectal cancer tissue treatment.
  • the scale is 100 ⁇ m, and the magnification is 100 times.
  • Figure 2 shows the cell mass obtained after primary culture of colorectal cancer tissue.
  • the scale is 100 ⁇ m, and the magnification is 100 times.
  • FIG. 3 is a HE staining diagram of colorectal cancer cell mass sections obtained after primary culture of colorectal cancer tissue.
  • the scale is 100 ⁇ m and 200 times magnification.
  • FIG. 4 is an immunofluorescence staining diagram of cancer cell masses obtained after primary culture of colorectal cancer tissue.
  • the scale is 50 ⁇ m and 200 times magnification.
  • Figure 5 is a copy number variation analysis (CNV) based on sequencing results showing the copy number of primary colorectal cancer cell cultures (P1, P2, P3, P4, P5) and primary colorectal cancer tumor tissue (Tumor) from each generation The variation is highly consistent.
  • CNV copy number variation analysis
  • Figure 6 shows single cells isolated from colorectal cancer ascites samples.
  • the scale is 100 ⁇ m.
  • FIG. 7 is a cell mass obtained by culturing primary tumor cells in a colorectal cancer ascites sample.
  • the scale is 200 ⁇ m.
  • FIG. 8 is a HE staining diagram of colorectal cancer cells obtained from primary culture of colorectal cancer ascites samples.
  • FIG. 9 is an immunofluorescence staining diagram of cancer cell masses obtained after primary culture of colorectal cancer ascites samples.
  • Figure 10 shows copy number variation analysis (CNV) based on sequencing results showing the copy number variation of primary cell cultures (P1, P2, P3, P4, P5) of colorectal cancer ascites and colorectal cancer ascites Highly consistent.
  • CNV copy number variation analysis
  • FIG. 11 is a result of in vitro drug sensitivity test of colorectal cancer ascites primary tumor cells cultured by the present invention.
  • FIG. 12 is a design diagram of a microplate chip of the present invention.
  • Example 1 Formulation of reagents for culturing primary cells of solid tumors of colorectal cancer
  • the sample cleaning solution should be prepared immediately.
  • sample dissociation solution is currently prepared and used.
  • the unit U of collagenase (the collagenase I or the collagenase IV) is defined by the protease activity: at 37 ° C, pH 7.5, treated with 1U protease Collagenase (the collagenase I or the collagenase IV) for 5 hours can release 1 ⁇ mol of L-leucine.
  • the digestion stop solution After the digestion stop solution is prepared, it can be stored at 4 ° C for one month.
  • the colorectal cancer solid tumor primary cell culture medium After the colorectal cancer solid tumor primary cell culture medium is prepared, it is sterilized by filtration with a 0.22 ⁇ M needle filter (Millipore SLGP033RS) and can be stored at 4 ° C for two weeks.
  • Table 9 the preparation of human recombinant protein stock solution is shown in Table 11-Table 15, the configuration of SB202190 stock solution is shown in Table 16, and the configuration of A83-01 stock solution is shown in Table 17, N-acetyl-L- The configuration of the cysteine stock solution is shown in Table 18, the configuration of the Nicotinamide stock solution is shown in Table 19, the formulation of the cortisol stock solution is shown in Table 20, and the formulation of the Y-27632 stock solution is shown in Table 21. The 100 ⁇ BSA solutions required to prepare these stock solutions are shown in Table 10.
  • Table 12 1000 ⁇ human recombinant protein bFGF stock solution (2.5mL)
  • Noggin stock solution After the 1000 ⁇ human recombinant protein Noggin stock solution is prepared, aliquot it with a 1.5 mL sterile centrifuge tube. The stock solution can be stored at -80 ° C for a long time.
  • N-acetyl-L-cysteine stock solution After the 1000 ⁇ N-acetyl-L-cysteine stock solution is prepared, aliquot it with a 0.5mL sterile centrifuge tube. The stock solution can be stored at -20 ° C for a long time.
  • Nicotinamide stock solution After the 1000 ⁇ Nicotinamide stock solution is prepared, aliquot it with a 0.5mL sterile centrifuge tube. The stock solution can be stored at -20 ° C for a long time.
  • the 1% methyl cellulose solution can be stored at 4 ° C for a long time after preparation.
  • the attending physician selects the patients according to the clinical indications specified in the medical guidelines, and selects suitable samples for in vitro culture according to the clinical indications during surgery.
  • the selection criteria for the samples are: primary colorectal cancer, pathological stage Stages II, III, or IV, various pathological types of colorectal cancer or colorectal cancer metastatic lesions, colorectal 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 and classification, and clinical diagnosis. Hide the patient's name, ID number and other information related to patient privacy, and replace it with a uniform experiment number.
  • the naming principle of the experiment number is the eight-digit date of the sample collected + the last four digits of the patient's hospital number. For example, the sample provided on January 1, 2018, the patient hospitalization number is T001512765, and the sample experiment number is 201801012765.
  • the surgical equipment used in the following operations need to be autoclaved and dried before use.
  • sample dissociation solution (Table 3) per mg of tissue
  • the dissociated single-cell suspension is mixed with a large number of other types of cells, such as red blood cells, lymphocytes, and fibroblasts.
  • other types of cells such as red blood cells, lymphocytes, and fibroblasts.
  • Example 5 Primary cell culture of solid tumors of colorectal cancer
  • the culture medium used is the primary cell culture medium for solid tumors of colorectal cancer in Table 1 (Table 9).
  • a well 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 .
  • cancer cells expanded in large numbers to form cell clumps with a diameter of 80 ⁇ m.
  • the total number of tumor cells can exceed 10 7 , and the number of other types of cells has significantly decreased or even disappeared.
  • the method has been tested on a large number of samples, and the success rate of primary tumor cell culture of solid tumors of colorectal cancer in vitro can reach 80%.
  • the medium used is the colorectal cancer solid tumor primary cell culture medium (Table 9) in Example 1.
  • the plate was plated at a density of 10 6 cells per well, and cultured in a cell incubator at 37 ° C and 5% CO 2 .
  • Primary cells of solid tumors of colorectal cancer in suspension culture can be frozen after 2-3 passages expansion:
  • Example 9 Identification of primary cells of solid tumors of colorectal cancer by HE staining
  • Reagent consumables used in the following examples:
  • Ginger slices were immersed in 95% ethanol and incubated at room temperature for 10 minutes. After repeated twice, rinse the slices twice with deionized water.
  • Figure 3 shows the HE staining effect of primary tumor cells of solid tumors of colorectal cancer obtained in vitro culture. It can be seen that these cells generally have high cytoplasmic ratio, deep nuclear staining, intranuclear chromatin aggregation, multinucleus, and uneven cell size. According to the characteristics of cancer cells, dozens to hundreds of tumor cells aggregate to form tumor cell clusters with a certain three-dimensional structure.
  • Paraformaldehyde (Beijing Chemical Reagent Company, analytical grade), dissolve paraformaldehyde powder with ultrapure water to make a 4% (4g / 100mL) paraformaldehyde solution;
  • Methanol, dimethyl sulfoxide, and 35% hydrogen peroxide are mixed at a ratio of 4: 4: 1 (volume ratio) to make a Dansei rinse;
  • Bovine serum albumin (Sigma, # A1933), dissolve bovine serum albumin with PBS solution to make a 3% (3g / 100mL) BSA solution;
  • Figure 4 shows the effect of immunofluorescence staining on primary tumor cell masses of solid tumors of colorectal cancer cultured in vitro. It can be seen that the cells that make up the cell mass are all CK8 / CK18 positive and are of epithelial origin, confirming this method The resulting tumor cells are of higher purity. Twenty primary cultures of colorectal cancer samples were identified by immunofluorescence staining. The statistical results showed that among the primary colorectal cancer solid tumor primary cells obtained by this method, the proportion of tumor cells reached 72% -95% (Table 24).
  • Example 11 Primary cell culture and primary tumor tissue of solid tumors of colorectal cancer
  • the DNA extraction procedure mentioned in the following examples was performed using the Tiangen Blood / Tissue / Cell Genome Extraction Kit (DP304).
  • the library building procedure mentioned in the following examples was performed using the NEB DNA sequencing library building kit (E7645).
  • the high-throughput sequencing mentioned in the following examples refers to the Illumina HiSeq X-ten sequencing platform.
  • CNV Copy number variation analysis
  • Example 12 Comparison of the success rate of colorectal cancer solid tumor primary cells cultured with different primary cell culture media
  • the primary cell culture medium After the primary cell culture medium is prepared, it is sterilized by filtration with a 0.22 ⁇ M needle filter (Millipore SLGP033RS), and it can be stored at 4 ° C for two weeks.
  • a 0.22 ⁇ M needle filter Millipore SLGP033RS
  • the primary cell culture medium has a great effect on the success rate of colorectal cancer primary cell culture.
  • the colorectal cancer solid tumor primary cell culture medium (Table 9) used in the present invention can stimulate colorectal cancer to the greatest extent.
  • the proliferation of cancer cells in solid tumor tissue samples improves the success rate of primary cell culture of solid tumors in colorectal cancer.
  • sample preservation solution has a greater impact on the success rate of primary cell culture of solid tumors of colorectal cancer.
  • the sample preservation solution (Table 1) used in the present invention can protect the colorectal cancer solid tumor tissue samples to the maximum The activity of cancer cells increases the success rate of culture.
  • the operation method flow of the primary culture of all the samples is completely the same (refer to the foregoing description), and only the sample dissociation liquid formula is different. See Table 29 for various sample dissociations tested.
  • Scheme D is the formula used in the present invention. See Table 3 for details.
  • sample dissociation solution has a great impact on the success rate of primary cell culture of solid tumors of colorectal cancer.
  • the sample dissociation solution (Table 3) used in the present invention can maximize the separation of colorectal cancer solid tumor tissues. Of cancer cells, and improve the success rate of primary cell culture of solid tumors of colorectal cancer.
  • Example 15 Comparison of the success rate of primary cells of colorectal cancer solid tumors passaged with different cell digestive fluids
  • the cell digestive juice (Table 7) used in the present invention can gently dissociate cancer cells in cell masses. Allows samples to be serially passaged while maintaining primary cell viability in solid tumors of colorectal cancer.
  • Example 16 Formulation of a reagent for culturing colorectal cancer ascites primary tumor cells
  • the cell isolation buffer After the cell isolation buffer is prepared, it can be stored at 4 ° C for 1 month.
  • the specific formula of the cell digestive fluid (10 mL) is shown in Table 7 (same as the cell digestive fluid formulation used for primary cell culture of solid tumors of colorectal cancer).
  • the specific formulation of the digestion termination solution (100 mL) is shown in Table 8 (same as the formulation of the digestion termination solution used for primary cell culture of solid tumors of colorectal cancer).
  • colorectal cancer ascites primary tumor cell culture medium 100 mL
  • Table 9 standard formula as colorectal cancer solid tumor primary cell culture medium used for colorectal cancer solid tumor primary cell culture.
  • the specific formulation of the cell cryopreservation solution is shown in Table 22 (same as the cell cryopreservation formulation used for primary cell culture of solid tumors of colorectal cancer).
  • the attending physician selects the patients according to the clinical indications specified in the medical guidelines, and selects suitable samples for in vitro culture according to the clinical indications during surgery.
  • the selection criteria of the samples are: primary colorectal cancer, pathological stage is Stage IV, the pathological classification is colorectal cancer or colorectal cancer metastatic lesions, patients with malignant ascites need to be excreted, the amount of excretion is not less than 50mL.
  • the attending doctor provides basic clinical information such as the patient's gender, age, medical history, family history, smoking history, pathological staging and classification, and clinical diagnosis. Hide the patient's name, ID number and other information related to patient privacy, and replace it with a uniform experiment number.
  • the naming principle of the experiment number is the eight-digit date of the sample collected + the last four digits of the patient's hospital number. For example, the sample provided on January 1, 2018, the patient hospitalization number is T001512765, and the sample experiment number is 201801012765.
  • the colorectal cancer ascites sample is left on ice for about 30 minutes to allow the clot and large insoluble solids in the sample to settle to the bottom of the sample tube.
  • a low surface adsorption of colorectal cancer ascites cell suspension culture of primary tumor, as an example of six-well plates, 106 per well were plated at a density of cells, at 37 °C, 5% CO 2 cell incubator conditions Cultivate.
  • cancer cells expanded in large numbers to form cell clumps with a diameter of 80 ⁇ m.
  • the total number of tumor cells can exceed 10 7 , and the number of other types of cells has significantly decreased or even disappeared.
  • the method has been tested on a large number of samples, and the success rate of primary tumor cells derived from ascites of colorectal cancer in vitro can reach 80%.
  • Example 21 Passage of primary tumor cells derived from ascites of colorectal cancer
  • a low surface adsorption of colorectal cancer primary cell culture an example in six-well plates, 106 per well were plated at a density of cells, at 37 °C, 5% CO 2 condition cultured in cell culture incubator.
  • Example 22 Cryopreservation of primary tumor cells derived from ascites of colorectal cancer
  • Primary tumor cells of colorectal cancer ascites in suspension culture can be frozen after 2-3 passages expansion:
  • Example 23 Resuscitation of primary tumor cells derived from ascites of colorectal cancer
  • Example 24 Identification of colorectal cancer ascites-derived primary tumor cells by HE staining
  • Reagent consumables used in the following examples:
  • Figure 8 shows the HE staining effect of colorectal cancer ascites-derived primary tumor cells obtained from in vitro culture. It can be seen that these cells generally have high nuclear-to-cytoplasm ratio, deep nuclear staining, nuclear agglutination, multinuclei, and uneven cell size. And other characteristics of cancer cells.
  • Example 25 Identification of colorectal cancer ascites-derived primary tumor cells by immunofluorescence staining
  • Paraformaldehyde (Beijing Chemical Reagent Company, analytical grade), dissolve paraformaldehyde powder with ultrapure water to make a 4% paraformaldehyde solution;
  • Methanol, dimethyl sulfoxide, and 35% hydrogen peroxide are mixed at a ratio of 4: 4: 1 to make a Danshin rinse;
  • Bovine serum albumin (Sigma, # A1933), dissolve bovine serum albumin with PBS solution to make a 3% BSA solution;
  • Figure 9 shows the effect of immunofluorescence staining of primary tumor cell clumps derived from ascites derived from colorectal cancer 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, confirming this method The resulting tumor cells are of higher purity. Immunofluorescence staining was performed on 20 colorectal cancer ascites primary cultures. The statistical results showed that the proportion of tumor cells in colorectal cancer ascites-derived primary tumor cells obtained by this method reached 72% -91% (Table 35). .
  • Example 26 Colorectal cancer ascites-derived primary tumor cell culture and primary tumor tissue
  • the DNA extraction procedure mentioned in the following examples was performed using the Tiangen Blood / Tissue / Cell Genome Extraction Kit (DP304).
  • the library building procedure mentioned in the following examples was performed using the NEB DNA sequencing library building kit (E7645).
  • the high-throughput sequencing mentioned in the following examples refers to the Illumina HiSeq X-ten sequencing platform.
  • Colorectal cancer ascites primary tumor cell culture medium (Table 9, where 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 10ng / mL; the final concentration of human recombinant protein Wnt-3a is 200ng / mL; the final concentration of human recombinant protein Noggin is 100ng / mL; the final concentration of SB202190 is 10 ⁇ M; the final concentration of A83-01 The final concentration is 1 ⁇ M; the final concentration of N-acetyl-L-cysteine is 1 mM; the final concentration of Nicotinamide is 10 mM; the final concentration of cortisol is 20 ng / mL; the final concentration of Y-27632 is 10 ⁇ M).
  • P0 generation cells Cell clumps with a diameter of 80 ⁇ m or more are recorded as P0 generation cells, and are then recorded as P1, P2, ..., Pn according to the number of passages.
  • P5 generation source of colorectal cancer ascites tumor cells in primary cultures from each 106 cells, DNA extraction, database and high-throughput genome sequencing (the WGS), sequencing depth 30X.
  • the sequencing results of each group were analyzed by copy number variation (CNV) to compare the copy number variation between cancer cells in colorectal cancer ascites and primary cell cultures of colorectal cancer ascites, as shown in Figure 10,
  • the primary cell culture (P1, P2, P3, P4, P5) of colorectal cancer ascites of each generation is highly consistent with the copy number variation of cancer cells in colorectal cancer ascites, so the colorectal cancer ascites source obtained by this method
  • Primary tumor cells can represent the true presence of cancer cells in the patient's ascites.
  • Example 27 Comparison of success rates of colorectal cancer-derived primary tumor cells cultured in different primary cell culture media
  • Table 36 Formulation of primary cell culture medium for testing (100 mL)
  • the primary cell culture medium After the primary cell culture medium is prepared, it is sterilized by filtration with a 0.22 ⁇ M needle filter (Millipore SLGP033RS), and it can be stored at 4 ° C for two weeks.
  • a 0.22 ⁇ M needle filter Millipore SLGP033RS
  • the primary cell culture medium has a great influence on the success rate of the primary cell culture of colorectal cancer ascites.
  • the colorectal cancer ascites primary tumor cell culture medium (Table 9) used in the present invention can stimulate the colorectum to the greatest extent. Cancer cell proliferation in cancer ascites samples improves the success rate of primary tumor cell culture in colorectal cancer ascites.
  • Example 28 Comparison of success rates of primary tumor cells in colorectal cancer ascites passaged by different cell digestive fluids
  • the cell digestive fluid (Table 7) used in the present invention can gently dissociate cancer cells from cell masses. Allows samples to be serially passaged while maintaining primary tumor cell activity in colorectal cancer ascites.
  • Example 29 Primary tumor cell culture of colorectal cancer ascites using cell culture consumables of different materials
  • PS Polystyrene
  • PC Polycarbonate
  • PMMA poly-methylmethacrylate
  • COC resin CycloOlefin Polymer
  • Abbreviation COP Abbreviation COP
  • LAS low-attachment-surface
  • Example 30 Culture of primary tumor cell of colorectal cancer ascites with cell culture consumables modified by CYTOP
  • the method of CYTOP modification is as follows: first, the cell culture vessel is subjected to pure oxygen etching, the etching condition is 20 W, and the etching time is 3 minutes. Then cover the surface of the petri dish or culture plate with an appropriate amount of 1% CYTOP solution (see Table 42 for a recipe, see Table 42). be usable.
  • the 1% CYTOP solution After the 1% CYTOP solution is prepared, it can be stored at room temperature for a long time.
  • Example 31 Drug sensitivity test using colorectal cancer ascites primary tumor cells
  • the chemotherapeutic drugs 5-Fluorouracil, Oxaliplatin, and Irinotecan used in this embodiment are all Selleck products.
  • the Celltiter-Glo cell viability detection kit mentioned in this example is a Promega product.
  • a micro-well plate chip used for culturing primary cells of the colorectal cancer of the present invention is obtained by processing using PMMA material (or materials such as PS, PC, COC, COP, LAS, etc.) by using injection molding processing.
  • the chip can be used for colorectal cancer primary cell culture and in vitro drug sensitivity detection experiments.
  • the microplate chip design drawing is shown in Figure 12.
  • the PMMA material (or materials such as PS, PC, COC, COP, LAS, etc.) is used to prepare the structure of the microplate chip shown in Figure 12 in the design drawing, and then the CYTOP modification method described above (see Implementation) Example 30) The surface was modified with CYTOP to obtain the microwell plate chip which can be used for primary cell culture of colorectal cancer.
  • the invention provides a method for extracting and culturing primary tumor cells of colorectal cancer from fresh colorectal cancer solid tumor tissue or colorectal cancer ascites, and a matching reagent.
  • the method has the following advantages: Tissue samples are small, and only about 20 mg of colorectal cancer surgery samples are needed. For colorectal cancer ascites, the sample is easy to obtain, making the best use of the ascites discharged during the routine treatment of colorectal cancer patients, without additional trauma and pain to the patient; the amount of sample is small, only about 50mL of colorectal cancer is needed Ascites samples; samples do not need to be processed immediately after collection. Processing of this method for up to 72 hours after ex vivo can ensure a cell viability of more than 90%.
  • the culture period is short, and it takes only 3-10 days to obtain 10 7 orders of colorectal cancer primary tumor cells.
  • the culture stability is high.
  • the success rate of in vitro culture of qualified solid and ascites specimens of colorectal cancer by this method is as high as 70% -80%.
  • the cell purity is high.
  • the proportion of cancer cells can reach 70% to 95%, and there is less interference from heterogeneous cells.
  • the colorectal cancer primary cell culture obtained by the method of the present invention can be used for in vitro experiments at multiple cell levels, second-generation sequencing, construction of animal models, construction of cell lines, and the like. It is foreseeable that this culture method has broad application prospects in the research and clinical diagnosis and treatment of colorectal cancer.

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Abstract

本发明公开了一种结直肠癌实体瘤原代细胞和结直肠癌腹水原代肿瘤细胞培养方法及配套试剂。本发明提供了一种结直肠癌实体瘤原代细胞和结直肠癌腹水原代肿瘤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结直肠癌实体瘤组织，用温和方法分离腹水中的结直肠癌细胞，最大程度的保证了癌细胞的活力；(2)配制特殊的无血清培养基，利用悬浮培养体系对结直肠癌实体瘤来源的肿瘤细胞进行体外培养，保证癌细胞正常扩增的同时最大限度的排除正常细胞的干扰。利用本发明方法得到的结直肠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，这种培养方法在结直肠癌的研究和临床诊断治疗领域具有广泛的应用前景。

Description

结直肠癌实体瘤原代细胞和结直肠癌腹水原代肿瘤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，具体涉及一种结直肠癌实体瘤原代细胞和结直肠癌腹水原代肿瘤细胞培养方法及配套试剂。
背景技术
结直肠癌是最常见的严重威胁人类的健康恶性肿瘤之一。在我国结直肠癌的发病率为9.24％，在所有恶性肿瘤中占第四位。而结直肠癌的死亡率为11.77％，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，结直肠癌的发病率还将呈不断上升的趋势。另外，结直肠癌复发转移风险高，超过50％的结直肠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。
尽管世界各国的科研和医疗机构对结直肠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。结直肠癌是一种复杂疾病，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。结直肠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。因此将结直肠癌实体瘤原代细胞培养物作为模型进行个体化精准研究是结直肠癌研究领域乃至结直肠癌诊断治疗领域的趋势。
对于错失手术时机的晚期结直肠癌患者，化疗和靶向治疗是最重要的治疗方法，初治患者首次化疗用药方案的选择对患者的病情控制尤为重要。随着二代测序技术的广泛应用，通过个体化基因测序和数据挖掘，但是仅有30％-50％的结直肠癌病人可以通过测序结果预测找到可能有效的药物，并且无法保证这些药物方案在实际化疗中一定能有效杀伤癌细胞。
传统方法用结直肠癌细胞系作为结直肠癌研究模型，很难代表成千上万不同结直肠癌患者体内癌细胞的真实情况，有很大的局限性。而代表了精准医学概念的PDX模型，则难以克服建模周期太长，无法指导临床治疗的弱点。中晚期结直肠癌的患者常常并发腹水需要及时排出体外，因此腹水是一种非常容易获得的临床样本，并且在腹水中经常可以找到脱落的结直肠癌细胞。将结直肠癌实体瘤原代细胞培养物和结直肠癌腹水原代肿瘤细胞培养物作为模型进行个体化精准研究是结直肠癌研究领域乃至结直肠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结直肠癌实体瘤原代细胞和结直肠癌腹水原代肿瘤细胞的培养技术及配套试剂，该技术的核心是：(1) 用温和细胞解离试剂处理结直肠癌实体瘤组织，最大程度的保证了组织中癌细胞的活力；(2)配制特殊的无血清培养基，利用悬浮培养体系对结直肠癌实体瘤来源的肿瘤细胞和结直肠癌腹水原代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结直肠癌原代细胞的培养基。
本发明所要求保护的用于培养结直肠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、人重组蛋白EGF、人重组蛋白bFGF、人重组蛋白HGF、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-carbothioamide)、Primocin TM、N-乙酰-L-半胱氨酸(N-acetyl-L-cysteine)、烟碱(Nicotinamide)、N-2Supplement、皮质醇、B27、ITS-X(Insulin,Transferrin,Selenium,Ethanolamine Solution)、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100-250ng/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)；所述人重组蛋白EGF的终浓度为10-100ng/mL(如20ng/mL或40ng/mL)；所述人重组蛋白bFGF的终浓度为10-50ng/mL(如20ng/mL)；所述人重组蛋白HGF的终浓度为5-25ng/mL(如20ng/mL)；所述人重组蛋白Wnt-3a的终浓度为200-300ng/mL(如300ng/mL)；所述人重组蛋白Noggin的终浓度为100-200ng/mL(如150ng/mL)；所述SB202190的终浓度为5-10μM(如10μM)；所述A83-01的终浓度为0.25-1.25μM(如0.5μM或1μM)；所述Primocin TM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(如1mM)；所述烟碱(Nicotinamide)的终浓度为5-10mM(如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(如20ng/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Y-27632的终浓度为5-20μM(如5μM或10μM)；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 PCTCN2019099245-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Primocin TM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-2Supplement为“N-2Supplement(100X)”(如Gibco#17502001，或与其组成相同的其他产品)。所述“N-2Supplement(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-27632 dihydrochloride(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-2Supplement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，所述用于培养结直肠癌原代细胞的培养基的存在形式可为两种：
其一，所述用于培养结直肠癌原代细胞的培养基为由所述抗菌抗真菌剂三抗、所述HEPES、所述GlutaMax、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-2Supplement、所述皮质醇、所述B27、所述ITS-X、所述Y-27632和所述Advanced DMEM/F12培养基混合而成的溶液。
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，所述用于培养结直肠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Wnt-3a、人重组蛋白Noggin可以储液(母液)形式存在(-80℃可长期保存)，具体可为1000倍储液(母液)。SB202190、N-acetyl-L-cysteine、Nicotinamide、皮质醇和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。
上述五种1000倍储液中，所述BSA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×Y-27632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结直肠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在所述样本解离液中的终浓度为150-250U/mL(如200U/mL)；所述胶原酶II在所述样本解离液中的终浓度为150-250U/mL(如200U/mL)；所述胶原酶IV在所述样本解离液中的终浓度为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，所述胎牛血清在所述样本保存液中的终浓度为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)中的青霉素在所述样本保存液中的终浓度为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)中的链霉素在所述样本保存液中的终浓度为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在所述样本保存液中的终浓度为250-500ng/mL(如250ng/mL)；所述HEPES在所述样本保存液中的终浓度为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 PCTCN2019099245-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)中的青霉素在所述样本清洗液中的终浓度为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)中的链霉素在所述样本清洗液中的终浓度为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在所述样本清洗液中的终浓度为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 PCTCN2019099245-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抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)、肝素钠和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)；所述肝素钠的终浓度为10IU/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 PCTCN2019099245-appb-000004
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，所述胎牛血清在所述消化终止液中的终浓度为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)中的青霉素在所述消化终止液中的终浓度为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)中的链霉素在所述消化终止液中的终浓度为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在所述消化终止液中的终浓度为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 PCTCN2019099245-appb-000005
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，可以将样本中的结直肠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，用于悬浮腹水中的细胞，为密度梯度分离细胞做准备。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，可以将结直肠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结直肠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结直肠癌实 体瘤原代细胞中的应用；
(B3)前文第二方面中中(A2)或(A4)所述的成套试剂在培养结直肠癌腹水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结直肠癌原代细胞的方法。
本发明所要求保护的培养结直肠癌原代细胞的方法，为方法A或方法B：
方法A：一种培养结直肠癌实体瘤原代细胞的方法，可包括如下步骤：
(a1)用前文第二方面中所述的样本解离液对结直肠癌实体瘤组织进行解离处理，获得结直肠癌实体瘤原代细胞；
(a2)利用前文第一方面中所述培养基悬浮培养步骤(a1)解离出来的结直肠癌实体瘤原代细胞。
方法B：一种培养结直肠癌腹水原代肿瘤细胞的方法，可包括如下步骤：
(b1)从结直肠癌腹水中分离获得结直肠癌腹水原代肿瘤细胞；
(b2)利用权利要求1所述培养基悬浮培养步骤(b1)分离出来的结直肠癌腹水原代肿瘤细胞。
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结直肠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，将剪碎后的所述结直肠癌实体瘤组织(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，可按照包括如下步骤的方法从结直肠癌腹水中分离获得结直肠癌腹水原代肿瘤细胞：用前文第二方面中所述的细胞分离缓冲液悬浮结直肠癌腹水中的细胞，然后通过密度梯度离心(利用Ficoll淋巴细胞分离液)获得结直肠癌腹水原代肿瘤细胞。
进一步地，步骤(a2)中，可按照包括如下步骤的方法用所述结直肠癌实体瘤原代细胞培养基悬浮培养所述结直肠癌实体瘤原代细胞：使用细胞培养容器M，利用所述培养基悬浮培养所述结直肠癌实体瘤原代细胞，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结直肠癌腹水原代肿瘤细胞：使用细胞培养容器M，利用所述培养基悬浮培养所述结直肠癌腹水原代肿瘤细胞，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结直肠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％(如75％)的乙醇清洗结直肠癌实体瘤组织样本表面10到30秒；用样本清洗液清洗所述结直肠癌实体瘤组织样本10-20次(如10次)，用无菌的PBS溶液清洗所述结直肠癌实体瘤组织样本5-10次(如5次)；然后除去所述结直肠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。
对所述结直肠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结直肠癌实体瘤组织样本的离体时间需为2小时以内，且在进行所述解离前处理之前一直保存于所述样本保存液中。
进一步地，在步骤(a1)中，用所述样本解离液对所述结直肠癌实体瘤组织进行解离处理后还可包括如下步骤：用8-15倍(如10倍)体积的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)之前还包括对结直肠癌腹水样本进行分离前处理的步骤：去除结直肠癌腹水样本中的杂质、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：待所述结直肠癌实体瘤原代细胞形成直径50-80μm(如80μm)的团块时，对所述结直肠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：待所述结直肠癌腹水原代肿瘤细胞形成直径50-80μm(如80μm)的团块时，对所述结直肠癌腹水原代 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结直肠癌实体瘤原代细胞或所述结直肠癌腹水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)结直肠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)结直肠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)结直肠癌腹水细胞分离缓冲液，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结直肠癌实体瘤组织中解离出结直肠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结直肠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结直肠癌腹水中分离出结直肠癌腹水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)一种从结直肠癌实体瘤组织中解离出结直肠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)一种保存结直肠癌实体瘤组织的方法，包括如下步骤：将刚刚离体的结直肠癌实体瘤组织置于前文第二方面所述的样本保存液中保存，保存时间为2小时以内。
(E3)一种从结直肠癌腹水中分离出结直肠癌腹水原代肿瘤细胞的方法，包括前文第四方面中的步骤(b1)。
在上述各方面中，所述结直肠癌可为原发性结直肠癌。病理分型为结直肠癌或结直肠癌转移病灶。病理分期为II期或III期或IV期。
进一步地，从结直肠癌实体瘤组织中分离结直肠癌原代细胞时采用的样本可为结直肠癌II期或III期或IV期样本。从结直肠癌腹水中分离结直肠癌原代细胞时采用的样本为结直肠癌IV期样本。
在上述各方面中，所述结直肠癌原代细胞可为结直肠癌实体瘤原代细胞或结直肠癌腹水原代肿瘤细胞。
在上述各方面中，所述结直肠癌原代细胞可分离自结直肠癌患者的手术样本(为实体瘤样本)、肠镜穿刺样本或者腹水样本。其中，手术样本获得的结直肠癌实体瘤组织标本最好重量超过20mg。腹水样本最好不少于50mL。肠镜穿刺样本(为实体瘤样本)不少于2块。
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为结直肠癌组织经过处理后得到的单细胞。标尺为100μm，100倍放大。
图2为结直肠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为结直肠癌组织原代培养后得到的结直肠癌细胞团块切片HE染色图。标尺为100μm，200倍放大。
图4为结直肠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)显示各代结直肠癌原代细 胞培养物(P1、P2、P3、P4、P5)与原发结直肠癌肿瘤组织(Tumor)的拷贝数变异情况高度一致。
图6为结直肠癌腹水样本中分离得到的单细胞。标尺为100μm。
图7为结直肠癌腹水样本中原代肿瘤细胞培养后得到的细胞团块。标尺为200μm。
图8为结直肠癌腹水样本原代培养后得到的结直肠癌细胞HE染色图。
图9为结直肠癌腹水样本原代培养后得到的癌细胞团块免疫荧光染色图。
图10为根据测序结果进行拷贝数变异分析(CNV)显示各代结直肠癌腹水原代细胞培养物(P1、P2、P3、P4、P5)与结直肠癌腹水中癌细胞的拷贝数变异情况高度一致。
图11为用本发明培养得到的结直肠癌腹水原代肿瘤细胞进行体外药物敏感性测试结果。
图12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。
实施例1、配制用于培养结直肠癌实体瘤原代细胞的试剂
1、样本保存液(100mL)
样本保存液(100mL)的具体配方如表1所示。
表1样本保存液(100mL)
Figure PCTCN2019099245-appb-000006
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 PCTCN2019099245-appb-000007
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 PCTCN2019099245-appb-000008
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 PCTCN2019099245-appb-000009
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 PCTCN2019099245-appb-000010
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 PCTCN2019099245-appb-000011
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 PCTCN2019099245-appb-000012
Figure PCTCN2019099245-appb-000013
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 PCTCN2019099245-appb-000014
消化终止液配制后，可在4℃保存一个月。
6、结直肠癌实体瘤原代细胞培养基(100mL)
结直肠癌实体瘤原代细胞培养基(100mL)的具体配方如表9所示。
表9结直肠癌实体瘤原代细胞培养基(100mL)
Figure PCTCN2019099245-appb-000015
结直肠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15所示，SB202190储液的配置 如表16所示，A83-01储液的配置如表17所示，N-acetyl-L-cysteine储液的配置如表18所示，Nicotinamide储液的配置如表19所示，皮质醇储液的配制如表20所示，Y-27632储液的配制如表21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 PCTCN2019099245-appb-000016
100×BSA溶液现配现用。
表11 1000×人重组蛋白EGF储液(5mL)
Figure PCTCN2019099245-appb-000017
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 PCTCN2019099245-appb-000018
1000×人重组蛋白bEGF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 PCTCN2019099245-appb-000019
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Wnt-3a储液(2.5mL)
Figure PCTCN2019099245-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15 1000×人重组蛋白Noggin储液(5mL)
Figure PCTCN2019099245-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16 1000×SB202190储液(1.51mL)
Figure PCTCN2019099245-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17 100000×A83-01储液(1.05mL)
Figure PCTCN2019099245-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18 1000×N-acetyl-L-cysteine储液(5mL)
Figure PCTCN2019099245-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19 1000×Nicotinamide储液(4mL)
Figure PCTCN2019099245-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20 1000×皮质醇储液(100mL)
Figure PCTCN2019099245-appb-000026
Figure PCTCN2019099245-appb-000027
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21 1000×Y-27632储液(3.125mL)
Figure PCTCN2019099245-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22所示。
表22细胞冻存液
Figure PCTCN2019099245-appb-000029
细胞冻存液现配现用。
表22中，1％甲基纤维素溶液的配制如表23所示。
表23 1％甲基纤维素溶液(10mL)
Figure PCTCN2019099245-appb-000030
1％甲基纤维素溶液配制后可在4℃长期保存。
实施例2、结直肠癌实体瘤术后标本的获取
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：原发性结直肠癌，病理分期为II期、III期或IV期，各种病理分型的结直肠癌或结直肠癌转移病灶，结直肠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(表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、结直肠癌实体瘤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(表2)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、结直肠癌实体瘤组织样本解离
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(表3)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(表8)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、用结直肠癌实体瘤原代细胞培养基(表9)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结直肠癌原代肿瘤细胞。
实施例5、结直肠癌实体瘤原代细胞培养
1、使用低吸附表面(low-attachment-surface)进行结直肠癌原代细胞悬浮培养，所用培养基即为实施例1中的结直肠癌实体瘤原代细胞培养基(表9)，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，结直肠癌实体瘤原代肿瘤细胞体外培养成功率 可以达到80％。
实施例6、结直肠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(表7)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(表8)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、用结直肠癌实体瘤原代细胞培养基(表9)重悬细胞沉淀，细胞计数。
7、使用低吸附表面(low-attachment-surface)进行结直肠癌原代细胞培养，所用培养基即为实施例1中的结直肠癌实体瘤原代细胞培养基(表9)，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、结直肠癌实体瘤原代细胞的冻存
悬浮培养的结直肠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(表7)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(表8)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(表22)，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、结直肠癌实体瘤原代细胞的复苏
液氮中保存的结直肠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、用结直肠癌实体瘤原代细胞培养基(表9)重悬细胞沉淀，使用低吸附表面进行结直肠癌原代细胞培养，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、结直肠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离心收集用结直肠癌腹水原代肿瘤细胞培养基(表9，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10ng/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10mM；皮质醇的终浓度为20ng/mL；Y-27632的终浓度为10μM)培养得到的悬浮结直肠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1分钟。
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1分钟。
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40秒。
10、吸去伊红染液，依次用75％、80％、90％、100％乙醇漂洗脱水20秒、20秒、40秒、40秒。
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结直肠癌实体瘤原代肿瘤细胞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、结直肠癌实体瘤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)；
按以下步骤对用结直肠癌腹水原代肿瘤细胞培养基(表9，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10ng/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10mM；皮质醇的终浓度为20ng/mL Y-27632的终浓度为10μM)培养得到的结直肠癌实体瘤原代肿瘤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展示了体外培养的结直肠癌实体瘤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个结直肠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结直肠癌实体瘤原代细胞中，肿瘤细胞的比例达到72％-95％(表24)。
表24结直肠癌样本原代培养物免疫荧光染色鉴定
Figure PCTCN2019099245-appb-000031
Figure PCTCN2019099245-appb-000032
实施例11、结直肠癌实体瘤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、取得结直肠癌实体瘤样本，进行体外培养操作之前，先取结直肠癌实体瘤样本10mg进行DNA提取，建库及全基因组高通量测序(WGS)，测序深度30X，剩余实体瘤样本用于结直肠癌原代细胞体外培养。
2、结直肠癌组织处理后经过一段时间的培养，形成直径80μm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代的结直肠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30X。
3、各组测序结果分别进行拷贝数变异分析(CNV)，比较原发结直肠癌肿瘤组织与各代结直肠癌原代细胞培养物之间的拷贝数变异，如图5所示，各代结直肠癌实体瘤原代细胞培养物(P1、P2、P3、P4、P5)与原发结直肠癌肿瘤组织(Tumor)的拷贝数变异情况高度一致，因此经本方法得到的结直肠癌实体 瘤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结直肠癌实体瘤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25。其中方案D为本发明中采用的配方，具体见表9。
表25测试用原代细胞培养基配方(100mL)
Figure PCTCN2019099245-appb-000033
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例结直肠癌实体瘤组织样本，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天后统计结直肠癌实体瘤原代细胞培养成功率如表26所示：
表26不同培养基培养情况
Figure PCTCN2019099245-appb-000034
可以看到，原代细胞培养基对结直肠癌原代细胞的培养成功率影响极大，本发明使用的结直肠癌实体瘤原代细胞培养基(表9)可以最大程度的刺激结直肠癌实体瘤组织样本中癌细胞增殖，提高结直肠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结直肠癌实体瘤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27。其中方案E为本发明中采用的配方，具体见表1。
表27测试用样本保存液配方(100mL)
Figure PCTCN2019099245-appb-000035
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例结直肠癌实体瘤样本，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天后统计结直肠癌实体瘤原代细胞培养成功率如表28：
表28不同样本保存液培养情况
Figure PCTCN2019099245-appb-000036
可以看到，样本保存液配方对结直肠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结直肠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结直肠癌实体瘤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29。其中方案D为本发明中采用的配方，具体见表3。
表29测试用样本解离液配方(10mL)
Figure PCTCN2019099245-appb-000037
Figure PCTCN2019099245-appb-000038
样本解离液现配现用。
选取20例结直肠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天后统计结直肠癌实体瘤原代细胞培养成功率如下表30：
表30不同样本解离液培养情况
Figure PCTCN2019099245-appb-000039
可以看到，样本解离液配方对结直肠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)可以最大程度分离结直肠癌实体瘤组织中的癌细胞，提高结直肠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结直肠癌实体瘤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。进行测试的各种细胞消化液见表31。其中方案D为本发明中采用的配方，具体见表7。
表31测试用细胞消化液配方(10mL)
Figure PCTCN2019099245-appb-000040
细胞消化液现配现用。
选取20例培养成功的结直肠癌实体瘤原代细胞样本，将培养得到的结直肠癌实体瘤原代细胞，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80μm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32：
表32不同细胞消化液培养情况
Figure PCTCN2019099245-appb-000041
Figure PCTCN2019099245-appb-000042
可以看到，细胞消化液配方对结直肠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结直肠癌实体瘤原代细胞活性。
实施例16、配制用于培养结直肠癌腹水原代肿瘤细胞的试剂
1、细胞分离缓冲液(100mL)
细胞分离缓冲液(100mL)的具体配方如表33所示：
表33细胞分离缓冲液(100mL)
Figure PCTCN2019099245-appb-000043
细胞分离缓冲液配制完成后，可于4℃保存1个月。
表33中，肝素钠溶液的配制见表34。
表34 1000×肝素钠(1mL)
Figure PCTCN2019099245-appb-000044
1000×肝素钠溶液现配现用。
2、细胞消化液(10mL)
细胞消化液(10mL)的具体配方如表7所示(同结直肠癌实体瘤原代细胞培养所用的细胞消化液配方)。
3、消化终止液(100mL)
消化终止液(100mL)的具体配方如表8所示(同结直肠癌实体瘤原代细胞培养所用的消化终止液配方)。
4、结直肠癌腹水原代肿瘤细胞培养基(100mL)
结直肠癌腹水原代肿瘤细胞培养基(100mL)的具体配方如表9所示(同结直肠癌实体瘤原代细胞培养所用的结直肠癌实体瘤原代细胞培养基配方)。
5、细胞冻存液
细胞冻存液的具体配方如表22所示(同结直肠癌实体瘤原代细胞培养所用的细胞冻存液配方)。
实施例17、结直肠癌腹水样本的获取
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，样本的选取标准为：原发性结直肠癌，病理分期为IV期，病理分型为结直肠癌或结直肠癌转移病灶，病人有恶性腹水需要排出，排出量不少于50mL。
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、由主治医生，用无菌的器材和容器采集腹水样本。样本离体后在冰上暂存，72小时内运输到实验室进行下一步操作。
实施例18、结直肠癌腹水样本预处理
下述实施例需要在冰上操作：
1、结直肠癌腹水样本冰上静置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。
3、2000g常温离心5分钟，弃去上清。
4、用细胞分离缓冲液(表33)重悬细胞沉淀，2000g常温离心5分钟，弃去上清。
5、用细胞分离缓冲液(表33)重悬细胞沉淀，调整细胞浓度至10 7-10 8个/mL。
实施例19、结直肠癌腹水样本密度梯度离心
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、用结直肠癌腹水原代肿瘤细胞培养基(表9)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6所示，从结直肠癌腹水样本中分离得到的单细胞悬液里，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结直肠癌原代肿瘤细胞。
实施例20、结直肠癌腹水来源原代肿瘤细胞的培养
1、使用低吸附表面进行结直肠癌腹水原代肿瘤细胞悬浮培养，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7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，结直肠癌腹水来源的原代肿瘤细胞体外培养成功率可以达到80％。
实施例21、结直肠癌腹水来源原代肿瘤细胞传代
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(表7)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(表8)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、用结直肠癌腹水原代肿瘤细胞培养基(表9)重悬细胞沉淀，细胞计数。
7、使用低吸附表面进行结直肠癌原代细胞培养，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22、结直肠癌腹水来源原代肿瘤细胞的冻存
悬浮培养的结直肠癌腹水原代肿瘤细胞经过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(表7)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(表8)终止反应，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(表22)，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23、结直肠癌腹水来源原代肿瘤细胞的复苏
液氮中保存的结直肠癌腹水来源原代肿瘤细胞可以进行复苏：
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、用结直肠癌腹水原代肿瘤细胞培养基(表9)重悬细胞沉淀，使用低吸附表面进行结直肠癌腹水来源原代肿瘤细胞培养，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24、结直肠癌腹水来源原代肿瘤细胞的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、将用结直肠癌腹水原代肿瘤细胞培养基(表9，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10ng/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10mM；皮质醇的终浓度为20ng/mL；Y-27632的终浓度为10μM)培养得到的悬浮结直肠癌腹水来源原代肿瘤细胞制成浓度为10 4/mL的细胞悬液，滴加10μL于阳离子防脱玻片上，自然晾干。
2、在风干的细胞上小心滴加50μL经4℃预冷过的甲醇/丙酮混合液(体积比1:1)，然后将玻片放入4℃冰箱固定10分钟。
3、取出固定细胞的玻片，室温自然晾干。
4、用200μL PBS清洗玻片两次。
5、待玻片上水分微干时加入100μL苏木精染液染色1分钟。
6、吸去苏木精染液，用200μL自来水清洗玻片3次。
7、滴加100μL分化液分化1分钟。
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40秒。
10、吸去伊红染液，依次用75％、80％、90％、100％乙醇漂洗脱水20秒、20秒、40秒、40秒。
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8展示了体外培养得到的结直肠癌腹水来源原代肿瘤细胞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。
实施例25、结直肠癌腹水来源原代肿瘤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％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％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)
按以下步骤对用结直肠癌腹水原代肿瘤细胞培养基(表9，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10ng/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10mM；皮质醇的终浓度为20ng/mL；Y-27632的终浓度为10μM)培养得到的结直肠癌腹水来源原代肿瘤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、收集培养皿中的腹水来源原代肿瘤细胞团块，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％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9展示了体外培养的结直肠癌腹水来源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个结直肠癌腹水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结直肠癌腹水来源原代肿瘤细胞中，肿瘤细胞的比例达到72％-91％(表35)。
表35结直肠癌腹水原代培养物免疫荧光染色鉴定
Figure PCTCN2019099245-appb-000045
实施例26、结直肠癌腹水来源原代肿瘤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、取得结直肠癌腹水样本，进行体外培养操作之前，先取20mL结直肠癌腹水样本进行离心，得到腹水细胞沉淀并进行DNA提取，建库及全基因组高通量测序(WGS)，测序深度30X，剩余结直肠癌腹水样本用于结直肠癌腹水原代肿瘤细胞体外培养(具体方法参照前述实施例进行)。
2、结直肠癌腹水样本处理后用结直肠癌腹水原代肿瘤细胞培养基(表9，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10ng/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10mM；皮质醇的终浓度为20ng/mL；Y-27632的终浓度为10μM)经过一段时间的培养，形成直径80μm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代的结直肠癌腹水来源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30X。
3、各组测序结果分别进行拷贝数变异分析(CNV)，比较结直肠癌腹水中的癌细胞与各代结直肠癌腹水原代细胞培养物之间的拷贝数变异，如图10所示，各代结直肠癌腹水原代细胞培养物(P1、P2、P3、P4、P5)与结直肠癌腹水中癌细胞的拷贝数变异情况高度一致，因此经本方法得到的结直肠癌腹水来源的原代肿瘤细胞能够代表患者腹水中存在癌细胞的真实情况。
实施例27、不同原代细胞培养基培养结直肠癌腹水来源的原代肿瘤细胞成功率比较
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36。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40ng/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Wnt-3a的终浓度为300ng/mL；人重组蛋白Noggin的终浓度为150ng/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20ng/mL Y-27632的终浓度为5μM)。
表36测试用原代细胞培养基配方(100mL)
Figure PCTCN2019099245-appb-000046
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例结直肠癌腹水样本，按实施例18、19、20中所述方法进行样本处理和培养操作，培养10天后统计结直肠癌腹水原代肿 瘤细胞培养成功率如表37所示：
表37不同培养基培养情况
Figure PCTCN2019099245-appb-000047
可以看到，原代细胞培养基对结直肠癌腹水原代细胞的培养成功率影响极大，本发明使用的结直肠癌腹水原代肿瘤细胞培养基(表9)可以最大程度的刺激结直肠癌腹水样本中的癌细胞增殖，提高结直肠癌腹水原代肿瘤细胞培养的成功率。
实施例28、不同细胞消化液传代结直肠癌腹水原代肿瘤细胞成功率比较
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。进行测试的各种细胞消化液见表38。其中方案D为本发明中采用的配方，具体见表7。
表38测试用细胞消化液配方(10mL)
Figure PCTCN2019099245-appb-000048
细胞消化液现配现用。
选取20例培养成功的结直肠癌腹水样本，将培养得到的结直肠癌腹水原代肿瘤细胞，分别用上述四种细胞消化液，按实施例21中所述方法进行连续传代操作。每当癌细胞扩增形成直径80μm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39：
表39不同细胞消化液培养情况
Figure PCTCN2019099245-appb-000049
Figure PCTCN2019099245-appb-000050
可以看到，细胞消化液配方对结直肠癌腹水原代肿瘤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结直肠癌腹水原代肿瘤细胞活性。
实施例29、用不同材质的细胞培养耗材进行结直肠癌腹水原代肿瘤细胞培养
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40)。
表40未经修饰的不同材质培养耗材对结直肠癌腹水原代肿瘤细胞培养的影响
Figure PCTCN2019099245-appb-000051
Figure PCTCN2019099245-appb-000052
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40可见：不同材质培养耗材对结直肠癌腹水原代肿瘤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30、用CYTOP修饰的细胞培养耗材进行结直肠癌腹水原代肿瘤细胞培养
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41)。
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(配方见表42)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41 CYTOP修饰的不同材质CYTOP修饰培养耗材对结直肠癌腹水原代肿瘤细胞培养的影响
Figure PCTCN2019099245-appb-000053
由表41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。
表42 1％CYTOP溶液(100mL)
Figure PCTCN2019099245-appb-000054
1％CYTOP溶液配制完成后，可于常温长期保存。
实施例31、用结直肠癌腹水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5-Fluorouracil、Oxaliplatin、Irinotecan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结直肠癌腹水原代肿瘤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11。该结果说明：本方法获得的结直肠癌腹水原代肿瘤细胞可以用于体外药物敏感性检测。
实施例32、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结直肠癌原代细胞所用的微孔板芯片。该芯片可以用于结直肠癌原代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12。
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12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30)对其表面进行CYTOP修饰，就得到了这里所说的可用于结直肠癌原代细胞培养的微孔板芯片。
实施例33、用肠镜穿刺微量样本进行结直肠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与结直肠癌实体瘤原代细胞培养方法完全一致(参照前文所述)，仅样本来源有所区别，统一为肠镜样本两块(表43)。
表43结直肠癌肠镜穿刺样本原代细胞培养情况
Figure PCTCN2019099245-appb-000055
由表43可见：肠镜穿刺得到的微量样本仍然可以以较高的成功率进行结直肠癌原代细胞培养。
工业应用
本发明提供了一种从新鲜结直肠癌实体瘤组织或者结直肠癌腹水中提取培养结直肠癌原代肿瘤细胞的方法和配套试剂，该方法具有以下优点：对于结直肠癌实体瘤组织而言，组织样本用量少，仅需20mg左右的结直肠癌手术样本。对于结直肠癌腹水而言，样本获取容易，最大限度的利用了结直肠癌病人常规治疗过程中排出的腹水，对患者无额外的创伤和痛苦；样本用量少，仅需50mL左右的结直肠癌腹水样本；样本采集后无需立即处理，样本离体后长达72小时的时间内按本方法进行处理均可以保证90％以上的细胞活率。培养周期短，仅需3-10天即可获得10 7数量级的结直肠癌原代肿瘤细胞。培养稳定性高，用本方法对合格的结直肠癌实体瘤和腹水标本进行体外培养的成功率高达70％-80％。细胞纯度高，利用本方法得到的结直肠癌原代细胞培养物中，癌细胞的比例可以达到70％-95％，杂细胞干扰少。利用本发明方法得到的结直肠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，这种培养方法在结直肠癌的研究和临床诊断治疗领域具有广泛的应用前景。

Claims (21)

  1. 一种用于培养结直肠癌原代细胞的培养基，由抗菌抗真菌剂三抗、HEPES、GlutaMax、人重组蛋白EGF、人重组蛋白bFGF、人重组蛋白HGF、人重组蛋白Wnt-3a、人重组蛋白Noggin、SB202190、A83-01、Primocin TM、N-乙酰-L-半胱氨酸、烟碱、N-2Supplement、皮质醇、B27、ITS-X、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100-250ng/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)；所述人重组蛋白EG0F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 TM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. 一种用于培养结直肠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在所述样本解离液中的终浓度为150-250U/mL；所述胶原酶II在所述样本解离液中的终浓度为150-250U/mL；所述胶原酶IV在所述样本解离液中的终浓度为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，所述胎牛血清在所述样本保存液中的终浓度为1-5％(体积百分含量)；所述抗菌抗真菌剂三抗中的青霉素在所述样本保存液中的终浓度为100-200U/mL；所述抗菌抗真菌剂三抗中的链霉素在所述样本保存液中的终浓度为100-200μg/mL；所述抗菌抗真菌剂三抗中的两性霉素B在所述样本保存液中的终浓度为250-500ng/mL；所述HEPES在所述样本保存液中的终浓度为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在所述样本清洗液中的终浓度为100-200U/mL；所述抗菌抗真菌剂三抗中的链霉素在所述样本清洗液中的终浓度为100-200μg/mL；所述抗菌 抗真菌剂三抗中的两性霉素B在所述样本清洗液中的终浓度为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抗菌抗真菌剂三抗、肝素钠和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；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，所述胎牛血清在所述消化终止液中的终浓度为8-12％(体积百分含量)；所述抗菌抗真菌剂三抗中的青霉素在所述消化终止液中的终浓度为100-200U/mL；所述抗菌抗真菌剂三抗中的链霉素在所述消化终止液中的终浓度为100-200μg/mL；所述抗菌抗真菌剂三抗中的两性霉素B在所述消化终止液中的终浓度为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结直肠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结直肠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结直肠癌腹水原代肿瘤细胞中的应用。
  4. 一种培养结直肠癌原代细胞的方法，为方法A或方法B：
    方法A：一种培养结直肠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结直肠癌实体瘤组织进行解离处理，获得结直肠癌实体瘤原代细胞；
    (a2)利用权利要求1所述培养基悬浮培养步骤(a1)解离出来的结直肠癌实体瘤原代细胞；
    方法B：一种培养结直肠癌腹水原代肿瘤细胞的方法，包括如下步骤：
    (b1)从结直肠癌腹水中分离获得结直肠癌腹水原代肿瘤细胞；
    (b2)利用权利要求1所述培养基悬浮培养步骤(b1)分离出来的结直肠癌腹水原代肿瘤细胞。
  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结直肠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，将剪碎后的所述结直肠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结直肠癌腹水中分离获得结直肠癌腹水原代肿瘤细胞的：用权利要求2中所述的细胞分离缓冲液悬浮结直肠癌腹水中的细胞，然后通过密度梯度离心获得结直肠癌腹水原代肿瘤细胞。
  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结直肠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结直肠癌实体瘤原代细胞，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结直肠癌腹水原代肿瘤细胞的：使用细胞培养容器M，利用所述培养基悬浮培养所述结直肠癌腹水原代肿瘤细胞，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，还包括如下对所述结直肠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％的乙醇清洗结直肠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溶液先后清洗所述结直肠癌实体瘤组织样本。
  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结直肠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结直肠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：待所述结直肠癌实体瘤原代细胞形成直径50-80μm的团块时，对所述结直肠癌实体瘤原代细胞进行传代；
    在步骤(b2)中，还包括如下步骤：待所述结直肠癌腹水原代肿瘤细胞形成直径50-80μm的团块时，对所述结直肠癌腹水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结直肠癌实体瘤原代细胞或所述结直肠癌腹水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)结直肠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)结直肠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)结直肠癌腹水细胞分离缓冲液，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结直肠癌实体瘤组织中解离出结直肠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结直肠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结直肠癌腹水中分离出结直肠癌腹水原代肿瘤细胞中的应用。
  17. 如下任一方法：
    (E1)一种从结直肠癌实体瘤组织中解离出结直肠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)一种保存结直肠癌实体瘤组织的方法，包括如下步骤：将刚刚离体的结直肠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)一种从结直肠癌腹水中分离出结直肠癌腹水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：所述结直肠癌为原发性结直肠癌。
  19. 根据权利要求1-18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：所述结直肠癌为结直肠癌或结直肠癌转移病灶。
  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：所述结直肠癌原代细胞为结直肠癌实体瘤原代细胞或结直肠癌腹水原代肿瘤细胞。
  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：所述结直肠癌原代细胞分离自结直肠癌患者的手术样本、肠镜穿刺样本或者腹水样本。
PCT/CN2019/099245 2018-06-13 2019-08-05 结直肠癌实体瘤原代细胞和结直肠癌腹水原代肿瘤细胞培养方法及配套试剂 WO2019238143A2 (zh)

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