Culture medium and method for constructing in-vitro 3D micro-tumor model of head and neck tumor

Abstract

The invention discloses a culture medium and a method for constructing a head and neck tumor in-vitro 3D micro-tumor model. The invention provides a culture medium for constructing an in-vitro 3D (three-dimensional) micro-tumor model of head and neck tumors. The culture medium is prepared from antibacterial and antifungal agents, namely three antibodies, HEPES, GlutaMax, MEM non-essential amino acid, human recombinant protein EGF, bFGF, HGF, MSP, Wnt-3a, Noggin, R-spondin 1, IL-2, IL-15, calomel, CHIR99021, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, nicotine, an N2 additive, B27, ITS-X, Y-27632 and a basal culture medium. The head and neck tumor in-vitro 3D micro-tumor model constructed by the invention can accurately reflect the original focus characteristics of a patient, and is a good scientific research experiment model and a preclinical experiment model in the field of precise tumor diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a culture medium and a method for constructing an in vitro 3D micro-tumor model of head and neck tumors. Background Art

[0002] Head and neck cancer is the sixth most common malignant tumor in the world, with approximately 890,000 new cases and nearly 450,000 deaths each year. The primary sites and pathological types of head and neck cancer are diverse, including three major categories: neck tumors, otolaryngology tumors, and oral and maxillofacial tumors. The most common neck tumors are thyroid tumors; the most common otolaryngology tumors are laryngeal cancer and paranasal sinus cancer; the most common oral and maxillofacial tumors are various oral cancers, such as tongue cancer, gingival cancer, cheek cancer, etc.

[0003] The important organs of the head and neck are relatively concentrated, the anatomy is relatively complex, and the blood supply and lymphatic drainage are very rich. Therefore, head and neck tumors are prone to invasion and metastasis, and the prognosis is poor. Early diagnosis and early treatment are recommended for head and neck tumors. However, the site of onset is relatively hidden, and patients are mostly in the middle and late stages when they seek medical treatment. Although there are a variety of comprehensive treatment methods such as surgery, radiotherapy, chemotherapy, targeted and immunotherapy, the 5-year overall survival rate of advanced patients is only 20%-35%. Therefore, how to formulate precise treatment plans based on individual differences in patients, prolong survival time and improve quality of life is a thorny problem faced by scholars at home and abroad, which has great scientific theoretical value and social practice significance.

[0004] To date, the translational application of in vitro tumor surrogate models is still limited, mainly due to the single composition of the cell model, the lack of simulation of the immune microenvironment, the species differences between animal models and humans, and the lack of consideration for personalized medical needs. In view of the current status of head and neck tumor diagnosis and treatment, it is urgent to develop and construct an in vitro 3D cell model with easy sample acquisition, good operation repeatability, high throughput, standardization, and full retention of the biological characteristics of the original tumor tissue, so as to promote preclinical effectiveness and safety evaluation, accelerate the development of new drugs, drive industrial development and benefit patients. Summary of the invention

[0005] The purpose of the present invention is to provide a culture medium and a method for constructing an in vitro 3D micro-tumor model of head and neck tumors.

[0006] In a first aspect, the present invention claims a culture medium for constructing an in vitro 3D micro-tumor model of head and neck tumors.

[0007] The culture medium for constructing an in vitro 3D micro-tumor model of head and neck tumors claimed in the present invention comprises three antibacterial and antifungal agents, HEPES, GlutaMax, MEM non-essential amino acid solution, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein MSP, human recombinant protein Wnt-3a, human recombinant protein Noggin, human recombinant protein R-spondin1, human recombinant protein IL-2, human recombinant protein IL-15, Forskolin (an adenylate cyclase agonist), CHIR99021, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, Nicotinamide, N2 supplement, B27, ITS-X (Insulin, Transferrin, Selenium, Ethanolamine Solution), Y-27632 and basal culture medium; wherein the three antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B.

[0008] In the culture medium, the final concentration of HEPES is 8-12 mM (e.g., 10 mM); the final concentration of GlutaMax is 0.8-1.2% (e.g., 1%) by volume; the final concentration of the MEM non-essential amino acid solution is 0.8-1.2% (e.g., 1%) by volume; the final concentration of the human recombinant protein EGF is 10-100 ng / mL (e.g., 50 ng / mL); the final concentration of the human recombinant protein bFGF is 10-50 ng / mL (e.g., 20 ng / mL); g / mL); the final concentration of the human recombinant protein HGF is 5-25ng / mL (such as 20ng / mL); the final concentration of the human recombinant protein MSP is 5-25ng / mL (such as 20ng / mL); the final concentration of the human recombinant protein Wnt-3a is 200-300ng / mL (such as 200ng / mL); the final concentration of the human recombinant protein Noggin is 100-200ng / mL (such as 100ng / mL); the final concentration of the human recombinant protein R-spondin The final concentration of 1 is 250-500 ng / mL (such as 400 ng / mL); the final concentration of the human recombinant protein IL-2 is 10-100 ng / mL (such as 20 ng / mL); the final concentration of the human recombinant protein IL-15 is 10-100 ng / mL (such as 10 ng / mL); the final concentration of forskolin is 2-10 μM (such as 5 μM); the final concentration of CHIR99021 is 1.5-6 μM (such as 2 μM); the concentration of SB202190 is 5-10 μM (such as 5 μM); the concentration of A83-01 is 0.2 5-1.25μM (such as 1μM); the concentration of the Primocin is 0.5mg / ml; the concentration of the N-acetyl-L-cysteine ​​is 0.5-2mM (such as 2mM); the concentration of the nicotine is 5-10mM (such as 10mM); the final concentration of the N2 additive is 1% by volume; the final concentration of the B27 is 1.5-2.5% (such as 2%) by volume; the final concentration of the ITS-X is 0.8-1.2% (such as 1%) by volume; the final concentration of the Y-27632 is 5-20μM (such as 7.5μM).

[0009] Furthermore, the solvent of the MEM non-essential amino acid solution is water, and the solutes and concentrations are as follows: glycine 10mM; L-alanine 10mM; L-asparagine 10mM; L-aspartic acid 10mM; L-glutamate 10mM; L-proline 10mM; L-serine 10mM. The GlutaMax is an advanced cell culture additive that can directly replace L-glutamine in cell culture medium. The GlutaMax is "GlutaMAX™ Supplement" (such as Gibco#35050061, or other products with the same composition). The ingredient of the "GlutaMAX™ Supplement" is L-alanyl-L-glutamine, which is a substitute for L-glutamine, with a concentration of 200nM, and the solvent is 0.85% NaCl solution. The A83-01 is "3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide" (such as Tocris#2939, or other products with the same composition). The SB202190 is "4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole" (such as Sigma#S7067, or other products with the same composition). The B27 is "B-27™ Supplement (50X), minus vitamin A" (such as Gibco#12587010, or other products with the same composition).The “B-27™ Supplement (50X), minus vitamin A” contains biotin, DL-α-tocopherol acetate, DL-α-tocopherol, BSA (fatty acidfree fraction V), catalase, human recombinant insulin, human transferrin, superoxide dismutase, corticosterone, D-galactose, ethanolamine hydrochloride, glutathione (reduced), L-carnitine hydrochloride, linoleic acid, linolenic acid, progesterone, putrescine2HCl, sodium selenite, triiodothyronine (T3 (triodo-I-thyronine)). The solvent of the ITS-X is EBSS solution (Earle's balanced salt solution), and the solutes and concentrations are as follows: insulin 1g / L; transferrin 0.55g / L; sodium selenite 0.00067g / L; ethanolamine 0.2g / L. 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 other products with the same composition). The Primocin 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, and has a killing effect on Gram-positive bacteria, Gram-negative bacteria, mycoplasma and fungi. The N-2 additive is "N-2 Supplement (100X)" (such as Gibco #17502001, or other products with the same composition).The "N-2 Supplement (100X)" contains 1mM human transferrin (Holo) at a final concentration, 500mg / L recombinant insulin full chain, 0.63mg / L progesterone, 10mM putrescine, and 0.52mg / L selenite. The CHIR99021 is a highly specific glycogen synthase kinase-3 (GSK-3) inhibitor that inhibits GSK-3α and GSK-3β, with IC50 values ​​of 10nM and 6.7nM, respectively.

[0010] Furthermore, the basic culture medium may be Advanced DMEM / F12 culture medium.

[0011] In the culture medium, the final concentration of penicillin in the antibacterial and antifungal agents is 100-200 U / mL (such as 100 U / mL), the final concentration of streptomycin is 100-200 μg / mL (such as 100 μg / mL), and the final concentration of amphotericin B is 200-250 ng / mL (such as 250 ng / mL).

[0012] Further, the antibacterial and antifungal triple antibiotic is "Antibiotic-Antimycotic, 100X" (such as Gibco #15240062, or other products with the same composition). The "Antibiotic-Antimycotic, 100X" contains 10,000 units of penicillin (base), 10,000 μg of streptomycin (base) and 25 μg of amphotericin B per milliliter, and uses penicillin G (sodium salt), streptomycin sulfate and amphotericin B in the form of 0.85% saline solution as antifungal agents.

[0013] Furthermore, the culture medium may exist in two forms: First, the culture medium is a solution formed by mixing the antibacterial and antifungal agents, the triple antibody, the HEPES, the GlutaMax, the MEM non-essential amino acid solution, the human recombinant protein EGF, the human recombinant protein bFGF, the human recombinant protein HGF, the human recombinant protein MSP, the human recombinant protein Wnt-3a, the human recombinant protein Noggin, the human recombinant protein R-spondin 1, the human recombinant protein IL-2, the human recombinant protein IL-15, the forskolin, the CHIR99021, the SB202190, the A83-01, the Primocin, the N-acetyl-L-cysteine, the nicotine, the N2 additive, the B27, the ITS-X, the Y-27632 and the basal culture medium.

[0014] After the culture medium is prepared, it needs to be filtered and sterilized using a 0.22 μM syringe filter (Millipore SLGP033RS) and can be stored at 4° C. for two weeks.

[0015] Secondly, each component in the culture medium exists independently and is prepared according to a formula when used.

[0016] Furthermore, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein MSP, human recombinant protein Wnt-3a, human recombinant protein Noggin, human recombinant protein R-spondin 1, human recombinant protein IL-2, and human recombinant protein IL-15 can exist in the form of a stock solution (mother solution), specifically a 1000-fold stock solution (mother solution). SB202190, Forskolin, N-acetyl-L-cysteine, Nicotinamide, and Y-27632 can exist in the form of a stock solution (mother solution), specifically a 1000-fold stock solution (mother solution). A83-01 can exist in the form of a stock solution (mother solution) (can be stored for a long time at -20°C), specifically a 100,000-fold stock solution (mother solution). CHIR99021 can exist in the form of a stock solution (mother solution) (can be stored for a long time at -20°C), specifically a 10,000-fold stock solution (mother solution).

[0017] The 1000× human recombinant protein EGF stock solution is composed of human recombinant protein EGF, BSA and PBS, wherein the final concentration of the human recombinant protein EGF is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0018] The 1000× human recombinant protein bFGF stock solution is composed of human recombinant protein bFGF, BSA and PBS, wherein the final concentration of the human recombinant protein bFGF is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0019] The 1000× human recombinant protein HGF stock solution consists of human recombinant protein HGF, BSA and PBS, wherein the final concentration of the human recombinant protein HGF is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

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

[0021] The 1000× human recombinant protein Wnt-3a stock solution consists of human recombinant protein Wnt-3a, BSA and PBS, wherein the final concentration of the human recombinant protein Wnt-3a is 200 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0022] The 1000× human recombinant protein Noggin stock solution consists of human recombinant protein Noggin, BSA and PBS, wherein the final concentration of the human recombinant protein Noggin is 100 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0023] The 1000× human recombinant protein R-spondin 1 stock solution consists of human recombinant protein R-spondin 1, BSA and PBS, wherein the final concentration of the human recombinant protein R-spondin 1 is 250 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0024] The 1000× human recombinant protein IL-2 stock solution consists of human recombinant protein IL-2, BSA and PBS, wherein the final concentration of the human recombinant protein IL-2 is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0025] The 1000× human recombinant protein IL-15 stock solution consists of human recombinant protein IL-15, BSA and PBS, wherein the final concentration of the human recombinant protein IL-15 is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

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

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

[0028] The 1000×SB202190 stock solution consists of SB202190 and DMSO, wherein the final concentration of the SB202190 is 10 mM and the remainder is DMSO.

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

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

[0031] 1000×Y-27632 is composed of Y-27632 and ultrapure water, wherein the final concentration of Y-27632 is 10 mM, and the remainder is ultrapure water.

[0032] The 10000×CHIR99021 stock solution consists of CHIR99021 and DMSO, wherein the concentration of CHIR99021 is 30 mM and the remainder is DMSO.

[0033] The 100000×A83-01 stock solution consists of A83-01 and DMSO, wherein the concentration of A83-01 is 25 mM and the remainder is DMSO.

[0034] In a second aspect, the present invention claims a set of reagents for constructing an in vitro 3D micro-tumor model of head and neck tumors.

[0035] The set of reagents claimed in the present invention for constructing an in vitro 3D micro-tumor model of head and neck tumors is composed of the culture medium described in the first aspect above and all or part of the following: sample dissociation solution, sample preservation solution, sample cleaning solution, cell digestion solution, digestion termination solution and cell freezing solution.

[0036] Furthermore, the sample dissociation solution consists of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of collagenase I is 150-250U / mL (such as 200U / mL); the final concentration of collagenase II is 150-250U / mL (such as 200U / mL); the final concentration of collagenase IV is 150-250U / mL (such as 200U / mL); and the remainder is PBS.

[0037] The unit U of collagenase (the collagenase I or the collagenase II or the collagenase IV) is defined by the enzymatic activity of the protease: at 37°C and pH 7.5, 1 U of protease is used to treat the collagenase (the collagenase I or the collagenase II or the collagenase IV) for 5 hours to release 1 μmol of L-leucine.

[0038] Furthermore, the sample preservation solution is composed of fetal bovine serum, three antibacterial and antifungal agents, HEPES and HBSS; wherein the three antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B; in the sample preservation solution, the final concentration of the fetal bovine serum is 1-5% (such as 2%) by volume; the final concentration of penicillin in the three antibacterial and antifungal agents is 100-200U / mL (such as 100U / mL), the final concentration of streptomycin is 100-200μg / mL (such as 100μg / mL), and the final concentration of amphotericin B is 200-250ng / mL (such as 250ng / mL); the final concentration of HEPES is 8-12mM (such as 10mM); and the remainder is HBSS.

[0039] Furthermore, the sample cleaning solution is composed of three antibacterial and antifungal agents and PBS; wherein the three antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B; in the sample cleaning solution, the final concentration of penicillin in the three antibacterial and antifungal agents is 100-200U / mL (such as 100U / mL), the final concentration of streptomycin is 100-200μg / mL (such as 100μg / mL), and the final concentration of amphotericin B is 200-250ng / mL (such as 250ng / mL); the remainder is PBS.

[0040] Furthermore, the cell digestion solution has the following composition: every 10 mL of the cell digestion solution contains 4-6 mL (eg, 5 mL) Accutase, EDTA with a final concentration of 5 mM, 1.5-2.5 mL (eg, 2 mL) TrypLE Express, and the remainder is PBS.

[0041] Wherein, the Accutase is "StemPro™ Accutase™ Cell Dissociation Reagent" (such as Gibco#A11105-01, or other products with the same composition). The Accutase is a single-component enzyme that is dissolved in D-PBS, 0.5mM EDTA solution. The TrypLE Express is "TrypLE™ Express Enzyme (1X), no phenol red" (such as Gibco#12604013, or other products with the same composition). The "TrypLE™ Express Enzyme (1X), no phenol red" contains 200mg / L KCl, 200mg / L KH2PO4, 8000mg / L NaCl, 2160mg / L Na2HPO4·7H2O, 457.6mg / L EDTA; and also contains recombinant protease.

[0042] Furthermore, the digestion stop solution is composed of fetal bovine serum, three antifungal antibodies and DMEM culture medium; wherein the three antibacterial and antifungal antibodies are penicillin, streptomycin and amphotericin B; in the digestion stop solution, the final concentration of the fetal bovine serum is 8-12% (such as 10%) by volume; the final concentration of penicillin in the three antibacterial and antifungal antibodies is 100-200U / mL (such as 100U / mL), the final concentration of streptomycin is 100-200μg / mL (such as 100μg / mL), and the final concentration of amphotericin B is 200-250ng / mL (such as 250ng / mL); the remainder is DMEM culture medium.

[0043] Furthermore, the cell freezing solution consists of Advanced DMEM / F12 culture medium, DMSO and 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM / F12 culture medium, the DMSO and the 1% methylcellulose solution is 20:2:(0.8-1.2), such as 20:2:1; and the 1% methylcellulose solution is a methylcellulose aqueous solution with a concentration of 1 g / 100 ml.

[0044] In a third aspect, the present invention claims the use of the culture medium described in the first aspect or the set of reagents described in the second aspect in constructing an in vitro 3D micro-tumor model of head and neck tumors.

[0045] The head and neck tumor can be a neck tumor, an otolaryngology tumor or an oral and maxillofacial tumor, which can be pathologically divided into squamous cell carcinoma and adenocarcinoma. In the embodiment of the present invention, the head and neck tumor is laryngeal cancer, nasopharyngeal cancer or thyroid cancer.

[0046] In a fourth aspect, the present invention claims a method for constructing an in vitro 3D micro-tumor model of head and neck tumors.

[0047] The method for constructing an in vitro 3D micro-tumor model of head and neck tumors claimed in the present invention may include the following steps: (a1) dissociating head and neck solid tumor tissue using the sample dissociation solution described in the second aspect above; (a2) Using the culture medium described in the first aspect above to suspend and culture the single cells dissociated in step (a1) to form cell clusters, thereby obtaining an in vitro 3D micro-tumor model of head and neck tumors.

[0048] Furthermore, in step (a1), the head and neck tumor solid tumor tissue can be dissociated with the sample dissociation solution according to a method comprising the following steps: the minced head and neck tumor solid tumor tissue is dissociated with the sample dissociation solution at 37° C., with 1 mL of the sample dissociation solution being no more than 0.5 mg of tissue, and the dissociation time is 15 minutes to 2 hours (e.g., 1 hour).

[0049] Furthermore, in step (a2), the cells dissociated from (a1) can be suspended and cultured using the culture medium according to a method comprising the following steps: using a cell culture container with a low adsorption surface, suspending and culture the cells dissociated from (a1) using the culture medium, and culturing the cells under conditions of 37° C. and 5% CO2.

[0050] The initial seeding density can be 10 5 Pieces / cm 2 The bottom area of ​​the container, taking a six-well plate as an example, is 10 per well. 6 The cells were plated at a density of 10 cells.

[0051] Furthermore, the culturing time in step (a2) is 3-5 days.

[0052] Furthermore, before step (a1), the following step of pre-dissociation treatment of the head and neck tumor solid tumor tissue may be included: washing the surface of the head and neck tumor solid tumor tissue sample with 70-75% (e.g., 75%) ethanol by volume for 10-30 seconds; washing the head and neck tumor solid tumor tissue sample with the sample washing solution described in the second aspect above for 5-10 times (e.g., 5 times), and washing the head and neck tumor solid tumor tissue sample with a sterile PBS solution for 5-10 times (e.g., 5 times); and then removing impurities, connective tissue, adipose tissue, necrotic tissue and other components that affect micro-tumor culture in the head and neck tumor solid tumor tissue sample.

[0053] The step of pre-dissociation treatment of the head and neck tumor solid tumor tissue needs to be performed on ice, and the entire operation step needs to be completed within 10 minutes.

[0054] Furthermore, in step (a1), after the head and neck tumor solid tumor tissue is dissociated with the sample dissociation solution, the following steps are also included: terminating the dissociation reaction with 8-15 times (such as 10 times) the volume of the digestion stop solution described in the second aspect of the above text, and collecting the cell suspension; filtering the cell suspension with a 100μm or 40μm sterile cell filter to remove tissue debris and adherent cells; centrifuging at 800-1000g (such as 800g) at room temperature for 10-15 minutes (such as 10 minutes), and discarding the supernatant; then resuspending the cells with 3-5mL (such as 5mL) of sterile PBS; centrifuging again at 800-1000g (such as 800g) at room temperature for 10-15 minutes (such as 10 minutes), and discarding the supernatant; and then resuspending the cell pellet with the culture medium described in the first aspect of the above text.

[0055] The head and neck solid tumor tissue sample subjected to the pre-dissociation treatment is isolated from the body within 12 hours, and is kept in the sample preservation solution described in the second aspect before the pre-dissociation treatment.

[0056] The head and neck tumor can be a neck tumor, an otolaryngology tumor or an oral and maxillofacial tumor, which can be pathologically divided into squamous cell carcinoma and adenocarcinoma. In the embodiment of the present invention, the head and neck tumor is laryngeal cancer, nasopharyngeal cancer or thyroid cancer.

[0057] In a fifth aspect, the present invention claims protection for an in vitro 3D micro-tumor model of head and neck tumors obtained using the method described in the fourth aspect above.

[0058] In a sixth aspect, the present invention claims a method for obtaining primary cells of head and neck solid tumors.

[0059] The method for obtaining primary solid tumor cells of head and neck tumors claimed in the present invention is to separate primary solid tumor cells of head and neck tumors from an in vitro 3D micro-tumor model of head and neck tumors obtained by the method described in the fourth aspect above.

[0060] The method may specifically include the following steps: (b1) digesting the in vitro 3D micro-tumor model of head and neck tumor using a cell digestion solution (such as the cell digestion solution described above) to obtain single cells; Furthermore, the method further comprises the step of terminating the digestion, such as using the digestion terminating solution as described above to terminate the digestion.

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

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

[0063] In the above aspects, the head and neck tumor can specifically be a primary head and neck tumor or a secondary head and neck tumor metastasis lesion of various pathological types, and the surgical specimen weight is more than 20 mg.

[0064] In the present invention, all of the above PBS can be 1×PBS, pH 7.3-7.5, and its specific composition is as follows: the solvent is water, and the solute and concentration are: KH2PO4 144 mg / L, NaCl 9000 mg / L, Na2HPO4·7H2O 795 mg / L.

[0065] The present invention provides a method and supporting reagents for extracting and culturing an in vitro 3D micro-tumor model of head and neck tumors from fresh head and neck tumor solid tumor tissues. The method has the following advantages: 1. Small amount of tissue sample is required, only about 20mg of head and neck tumor surgical sample is needed; 2. The cultivation cycle is short, and 10 can be obtained in just 2-3 days. 4 -10 6 An in vitro 3D micro-tumor model of head and neck tumors of various magnitudes; 3. High culture stability. The success rate of in vitro culture of qualified head and neck tumor surgical specimens using this method is as high as 90%; 4. Rich in cell types, the in vitro 3D micro-tumor model of head and neck tumors can preserve multiple cell types such as tumor cells, stromal cells and immune cells in the original lesions, and reproduce the tumor microenvironment well; 5. The in vitro 3D micro-tumor model of head and neck tumors can accurately reproduce the pathological subtypes of the original lesions; 6. The in vitro 3D micro-tumor model of head and neck tumors can accurately reproduce the genetic background of the original lesion.

[0066] The in vitro 3D micro-tumor model of head and neck tumors obtained by the method of the present invention can accurately reflect the various characteristics of the original lesions of patients, and is a good scientific research experimental model and preclinical experimental model in the field of tumor precision diagnosis and treatment. It can be foreseen that this culture method has broad application prospects in the research and clinical diagnosis and treatment of head and neck tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Bright field images of the in vitro 3D micro-tumor model formation process of head and neck tumors (0-48h). The scale bar is 200μm, 100 times magnification.

[0068] Figure 2 This is a comparison of the HE staining effects of the in vitro 3D micro-tumor model of head and neck tumors and its corresponding original lesion tissue sample. The scale is 50μm, 400 times magnification.

[0069] Figure 3This is a comparison of the multiple immunofluorescence staining effects of the in vitro 3D micro-tumor model of head and neck tumors. The scale is 50μm, 400 times magnification.

[0070] Figure 4 This is a bright field image of primary tumor cells of head and neck tumors isolated and purified from an in vitro 3D microtumor model of head and neck tumors.

[0071] Figure 5 Comparison results of copy number variation analysis (CNV) of in vitro 3D micro-tumor models of head and neck tumors and their corresponding original lesion tissue samples obtained by culturing in three culture media of Schemes A, B, and C. T represents original lesion tissue sample; PTC represents in vitro 3D micro-tumor model of head and neck tumors. DETAILED DESCRIPTION

[0072] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0073] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0074] Example 1: Preparation of reagents for constructing an in vitro 3D micro-tumor model of head and neck tumors 1. Sample preservation solution The specific formula of the sample preservation solution (100 mL) is shown in Table 1.

[0075]

[0076] After the sample preservation solution is prepared, it is divided into 15mL centrifuge tubes, 5mL per tube. After division, it can be stored at 4℃ for 1 month.

[0077] 2. Sample cleaning solution The specific formula of the sample cleaning solution (100 mL) is shown in Table 2.

[0078]

[0079] The sample cleaning solution must be prepared and used immediately.

[0080] 3. Sample dissociation solution The specific formula of the sample dissociation solution (10 mL) is shown in Table 3.

[0081]

[0082] Note: The sample dissociation solution should be prepared and used immediately.

[0083] In Table 3, the preparations of the three collagenase stock solutions are shown in Tables 4 to 6.

[0084]

[0085] After the 10× collagenase I stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored at -20°C for a long time.

[0086]

[0087] After the 10× collagenase II stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored at -20°C for a long time.

[0088]

[0089] After the 10× collagenase IV stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored at -20°C for a long time.

[0090] In Tables 4, 5 and 6, the unit U of collagenase (the collagenase I or the collagenase II or the collagenase IV) is defined by the enzymatic activity of the protease: at 37° C., pH 7.5, 1 U of protease is used to treat the collagenase (the collagenase I or the collagenase II or the collagenase IV) for 5 hours to release 1 μmol of L-leucine.

[0091] 4. Cell digestion fluid The specific formula of the cell digestion solution (10 mL) is shown in Table 7.

[0092]

[0093] The cell digestion solution is prepared and used immediately.

[0094] 5. Digestion stop solution The specific formula of the digestion stop solution (100 mL) is shown in Table 8.

[0095]

[0096] After the digestion stop solution is prepared, it can be stored at 4°C for one month.

[0097] 6. In vitro 3D micro-tumor model culture medium for head and neck tumors The specific formula of the head and neck tumor in vitro 3D micro-tumor model culture medium (100 mL) is shown in Table 9.

[0098]

[0099] After the preparation of the culture medium for the in vitro 3D micro-tumor model of head and neck tumors is completed, it is sterilized by filtration using a 0.22 μM syringe filter (Millipore SLGP033RS) and can be stored at 4°C for two weeks.

[0100] In Table 9, the preparations of 9 human recombinant protein stock solutions are shown in Tables 11 to 19 (the preparation of BSA stock solution is shown in Table 10), the preparation of Forskolin stock solution is shown in Table 20, the preparation of CHIR99021 stock solution is shown in Table 21, the preparation of SB202190 stock solution is shown in Table 22, the preparation of A83-01 stock solution is shown in Table 23, the preparation of N-acetyl-L-cysteine ​​stock solution is shown in Table 24, the preparation of Nicotinamide stock solution is shown in Table 25, and the preparation of Y-27632 stock solution is shown in Table 26.

[0101]

[0102] 100× BSA solution is prepared and used immediately.

[0103]

[0104] After the 1000× human recombinant protein EGF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0105]

[0106] After the 1000× human recombinant protein bEGF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0107]

[0108] After the 1000× human recombinant protein HGF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0109]

[0110] After the 1000× human recombinant protein MSP stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0111]

[0112] After the 1000× human recombinant protein Wnt-3a stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0113]

[0114] After the 1000× human recombinant protein Noggin stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0115]

[0116] After the 1000× human recombinant protein R-spondin 1 stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0117]

[0118] After the 1000× human recombinant protein IL-2 stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0119]

[0120] After the 1000× human recombinant protein IL-15 stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0121]

[0122] After the 1000× Forskolin stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0123]

[0124] After the 10000×CHIR99021 stock solution is prepared, it is packaged in 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0125]

[0126] After the 1000×SB202190 stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0127]

[0128] After the 100000×A83-01 stock solution is prepared, it is packaged in 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0129]

[0130] After the 1000× N-acetyl-L-cysteine ​​stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0131]

[0132] After the 1000× Nicotinamide stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0133]

[0134] After the 1000×Y-27632 stock solution is prepared, it is packaged in 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0135] 7. Cell Cryopreservation Solution The specific formula of the cell freezing solution is shown in Table 27.

[0136]

[0137] Cell freezing solution is prepared and used immediately.

[0138] In Table 27, the preparation of 1% methylcellulose solution is shown in Table 28.

[0139]

[0140] 1% methylcellulose solution can be stored at 4°C for a long time after preparation.

[0141] Example 2: Obtaining specimens after head and neck tumor surgery 1. Cooperate with tertiary hospitals to obtain samples through researcher-initiated clinical research, and the cooperation has passed formal medical ethics review.

[0142] 2. The attending physician selects patients for inclusion in the study according to the clinical indications specified in the medical guidelines, and selects appropriate specimens for in vitro culture based on the intraoperative clinical indications. The sample selection criteria are: head and neck tumors, and specimens weighing more than 20 mg.

[0143] 3. All included cases are uniformly replaced by experimental numbers. The naming principle of the experimental number is the eight-digit date of sample collection + the last four digits of the patient's hospitalization number. When the hospital provides samples, it also provides the patient's pathological staging, clinical diagnosis and other basic clinical information. The naming principle of the experimental number is the eight-digit date of sample collection + the last four digits of the patient's hospitalization number. For example, for samples provided on January 1, 2020, the patient's hospitalization number is T001537474, then the sample experimental number is 202001017474.

[0144] 4. After the tumor tissue is removed from the body during surgery, a sample collection specialist collects fresh specimens in the sterile environment of the operating room. Samples should be collected from areas with rich fresh blood vessels, avoiding areas with poor cell activity such as necrotic tissue, adipose tissue, and fibrotic tissue. The collected samples are placed in a sample preservation solution (see Example 1) pre-cooled at 4°C. The sample preservation tube containing the sample is temporarily stored on ice and transported to the laboratory for the next step within 12 hours. The temperature during transportation is controlled at 2-8°C.

[0145] Example 3: Pre-dissociation treatment of head and neck tumor tissue samples The following operations need to be performed on ice and the entire operation should be completed within 10 minutes.

[0146] The surgical instruments used in the following operations must be sterilized with high-temperature steam (120°C, 20 minutes) and dried before use.

[0147] 1. After weighing the sample, clean the sample surface with medical alcohol (75% by volume) for 10 to 30 seconds.

[0148] 2. Wash the sample 5 times with sample cleaning solution and 5 times with sterile PBS solution.

[0149] 3. Use ophthalmic scissors, ophthalmic tweezers, scalpel and other instruments to carefully peel off the fat tissue, connective tissue and necrotic tissue in the sample.

[0150] Example 4: Dissociation of head and neck tumor tissue samples The surgical instruments used in the following embodiments must be sterilized by high-temperature steam (120°C, 20 minutes) and dried before use.

[0151] 1. Use ophthalmic scissors to cut the tissue into 0.5mm pieces. 3 Small pieces around.

[0152] 2. Treat the tissue with sample dissociation solution (see Example 1). For tissues with a sample size not exceeding 0.5 mg, use 1 mL of sample dissociation solution. For tissues with a sample size exceeding 0.5 mg, add 0.1 mL of sample dissociation solution for every 0.1 mg increase in tissue weight. The sample dissociation solution treatment condition is 37°C, and the dissociation time is 1 hour. During the dissociation process, observe the dissociation of the sample under a microscope every 15 minutes until most cells are observed to fall off from the tissue.

[0153] 3. Use 10 times the volume of digestion stop solution (see Example 1) to terminate the dissociation reaction. Filter the cell suspension through a 100 μm sterile cell strainer to remove tissue debris and adherent cells, centrifuge at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0154] 4. Resuspend the cells in 5 mL of sterile PBS, centrifuge at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0155] 5. Resuspend the cell pellet in the head and neck tumor in vitro 3D micro-tumor model culture medium (see Table 9 in Example 1), count the cells, and determine the cell viability by trypan blue staining. If the viability of the isolated cells is greater than 70%, the cells can be inoculated and cultured.

[0156] Example 5: In vitro 3D micro-tumor model cultivation of head and neck tumors 1. Use a vessel with a low adsorption surface to perform suspension culture of an in vitro 3D micro-tumor model of head and neck tumors. The culture medium used is the in vitro 3D micro-tumor model culture medium of head and neck tumors in Table 9 of Example 1 (wherein the final concentration of the antibacterial and antifungal agent triple antibody is 1% by volume; the final concentration of the HEPES is 10 mM; the final concentration of the GlutaMax is 1% by volume; the final concentration of the MEM non-essential amino acid solution is 1% by volume; the final concentration of the human recombinant protein EGF is 50 ng / mL; the final concentration of the human recombinant protein bFGF is 20 ng / mL; the final concentration of the human recombinant protein HGF is 20 ng / mL; the final concentration of the human recombinant protein MSP is 20 ng / mL; the final concentration of the human recombinant protein Wnt-3a is 200 ng / mL; the final concentration of the human recombinant protein Noggin is 100 ng / mL; the final concentration of the human recombinant protein R-spondin The final concentration of 1 is 400ng / mL; the final concentration of the human recombinant protein IL-2 is 20ng / mL; the final concentration of the human recombinant protein IL-15 is 10ng / mL; the final concentration of forskolin is 5μM; the final concentration of CHIR99021 is 2μM; the concentration of SB202190 is 5μM; the concentration of A83-01 is 1μM; the final concentration of Primocin is 1% volume percentage (i.e. 0.5mg / ml); the concentration of N-acetyl-L-cysteine ​​is 2mM; the concentration of nicotine is 10mM; the final concentration of N2 additive is 1% volume percentage; the final concentration of B27 is 2% volume percentage; the final concentration of ITS-X is 1% volume percentage; the final concentration of Y-27632 is 7.5μM; the rest are Advanced DMEM / F12 culture medium), taking a six-well plate as an example, 10 per well 6 The cells were plated at a density of 100 cells / well, with 2-3 mL of culture medium per well. The inoculated cells were cultured in a cell culture incubator at 37°C and 5% CO2.

[0157] 2. Observe the cell status every day until the cells form clumps with a diameter of about 100-300 μm, and then replace the culture medium every 2-3 days to maintain the growth state of head and neck tumor microtumors.

[0158] like Figure 1 As shown in Figure 2, within the first 48 hours of culture, various cell types derived from head and neck tumor tissues spontaneously aggregated and self-assembled into cell clusters of 100-300 μm in size, which we call microtumor models. The total number of microtumor cell clusters can reach 10 4 -10 6 This method has been tested on a large number of samples, and the success rate of culturing micro-tumor models of surgical samples of different head and neck tumors (laryngeal cancer, nasopharyngeal carcinoma and thyroid cancer) can reach 90%.

[0159] Example 6. Comparative identification of in vitro 3D micro-tumor model of head and neck tumor and HE staining of the original lesion Description of reagents and consumables used in the following examples: Hematoxylin and eosin (HE) staining kit (Beijing Solebow Biotechnology Co., Ltd., #G1120); 1. Take fresh samples, fix them, embed them in paraffin wax and slice them into 3-8um slices; 2. Dewax in xylene twice, 5-10 min each time, rehydrate in a series of ethanol (100%, 95%, 85%, 75%), 3 min each gradient, and soak in distilled water for 2 min; 3. Dye with hematoxylin solution for 2-20 minutes, and wash away the floating color with distilled water; 4. Differentiate in differentiation solution for 10-60 seconds, add tap water or soak twice, each time for 3-5 minutes; 5. Place in eosin stain solution for 30s-2min, pour off excess stain solution and dehydrate quickly; 6. Dehydration, transparency, and sealing: 75% ethanol, 85% ethanol, 95% ethanol, and 100% ethanol (I) were used for 2-3 seconds each, 100% ethanol (II) was used for 1 minute, xylene was used for transparency twice, each time for 1 minute, and neutral resin was used for sealing; 7. Observe the staining effect under the microscope and take photos.

[0160] Figure 2 The comparison chart of HE staining effect of the head and neck in vitro 3D tumor micro-tumor model and its corresponding original lesion tissue sample cultured in Example 5 is shown. Head and neck tumors are divided into squamous cell carcinoma and adenocarcinoma, and it can be seen that the pathological tissue structure of the original lesion is reproduced to the greatest extent.

[0161] Example 7: Multiple immunofluorescence staining of in vitro 3D microtumor model of head and neck tumors and identification of primary lesions The reagents used in the following examples are described: Paraformaldehyde (Beijing Chemical Reagent Company, analytical grade) was prepared by dissolving paraformaldehyde powder in ultrapure water to prepare a 4% (4 g / 100 mL) paraformaldehyde solution; Methanol and dimethyl sulfoxide (Beijing Chemical Reagent Company, analytical grade); Hydrogen peroxide (Beijing Chemical Reagent Company, 35%); Methanol, dimethyl sulfoxide, and 35% hydrogen peroxide were mixed in a ratio of 4:4:1 (volume ratio) to prepare Dan's rinse solution; Bovine serum albumin (Sigma, #A1933) was dissolved in PBS to prepare a 3% (3 g / 100 mL) BSA solution; Primary antibodies for immunofluorescence (anti-PanCK antibody: Abcam#ab215838; anti-Vim antibody: abclonal#A19607; anti-CD3 antibody: Biolegend#300434); Secondary antibodies for immunofluorescence (CST#4408s, CST#8889s); Hoechst stain (Beijing Solebow Biotechnology Co., Ltd., #C0021); Using the paraffin sections obtained in Example 6 as materials, multiple immunofluorescence staining was performed according to the following steps. The three antibodies respectively labeled pan-CK to characterize epithelial tumor cells, Vimentin to label stromal cells, and CD3 to label T cells: 1. After dewaxing and rehydration of paraffin sections, wash them with PBS and treat them with pre-cooled methanol solution for 1 hour.

[0162] 2. Treat with Dan's rinse solution at room temperature for 2 hours, and then treat with 75%, 50%, and 25% (volume percentage) methanol solution diluted with PBS in sequence, each time for 10 minutes.

[0163] 3. Block with 3% BSA solution at room temperature for 2 hours.

[0164] 4. Incubate the primary antibody in mixed dilution solution (3% BSA solution) at 4°C overnight.

[0165] 5. Wash the sections with PBS solution 5 times, 20 minutes each time.

[0166] 6. Add secondary antibody mixed dilution (diluted with 3% BSA solution at a ratio of 1:2000) and incubate the secondary antibody at room temperature for 2 hours.

[0167] 7. Wash the sections with PBS solution 5 times, 20 minutes each time.

[0168] 8. Hoechst staining solution, staining at room temperature for 20 minutes.

[0169] 9. Seal the slides with quenching sealing medium and use a laser confocal microscope to observe the staining and take pictures.

[0170] Figure 3The effect diagram of multiple immunofluorescence staining of the in vitro 3D micro-tumor model of head and neck tumors and their corresponding original lesion tissue samples is shown. It can be seen that the in vitro 3D micro-tumor model of head and neck tumors not only retains the panCK-positive (green fluorescence) tumor cells in the original lesions, but also preserves the Vimentin-positive stromal cells (white) and immune cells (T cells red), reproducing the cell diversity and microenvironment of the original lesions to the greatest extent. It confirms the high similarity in cell composition between the micro-tumor model cultured by this method and the original lesions. We performed multiple immunofluorescence staining identification on 10 in vitro 3D micro-tumor samples of head and neck tumors (laryngeal cancer, nasopharyngeal carcinoma and thyroid cancer) and their corresponding original lesion tissue blocks. The statistical results are shown in Table 29.

[0171]

[0172] Example 8: Isolation of primary head and neck tumor cells from an in vitro 3D micro-tumor model of head and neck tumors The cell magnetic bead sorting process mentioned in the following examples uses the Miltenyi CD326 magnetic bead positive selection kit (Miltenyi #130-061-101).

[0173] 1. Collect the in vitro 3D micro-tumor model of head and neck tumor cultured in suspension in Example 5, centrifuge at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0174] 2. Wash the cell pellet once with sterile PBS solution, centrifuge at 800g for 10 minutes at room temperature, and discard the supernatant.

[0175] 3. Reselect the cell pellet with cell digestion solution (see Example 1) and digest at 37°C for 5-30 minutes. During the digestion process, observe the digestion of cell clumps under a microscope every 5 minutes until most cell clumps are digested into single cells.

[0176] 4. Terminate the digestion with 10 times the volume of the digestion stop solution (see Example 1), centrifuge the cell suspension at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0177] 5. The single cell suspension obtained by digestion was sorted with Miltenyi CD326 magnetic bead positive selection kit to obtain CD326 positive tumor cells: resuspend the cell pellet with sorting buffer (formula is Table 30), and the cell amount should be less than 10 7 Add 20 μL CD326 magnetic beads to the system and incubate on ice for 30 minutes. Wash the cell pellet with 2 mL sorting buffer. Wash the sorting column with 5 mL sorting buffer and place the sorting column on the magnetic rack. Pass the cell suspension through the cell sorting column and rinse the sorting column three times with 3 mL sorting buffer to elute the CD326 negative cells. Remove the sorting column from the magnetic rack and rinse the sorting column once with 5 mL sorting buffer to elute the CD326 positive cells.

[0178]

[0179] Note: Prepare and use immediately.

[0180] 6. Centrifuge the sorted CD326 positive cells at 800g for 10 minutes at room temperature and discard the supernatant. 5 / 3.5cm cell culture dish for inoculation and adherence culture.

[0181] Example 9: Passaging of primary tumor cells of head and neck tumors 1. For the adherently cultured primary head and neck tumor cells, remove the culture medium and wash the cells with sterile PBS solution.

[0182] 2. Digest the cells with 0.05% trypsin at room temperature for 30-300 seconds, and observe the cell status during the process until most of the cells are digested into spherical shapes.

[0183] 3. Terminate the digestion reaction with 10 times the volume of DMEM medium containing 10% serum, collect the cell suspension and centrifuge at 800g for 10 minutes at room temperature, and discard the supernatant.

[0184] 4. Centrifuge at 800 g for 10 minutes at room temperature and discard the supernatant.

[0185] 5. Resuspend the cell pellet in DMEM medium containing 10% serum and count the cells. 5 The cells were inoculated and cultured at a density of 1 / 3.5 cm cell culture dish (using the culture medium in Table 9 plus 10% FBS).

[0186] After the purification step of Example 8 and the above-mentioned subculture, high-purity primary tumor cells of head and neck tumors can be obtained ( Figure 4 ).

[0187] Example 10. Cryopreservation of primary head and neck tumor cells The primary head and neck tumor cells purified and cultured in Example 8 can be cryopreserved after 2-3 passages and amplification: 1. For the adherently cultured primary head and neck tumor cells, remove the culture medium and wash the cells with sterile PBS solution.

[0188] 2. Digest the cells with 0.05% trypsin at room temperature for 30-300 seconds, and observe the cell status during the process until most of the cells are digested into spherical shapes.

[0189] 3. Terminate the digestion reaction with 10 times the volume of DMEM medium containing 10% serum, collect the cell suspension and centrifuge at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0190] 4. Centrifuge at 800 g for 10 minutes at room temperature and discard the supernatant.

[0191] 5. Use cell freezing solution (see Example 1) at 10 6 Resuspend the cell pellet at a density of 1 mL / mL and add 1 mL of cell suspension to each 2 mL cryovial. Cryopreservate in a gradient cooling box overnight and then transfer to liquid nitrogen for long-term storage.

[0192] Example 11: Recovery of primary head and neck tumor cells Primary tumor cells from head and neck tumors stored in liquid nitrogen can be resuscitated: 1. Prepare 37℃ sterile water five minutes in advance.

[0193] 2. Take out the cryovial from liquid nitrogen and thaw the cells quickly in sterile water at 37°C.

[0194] 3. Transfer the cells in the cryopreservation tube to a 15 ml centrifuge tube, add ten times the volume of DMEM medium containing 10% serum and mix thoroughly, centrifuge at 800g for 10 minutes at room temperature, and discard the supernatant.

[0195] 4. Resuspend the cell pellet in DMEM medium containing 10% serum and count the cells. 5 / 3.5cm cell culture dish for inoculation and adherence culture.

[0196] Example 12 Comparison of the ability of different culture media to form 3D micro-tumor structures in vitro for head and neck tumors The operation method and process of culturing the micro-tumor model of all samples in this embodiment are completely consistent (refer to the above-mentioned Example 5), and only the culture medium formula is different. The various culture media tested are shown in Table 31. Among them, Scheme A is the formula used in the present invention, and the specific ones are shown in Table 9 (the specific formula in Example 5).

[0197]

[0198] Twenty head and neck tumor tissue samples (6 cases of laryngeal cancer, all of which were squamous cell carcinomas; 10 cases of nasopharyngeal carcinomas, all of which were squamous cell carcinomas; and 4 cases of thyroid cancers, all of which were adenocarcinomas) were treated with each of the five culture medium schemes. Sample processing and PTC culture operations were performed according to the methods described in Examples 3, 4, and 5. After 3 days of culture, the formation of cell cluster structures (i.e., microtumor models) in the culture system was observed as shown in Table 32.

[0199]

[0200] It can be seen that the three culture media of Scheme A, B, and C can all be used to culture the in vitro 3D micro-tumor model of head and neck tumors, while the culture media of Scheme D and E are not suitable for the formation of in vitro 3D micro-tumor models of head and neck tumors. The in vitro 3D micro-tumor model of head and neck tumors formed in the culture medium of Scheme A is larger in volume, which is slightly better than the culture media of Schemes B and C.

[0201] Example 13. Comparison of genetic backgrounds of in vitro 3D micro-tumor models of head and neck tumors formed in different culture media The DNA extraction process mentioned in the following examples was performed using the Tiangen Blood / Tissue / Cell Genomic Extraction Kit (DP304).

[0202] The library construction process mentioned in the following examples was performed using the NEB DNA sequencing library construction kit (E7645).

[0203] The high-throughput sequencing mentioned in the following examples refers to the Illumina HiSeq X-ten sequencing platform.

[0204] The present invention obtains the in vitro 3D micro-tumor model of head and neck tumor and the primary lesion sample of head and neck tumor obtained by culturing the three culture media of Schemes A, B and C obtained in Example 12, and performs DNA extraction, whole genome sequencing and copy number variation analysis methods as follows: 1. Take 10 mg of the original lesion tissue of the head and neck tumor and culture it with the three culture media of Scheme A, B and C obtained in Example 12 to obtain an in vitro 3D micro-tumor model of the head and neck tumor of about 10 mg. 5 DNA was extracted from a number of cell clusters, and libraries were constructed and whole genome high-throughput sequenced (WGS) was performed with a sequencing depth of 30×.

[0205] 2. The sequencing results of the two groups of original lesions and in vitro 3D micro-tumor models of head and neck tumors were analyzed for copy number variation (CNV) respectively, and the copy number variation between the primary head and neck tumor tissue samples and the in vitro 3D micro-tumor models of head and neck tumors of each generation was compared, such as Figure 5 The results showed that the in vitro 3D micro-tumor models of head and neck tumors cultured in the three culture media A, B, and C can well retain the copy number variation characteristics of the primary lesions of head and neck tumors.

[0206] Example 14 Comparison of cell composition of in vitro 3D micro-tumor models of head and neck tumors formed in different culture media The present invention obtained the in vitro 3D micro-tumor model of head and neck tumor and the primary lesion sample of head and neck tumor cultured in the three culture media of Schemes A, B and C obtained in Example 12, and performed multiple immunofluorescence staining to identify their cell composition (the method is shown in Example 7). The results are shown in Table 33.

[0207]

[0208] It can be seen that culture media B and C can well preserve the tumor cells in the original lesions, and the in vitro 3D micro-tumor model of head and neck tumors formed by them is formed with tumor cells of higher purity. In addition to maintaining the activity of tumor cells in the original lesions, culture media A can also well preserve the fibroblasts and T cells in the tissue, and has greater advantages than culture media B and C in maintaining cell diversity.

[0209] Therefore, the head and neck tumor in vitro 3D micro-tumor model culture medium (Table 9) used in the present invention can protect the various cell types in the original lesion to the greatest extent, and promote them to spontaneously form a multi-cellular structure under suspension conditions, thereby constructing an in vitro micro-tumor model that is extremely close to the characteristics of the original lesion.

[0210] Example 15: Comparison of the success rate of culture in different sample dissociation solutions The operation method and process of primary culture of all samples in this embodiment are completely consistent (refer to the above description), and only the sample dissociation solution formula is different. The various sample dissociation solutions tested are shown in Table 34, among which Scheme D is the formula used in the present invention, see Table 34 for details.

[0211]

[0212] The sample dissociation solution is prepared and used immediately.

[0213] 10 head and neck solid tumor tissue samples with a weight of more than 100 mg were selected and divided into four equal parts. The four sample dissociation solutions were used respectively to perform sample processing and culture operations according to the methods described in Examples 3, 4, and 5. After 3 days of culture, the formation of cell cluster structures in the culture system was observed as shown in Tables 35 and 36:

[0214]

[0215] It can be seen that the sample dissociation solution formula has a certain influence on the formation ability of the in vitro 3D micro-tumor model of head and neck tumors and the size of the micro-tumor cell clusters. The sample dissociation solution (Table 3) used in the present invention can gently dissociate cells in the tissue, maintain cell activity to the greatest extent, and improve the formation efficiency and quality of the micro-tumor model.

[0216] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A culture medium for constructing an in vitro 3D micro-tumor model of head and neck tumors, characterized in that: The culture medium is composed of three antibiotics of antibacterial and antifungal agents, HEPES, GlutaMax, MEM non-essential amino acid solution, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein MSP, human recombinant protein Wnt-3a, human recombinant protein Noggin, human recombinant protein R-spondin 1, human recombinant protein IL-2, human recombinant protein IL-15, forskolin, CHIR99021, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, nicotine, N2 additive, B27, ITS-X, Y-27632 and basal culture medium; wherein the three antibiotics of antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B; In the culture medium, the final concentration of HEPES is 8-12 mM; the final concentration of GlutaMax is 0.8-1.2% by volume; the final concentration of the MEM non-essential amino acid solution is 0.8-1.2% by volume; the final concentration of the human recombinant protein EGF is 10-100 ng / mL; the final concentration of the human recombinant protein bFGF is 10-50 ng / mL; the final concentration of the human recombinant protein HGF is 5-25 ng / mL; the final concentration of the human recombinant protein MSP is 5-25 ng / mL; the final concentration of the human recombinant protein Wnt-3a is 200-300 ng / mL; the final concentration of the human recombinant protein Noggin is 100-200 ng / mL; the final concentration of the human recombinant protein R-spondin is 100-200 ng / mL. The final concentration of 1 is 250-500 ng / mL; the final concentration of the human recombinant protein IL-2 is 10-100 ng / mL; the final concentration of the human recombinant protein IL-15 is 10-100 ng / mL; the final concentration of forskolin is 2-10 μM; the final concentration of CHIR99021 is 1.5-6 μM; the concentration of SB202190 is 5-10 μM; the concentration of A83-01 is 0.25-1.25 μM; the concentration of Primocin is 0.5mg / ml; the concentration of N-acetyl-L-cysteine ​​is 0.5-2mM; the concentration of nicotine is 5-10mM; the final concentration of the N2 additive is 1% by volume; the final concentration of B27 is 1.5-2.5% by volume; the final concentration of ITS-X is 0.8-1.2% by volume; and the final concentration of Y-27632 is 5-20μM.

2. The culture medium according to claim 1, characterized in that: The basic culture medium is Advanced DMEM / F12 culture medium; and / or In the culture medium, the final concentration of penicillin in the antibacterial and antifungal agents is 100-200 U / mL, the final concentration of streptomycin is 100-200 μg / mL, and the final concentration of amphotericin B is 200-250 ng / mL.

3. A set of reagents for constructing an in vitro 3D micro-tumor model of head and neck tumors, comprising the culture medium of claim 1 and all or part of the following: Sample dissociation solution, sample preservation solution, sample cleaning solution, cell digestion solution, digestion termination solution and cell freezing solution.

4. The reagent set according to claim 3, characterized in that: The sample dissociation solution consists of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of collagenase I is 150-250 U / mL; the final concentration of collagenase II is 150-250 U / mL; the final concentration of collagenase IV is 150-250 U / mL; the remainder is PBS; and / or The sample preservation solution is composed of fetal bovine serum, three antibiotics of antibacterial and antifungal agents, HEPES and HBSS; wherein the three antibiotics of antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B; in the sample preservation solution, the final concentration of the fetal bovine serum is 1-5% by volume; the final concentration of penicillin in the three antibiotics of antibacterial and antifungal agents is 100-200U / mL, the final concentration of streptomycin is 100-200μg / mL, and the final concentration of amphotericin B is 200-250ng / mL; the final concentration of HEPES is 8-12mM; the remainder is HBSS; and / or The sample cleaning solution is composed of three antibiotics of antibacterial and antifungal agents and PBS; wherein the three antibiotics of antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B; in the sample cleaning solution, the final concentration of penicillin in the three antibiotics of antibacterial and antifungal agents is 100-200U / mL, the final concentration of streptomycin is 100-200μg / mL, and the final concentration of amphotericin B is 200-250ng / mL; the rest is PBS; and / or The cell digestion solution is composed of: 4-6 mL Accutase, EDTA with a final concentration of 5 mM, 1.5-2.5 mL TrypLE Express, and the balance is PBS per 10 mL of the cell digestion solution; and / or The digestion stop solution is composed of fetal bovine serum, three antifungal antibodies and DMEM culture medium; wherein the three antibacterial and antifungal antibodies are penicillin, streptomycin and amphotericin B; in the digestion stop solution, the final concentration of the fetal bovine serum is 8-12% by volume; the final concentration of penicillin in the three antibacterial and antifungal antibodies is 100-200 U / mL, the final concentration of streptomycin is 100-200 μg / mL, and the final concentration of amphotericin B is 200-250 ng / mL; the remainder is DMEM culture medium; and / or The cell freezing solution consists of Advanced DMEM / F12 culture medium, DMSO and 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM / F12 culture medium, the DMSO and the 1% methylcellulose solution is 20:2:(0.8-1.2); and the 1% methylcellulose solution is a methylcellulose aqueous solution with a concentration of 1g / 100ml.

5. Use of the culture medium according to claim 1 or 2 or the reagent set according to claim 3 or 4 in constructing an in vitro 3D micro-tumor model of head and neck tumors.

6. The use according to claim 5, characterized in that: The head and neck tumor is laryngeal cancer, nasopharyngeal cancer or thyroid cancer.

7. A method for constructing an in vitro 3D micro-tumor model of head and neck tumors, comprising the following steps: (a1) dissociating head and neck solid tumor tissue using the sample dissociation solution described in claim 3 or 4; (a2) Using the culture medium of claim 1 or 2 to suspend and culture the single cells dissociated in step (a1) to form cell clusters, thereby obtaining an in vitro 3D micro-tumor model of head and neck tumors.

8. The method according to claim 7, characterized in that: The head and neck tumor is laryngeal cancer, nasopharyngeal cancer or thyroid cancer.

9. An in vitro 3D micro-tumor model of head and neck tumor constructed by the method of claim 7 or 8.

10. A method for obtaining primary cells of head and neck solid tumors, characterized in that: The method is to separate primary solid tumor cells of head and neck tumor from an in vitro 3D micro-tumor model of head and neck tumor constructed by the method of claim 7 or 8.

Images