Head and neck malignant tumor organoid culture method and culture medium for reconstructing tumor microenvironment

Abstract

The invention relates to a head and neck malignant tumor organoid culture method and culture medium for reconstructing a tumor microenvironment, and the culture method comprises the following steps: adding a reconstructed collagen solution into an inner chamber, and solidifying to form a bottom gel layer; resuspending the treated malignant tumor tissues of the head and the neck by adopting a reconstructed collagen solution to obtain collagen gel containing the tumor tissues; adding collagen gel containing tumor tissues into the bottom gel layer, and solidifying to obtain an upper tissue gel layer; culturing the tumor organs; and after passage, obtaining the head and neck malignant tumor organoid system. According to the invention, matrix components such as tumor cells, tumor-related fibroblasts and the like and a plurality of comprehensive immune elements such as T lymphocytes and the like in the original head and neck malignant tumors can be retained, and the complex microenvironment of the head and neck malignant tumors can be effectively restored; a reliable and stable preclinical model is provided for deeply researching interaction among different components in a tumor microenvironment and realizing personalized precise treatment of clinical transformation of a patient with head and neck cancer.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method and culture medium for culturing head and neck malignant tumor organoids for reconstructing a tumor microenvironment. Background Art

[0002] Head and neck cancer is a highly invasive solid tumor, with about 350,000 deaths each year and a 5-year mortality rate of about 50%. 90% of them are squamous cell carcinoma, an immunosuppressive tumor and the sixth most common cancer in the world. For patients with advanced metastasis or recurrence, comprehensive treatment including surgery, chemotherapy and radiotherapy is currently used. Despite significant progress in these areas, the 5-year overall survival rate of patients with head and neck cancer is still around 50%.

[0003] Malignant tumors are complex structures composed of multiple types of cells, such as tumor cells, tumor stem cells, endothelial cells, pericytes, immune inflammatory cells, cancer-associated fibroblasts, stem cells and progenitor cells in the tumor stroma, which can create an evolving immunosuppressive microenvironment regulated by multiple cells, hormones and inflammatory responses.

[0004] In recent years, immunotherapy by inhibiting checkpoint molecules has become an important part of the successful treatment of head and neck tumors, bringing significant clinical benefits to some tumor patients. However, the number of patients suitable for this treatment is still very limited, mainly because patients may have innate resistance to immunotherapy. In addition, traditional preclinical models used for tumor research often cannot truly reflect the complex tumor immune microenvironment, let alone the interaction between tumor cells and other tumor-related immune components. Therefore, the in vitro experimental accuracy and clinical conversion rate of related immunotherapy drug sensitivity detection are low, especially for head and neck malignant tumors with low immunotherapy response rates. There is still no stable and reliable preclinical model for immunotherapy drug research and prediction.

[0005] Therefore, it is necessary to establish a new preclinical model that truly simulates the human head and neck tumor immune microenvironment for basic and clinical translational research on tumor immunity. Summary of the invention

[0006] Technical problems to be solved: In order to solve the technical problems existing in the prior art such as the inability to restore the microenvironment of head and neck malignant tumors and retain the immune factors of tumor homology, the present invention provides a head and neck malignant tumor organoid culture method and culture medium for reconstructing the tumor microenvironment, which can retain the matrix, T cells, fibroblasts and various comprehensive immune elements of the original head and neck malignant tumors, and can effectively restore the complex microenvironment of head and neck malignant tumors.

[0007] Technical solution: One of the purposes of the present invention is to provide a method for culturing head and neck malignant tumor organoids for reconstructing a tumor microenvironment. The culturing method is based on a gas-liquid interface double-disc culturing system, which comprises an outer chamber and an inner chamber disposed inside the outer chamber. The outer chamber is a sterile cell culture dish, and the inner chamber is an internal insert cell culture dish with a hydrophilic permeable membrane at the bottom. The inner chamber has a smaller inner diameter than the outer chamber. The specific steps of the method for culturing head and neck malignant tumor organoids for reconstructing a tumor microenvironment are as follows:

[0008] S1, adding the reconstructed collagen solution into the inner chamber, and forming a bottom gel layer without tumor tissue in the inner chamber of the air-liquid interface double-disc culture system after solidification;

[0009] S2, processing the obtained head and neck malignant tumor tissue, resuspending it with a reconstructed collagen solution, and obtaining a collagen gel containing the tumor tissue;

[0010] S3, adding collagen gel containing tumor tissue to the bottom gel layer, and obtaining an upper tissue gel layer after solidification;

[0011] S4. Cultivate tumor organoids by placing an air-liquid interface culture medium in the outer chamber. The liquid level of the culture medium should submerge the bottom gel layer and contact the lower interface of the upper tissue gel layer but should not submerge the upper tissue gel layer to form an air-liquid interface. Cultivate the cells under the conditions of 37°C, 5% carbon dioxide volume concentration, and replace the culture medium twice a week.

[0012] S5. After culturing for 14-30 days and passaging, a head and neck malignant tumor organoid system is obtained, wherein the head and neck malignant tumor organoid system comprises head and neck malignant tumor organoids, T cells and fibroblasts.

[0013] Preferably, the preparation method of the reconstructed collagen solution in step S1 is as follows: by mixing the collagen matrix, 10× concentrated sterile culture medium and sterile reconstitution buffer in a volume ratio of 8:1:1 on ice, first mixing the collagen matrix and 10× concentrated sterile culture medium and then adding the sterile reconstitution buffer and mixing again to prevent the generation of bubbles. It needs to be placed on ice before use to prevent the formation of gel.

[0014] Preferably, in step S1, after adding the reconstructed collagen solution into the inner chamber, the solution is placed at 37°C for 30 minutes to obtain a bottom gel layer after solidification; in step S3, after adding the collagen gel containing tumor tissue, the solution is placed at 37°C for 30 minutes to obtain an upper tissue gel layer; the volume ratio of the reconstructed collagen solution taken in step S1 and the reconstructed collagen solution resuspended in step S2 is 1:1.

[0015] Preferably, the source of the head and neck malignant tumor tissue in step S2 includes but is not limited to squamous epithelial-derived malignant tumors (squamous cell carcinoma) and glandular epithelial-derived malignant tumors (adenoid cystic carcinoma or ductal carcinoma), and the processing steps include cutting, soaking, shearing, and washing steps.

[0016] Preferably, the pretreatment step is as follows: fresh tumor specimens are cut and immersed in a sample collection solution, which is a DMEM medium (Nanjing KeyGen Biotech) containing 20X penicillin-streptomycin-amphotericin solution, washed twice with DPBS, and then minced on ice in an Advance DMEM / F12 medium containing Normocin. The minced tumor tissue should be less than 0.3 mm 3 , and then centrifuged at 1500 rpm, 4°C, and 5 min; then ADMEM / F12 containing Normocin was added for resuspending and washing, and centrifuged at 1500 rpm, 4°C, and 5 min again. The washing operation was repeated twice to obtain the treated head and neck malignant tumor tissue.

[0017] Preferably, in the internal insert cell culture dish whose bottom is a hydrophilic permeable membrane, the hydrophilic permeable membrane is a hydrophilic porous PTFE membrane with a pore size of 0.4 μm.

[0018] Preferably, the passaging in step S5 is specifically as follows: transfer the tissue gel layer in the inner chamber to a new sterile culture dish, cut the tissue gel layer into pieces, add dissociation solution to the sterile culture dish, transfer to a centrifuge tube, dissociate at 37°C for 30 minutes, shake once every 10 minutes, and then centrifuge at 1500rpm, 4°C, 5min, and discard the supernatant; add DMEM culture medium containing 10% serum, blow it thoroughly, centrifuge at 1500rpm, 4°C, 5min, and discard the supernatant, repeat 3 times, and then perform 1:2 passaging.

[0019] The dissociation solution is prepared with 200 units / ml of type IV collagenase and 10 ug / ml of DNase I in serum-free DMEM as solvent.

[0020] The second object of the present invention is to provide a head and neck malignant tumor organoid gas-liquid interface culture medium for the above-mentioned head and neck malignant tumor organoid culture method for reconstructing the tumor microenvironment, wherein the head and neck malignant tumor organoid gas-liquid interface culture medium is composed of: Advanced DMEM / F12 basal culture medium, Glutamax additive, HEPES, Pen-Strep Glutamine, nicotinamide, N-Acetylcysteine, B27, p38MAPK inhibitor SB202190, A8301, R-spondin1 recombinant protein, Noggin protein, EGF, Gastrin I, Wnt 3A, IL-2, wherein the concentration of Glutamax additive is 1×, the concentration of HEPES is 10 mmol / L, the concentration of N-acetylcysteine ​​is 1 mmol / L, the concentration of nicotinamide is 10 mmol / L, and the concentration of Pen-Strep The concentration of Glutamine was 1×, the concentration of B27 was 1×, the concentration of p38MAPK inhibitor SB202190 was 10umol / L, the concentration of A8301 was 0.5umol / L, the concentration of R-spondin1 recombinant protein was 0.05-0.25ug / ml, the concentration of recombinant Human Noggin protein was 0.05-0.2ug / ml, the concentration of EGF was 50ng / mL, the concentration of Gastrin I was 10nmol / L, the concentration of Human Wnt-3A was 0.05-0.2ug / ml, the concentration of IL-2 was 500IU / mL, and the Advanced DMEM / F12 basal medium was supplemented to the required volume of 50ml.

[0021] The third object of the present invention is to provide an application of head and neck malignant tumor organoids prepared based on the above-mentioned head and neck malignant tumor organoid culture method for reconstructing the tumor microenvironment in screening drugs for treating tumors.

[0022] The air-liquid interface double-disc culture system is a special tumor organoid culture model. Compared with the co-culture model, which is insufficient in evaluating the tumor microenvironment, in this system, tumor cells can grow normally to form organoids, retaining the pathological characteristics and genetic changes of the original tumor. The immune cells and fibroblast matrix of the tumor tissue can be maintained for a certain period of time, better simulating the growth environment of the tumor in vivo. This method provides an overall strategy for in vitro immune TME modeling, which can explore the complex crosstalk between multiple different cell populations.

[0023] Beneficial effects: (1) The method for culturing organoids of head and neck malignant tumors for reconstructing the tumor microenvironment of the present invention can retain the fibroblast matrix and various comprehensive immune elements of the original tumor, and can effectively restore the complex tumor microenvironment. As a drug screening platform for precision medicine of patients with head and neck malignant tumors, such as tumor-targeted drugs, it is more reliable than other in vitro models; it can also be used as an in vitro model platform for studying the interactions between various components in the tumor microenvironment.

[0024] (2) The method for culturing head and neck malignant tumor organoids for reconstructing the tumor microenvironment of the present invention can obtain organoids containing naturally homologous, autologous tumor-reactive tumor-infiltrating lymphocytes, which can directly simulate the in vivo tumor immune microenvironment without the need to add additional T cells for co-culture of organoids. In addition, immunofluorescence and flow cytometry are used to verify that the head and neck malignant tumor organoid system can maintain tumor-homologous T cells and other tumor-infiltrating lymphocytes in vitro for a long time, and can be subsequently used for personalized precision medicine as a drug screening platform for the sensitivity of immune checkpoint inhibitors.

[0025] (3) The method for culturing organoids of head and neck malignant tumors for reconstructing the tumor microenvironment of the present invention uses a simple mechanical method to break up the tumor instead of using an enzymatic method to digest the tumor tissue into single cells or break it into a homogenate, thereby retaining the activity of the tumor cells to the greatest extent. This allows the individual cases where it is difficult to construct organoids using the matrix gel method after clinical radiotherapy and chemotherapy to be able to culture a preclinical organoid drug sensitivity model in vitro.

[0026] (4) The method for culturing head and neck malignant tumor organoids for reconstructing the tumor microenvironment of the present invention uses recombinant collagen gel, wherein Cellmatrix Type IA can reconstruct a hard and highly consistent gel more quickly, so that the organoids are fixed in the gel before sinking to the bottom, making it easier to maintain three-dimensional growth; cells and organoids can grow and develop in the gel, and the culture results are highly reproducible; cells cultured using the gel can proliferate in a three-dimensional morphology similar to that in vivo, avoiding the instability of organoid culture caused by differences between different batches of Matrigel.

[0027] (5) The culture medium of the present invention selects a variety of cytokine components according to the culture and growth characteristics of maxillofacial malignant tumors and components in the tumor microenvironment, and compound them in a specific ratio so that the obtained culture medium contains appropriate amounts of cytokines and signal pathway regulatory factors. The various cytokines and regulatory factors directly and closely influence each other and cooperate with each other to increase the proportion of living cells, thereby accelerating the expansion of organoids and shortening the culture time, so that the organoids cultured by the gas-liquid interface method can maintain the same tissue morphology and characteristics as the original tumor; at the same time, commercial products are used to avoid the component errors caused by traditional tumor organoid culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the microscopic image of the primary culture of parotid ductal carcinoma organoids by air-liquid interface culture method on the 19th day (the scale bar in the figure is 50μm);

[0029] Figure 2 (a) HE staining image of the slices prepared from the primary parotid duct carcinoma organoids cultured by air-liquid interface method on the 28th day; Figure 2 (b) HE staining image of the second generation slice of parotid duct carcinoma organoid cultured by air-liquid interface method; Figure 2 (c) HE staining image of the third generation slice of parotid duct carcinoma organoid cultured by air-liquid interface method; Figure 2 (d) HE staining image of the patient's tumor sample section;

[0030] Figure 3 This is an immunofluorescence (IF) image of a slice prepared from a primary air-liquid interface parotid duct carcinoma organoid on day 28. The pixel is 50px. In the image, CK7 is a specific marker for ductal carcinoma (red), Vimentin is a specific marker for CAF cells (yellow), and CD3 is a specific marker for T cells (green). Figure 3 It can be seen that the IF identification results of the ductal carcinoma organoid system cultured in this example all contain T lymphocytes and CAFs;

[0031] Figure 4 Immunohistochemical images of protein expression (CK7, CK19, EMA) of parotid duct carcinoma organoid sections from primary to fourth generation by air-liquid interface method (the scale bar in the figure is 100 μm). From top to bottom, each row in the figure represents the first generation (primary day 28), second generation (48th day), third generation (68th day) and fourth generation (88th day), and from left to right, each column represents the expression of CK7, CK19 and EMA proteins;

[0032] Figure 5 Schematic diagram of the gas-liquid interface double-dish culture system. (a) is a schematic diagram of the structure of the gas-liquid interface double-dish culture system; (b) is a cross-sectional view of the gas-liquid interface double-dish culture system; (c) is a physical picture of the gas-liquid interface double-dish culture system:

[0033] The numbers in the figure represent the following: 1, inner chamber, 2, outer chamber, 3, air-liquid interface culture medium, 4, upper tissue gel layer, 5, bottom gel layer, 6, PTFE membrane. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] The embodiment of the present invention provides a method for culturing head and neck malignant tumor organoids for reconstructing a tumor microenvironment. The culturing method is based on a gas-liquid interface double-disc culturing system. The gas-liquid interface double-disc culturing system comprises an outer chamber and an inner chamber arranged inside the outer chamber. The outer chamber is a sterile cell culture dish, and the inner chamber is an internal insert cell culture dish with a hydrophilic permeable membrane at the bottom. The inner chamber has a smaller inner chamber diameter than the outer chamber. The method for culturing head and neck malignant tumor organoids for reconstructing a tumor microenvironment has the following specific steps:

[0036] S1, adding the reconstructed collagen solution into the inner chamber, and forming a bottom gel layer without tumor tissue in the inner chamber of the air-liquid interface double-disc culture system after solidification;

[0037] S2, processing the obtained head and neck malignant tumor tissue, resuspending it with a reconstructed collagen solution, and obtaining a collagen gel containing the tumor tissue;

[0038] S3, adding collagen gel containing tumor tissue to the bottom gel layer, and obtaining an upper tissue gel layer after solidification;

[0039] S4. Cultivate tumor organoids by placing an air-liquid interface culture medium in the outer chamber. The liquid level of the culture medium should submerge the bottom gel layer and contact the lower interface of the upper tissue gel layer without submerging the upper tissue gel layer to form an air-liquid interface. Cultivate the cells under the conditions of 37°C and 5% carbon dioxide volume concentration. Replace the culture medium twice a week.

[0040] S5. After culturing for 14-30 days and passaging, a head and neck malignant tumor organoid system is obtained, wherein the head and neck malignant tumor organoid system comprises head and neck malignant tumor organoids, T cells and fibroblasts.

[0041] As one of the preferred embodiments, the preparation method of the reconstructed collagen solution in step S1 is as follows: by mixing the collagen matrix, 10× concentrated sterile culture medium and sterile reconstitution buffer in a volume ratio of 8:1:1 on ice, first mixing the collagen matrix and 10× concentrated sterile culture medium and then adding the sterile reconstitution buffer and mixing again to prevent the generation of bubbles. It needs to be placed on ice before use to prevent the formation of gel.

[0042] As one of the preferred embodiments, in step S1, after adding the reconstructed collagen solution into the inner chamber, the solution is placed at 37°C for 30 minutes to obtain a bottom gel layer after solidification; in step S3, after adding the collagen gel containing tumor tissue, the solution is placed at 37°C for 30 minutes to obtain an upper tissue gel layer; the volume ratio of the reconstructed collagen solution taken in step S1 and the reconstructed collagen solution resuspended in step S2 is 1:1.

[0043] As one of the preferred embodiments, the source of the head and neck malignant tumor tissue in step S2 includes but is not limited to squamous epithelial-derived malignant tumors (squamous cell carcinoma) and glandular epithelial-derived malignant tumors (adenoid cystic carcinoma or ductal carcinoma), and the processing steps include cutting, soaking, shearing, and washing steps.

[0044] As one of the preferred embodiments, the pretreatment step is specifically as follows: fresh tumor specimens are cut and immersed in a sample solution (the sample solution is a DMEM culture medium (Nanjing KeyGen Biotech) containing 20X penicillin-streptomycin-amphotericin solution), washed twice with DPBS, and then minced on ice in an Advance DMEM / F12 culture medium containing Normocin. The minced tumor tissue should be less than 0.3 mm 3 , then centrifuged at 1500rpm, 4℃, 5min; then added ADMEM / F12 containing Normocin for resuspending and washing, centrifuged at 1500rpm, 4℃, 5min again, and repeated the washing operation twice to obtain the treated head and neck malignant tumor tissue. After the fresh tumor specimen is cut, cutting the tissue in Advance DMEM / F12 medium containing Normocin is helpful to reduce the risk of contamination in subsequent primary organoid culture.

[0045] As one of the preferred embodiments, in the internal insert cell culture dish whose bottom is a hydrophilic permeable membrane, the hydrophilic permeable membrane is a hydrophilic porous PTFE membrane with a pore size of 0.4 μm.

[0046] As one of the preferred embodiments, the specific passaging in step S5 is as follows: transfer the tissue gel layer in the inner chamber to a new sterile culture dish, cut the tissue gel layer into pieces, add dissociation solution to the sterile culture dish, transfer to a centrifuge tube, dissociate at 37°C for 30 minutes, shake once every 10 minutes, and then centrifuge at 1500rpm, 4°C, 5min, and discard the supernatant; add DMEM culture medium containing 10% serum, blow it thoroughly, centrifuge at 1500rpm, 4°C, 5min, and discard the supernatant, repeat 3 times, and then perform 1:2 passaging.

[0047] The dissociation solution is prepared with 200 units / ml of type IV collagenase and 10 ug / ml of DNase I in serum-free DMEM as solvent.

[0048] The present invention also provides a head and neck malignant tumor organoid gas-liquid interface culture medium based on the above-mentioned head and neck malignant tumor organoid culture method for reconstructing a tumor microenvironment, wherein the head and neck malignant tumor organoid gas-liquid interface culture medium is composed of: Advanced DMEM / F12 basal culture medium, Glutamax additive, HEPES, Pen-StrepGlutamine, nicotinamide, N-Acetylcysteine, B27, p38MAPK inhibitor SB202190, A8301, R-spondin1 recombinant protein, Noggin protein, EGF, Gastrin I, Wnt 3A, IL-2, wherein the concentration of the Glutamax additive is 1×, the concentration of HEPES is 10 mmol / L, the concentration of N-acetylcysteine ​​is 1 mmol / L, the concentration of nicotinamide is 10 mmol / L, and the concentration of Pen-Strep The concentration of Glutamine was 1×, the concentration of B27 was 1×, the concentration of p38 MAPK inhibitor SB202190 was 10umol / L, the concentration of A8301 was 0.5umol / L, the concentration of R-spondin1 recombinant protein was 0.05-0.25ug / ml, the concentration of recombinant Human Noggin protein was 0.05-0.2ug / ml, the concentration of EGF was 50ng / mL, the concentration of Gastrin I was 10nmol / L, the concentration of Human Wnt-3A was 0.05-0.2ug / ml, the concentration of IL-2 was 500IU / mL, and the Advanced DMEM / F12 basal medium was supplemented to the required volume of 50ml.

[0049] The application of head and neck malignant tumor organoids prepared by the above-mentioned head and neck malignant tumor organoid culture method for reconstructing the tumor microenvironment in screening drugs for treating tumors.

[0050] It should be noted that the sample of the present invention is a tumor tissue obtained through legal means, and the tumor tissue is derived from humans. The source of the sample is a clinically confirmed tumor tissue obtained in cooperation with a hospital, and the tumor tissue is a sample that has passed ethical approval. In this field, technicians with experimental conditions can obtain the above-mentioned tumor tissue.

[0051] The specific embodiments are as follows:

[0052] Example 1 Preparation of malignant tumor tissue organoids from patients with parotid duct carcinoma

[0053] 1. Tissue acquisition and processing:

[0054] The fresh tumor specimens used were derived from tumor tissue specimens obtained by surgical resection or biopsy of clinical patients with head and neck malignancies. In this embodiment, the project was approved by the Medical Ethics Committee of Xuzhou Central Hospital, and was specifically taken from the Department of Stomatology of Xuzhou Central Hospital. A 68-year-old male patient with parotid duct carcinoma was preoperatively diagnosed with parotid duct carcinoma based on clinical manifestations, imaging examinations, and auxiliary examinations such as fine needle aspiration biopsy. The patient and his family were fully informed and agreed before the operation, and an informed consent form was signed. Rapid intraoperative pathology and postoperative routine pathology were diagnosed as parotid duct carcinoma.

[0055] The patient's tumor is biopsied during surgery, and a sample of about 1cm*1cm*1cm in size should be taken from the lesion or the junction of the lesion and normal tissue, avoiding the necrotic area of ​​the tumor center.

[0056] After washing three times with physiological saline, the cells were immersed in the sample solution, stored at low temperature and transported to the laboratory. The cells were immersed for 1 hour and transported to the laboratory on ice or at low temperature (the sample solution was DMEM culture medium (Nanjing KeyGen Biotech) containing 20X penicillin-streptomycin-amphotericin solution).

[0057] 2. Preparation of recombinant collagen solution

[0058] First, prepare 4 ml of reconstructed collagen solution: mix collagen matrix (Cellmatrix IA), 10× concentrated sterile culture medium (Ham's F12) and sterile reconstitution buffer (2.2 g NaHCO3, 100 mL 0.05 N NaOH and 200 mmol / L HEPES) in a volume ratio of 8:1:1 on ice (or at low temperature). First mix Cellmatrix IA and 10× concentrated sterile culture medium (Ham's F12) and then add sterile reconstitution buffer and mix again to avoid bubbles. Keep on ice before use to prevent gel formation.

[0059] The collagen matrix (Cellmatrix IA) was purchased from Wako; the sterile reconstitution buffer (2.2 g NaHCO 3 , 100 mL 0.05 N NaOH and 200 mmol / L HEPES) was purchased from Saiguo Company; and the Ham's F12 nutrient mixture powder was purchased from ThermoFisher Company.

[0060] Specimen processing and construction of an air-liquid interface double-dish system to culture organoids of head and neck malignant tumors

[0061] See also Figure 5The gas-liquid interface double-disc culture system comprises an outer chamber 2 and an inner chamber 1 disposed inside the outer chamber, wherein the outer chamber 2 is a cell culture dish, and the inner chamber 1 is an internal insert cell culture dish having a permeable bottom membrane at the bottom, and the inner diameter of the inner chamber 1 is smaller than the inner diameter of the outer chamber 2. In this embodiment, the permeable bottom membrane is a hydrophilic porous PTFE membrane 6 (polytetrafluoroethylene filter membrane) with a pore size of 0.4 μm. The inner diameter of the inner chamber 1 is 30 mm, and the inner diameter of the outer chamber 2 is 60 mm.

[0062] Place the inner insert cell culture dish with a hydrophilic PTFE membrane with a pore size of 0.4 μm at the bottom in a sterile culture dish (60 mm*15 mm), and evenly spread 1 ml of the reconstructed collagen solution on the PTFE membrane 6 in the inner insert cell culture dish. Do not generate bubbles during the process. Then place the double-disc system in a 37°C sterile incubator for more than 30 minutes to allow the reconstructed collagen solution to solidify, so that a bottom gel layer 5 without tumor tissue is formed in the inner chamber of the air-liquid interface double-disc culture system.

[0063] Take out the specimen processed in step 1 from the biosafety cabinet, rinse it three times with sterile PBS, add 7-8 ml of DMEM / F12 with Normocin to a sterile culture dish (60 mm*15 mm), and use a sterile scalpel and tissue scissors to mince the tissue into pieces less than 3 mm. 3 Size, pipette into a sterile centrifuge tube, and centrifuge at low temperature: 1500rpm, 4℃, 5min.

[0064] Discard the supernatant, add 7 ml of DMEM / F12 containing Normocin again, and centrifuge again at 1500 rpm, 4°C, 5 min. Repeat this step three times.

[0065] 4 ml of reconstructed collagen solution was prepared again, and the tumor tissue fragments discarded from the supernatant after washing were resuspended in the reconstructed collagen solution to obtain collagen gel containing tumor tissue; 1 ml of the collagen gel containing tumor tissue was added to the bottom gel layer 5 in the inserted cell culture dish inside the double-disc system, and the dish was placed in a sterile incubator at 37°C for 30 minutes to obtain an upper tissue gel layer 4 after solidification.

[0066] After the preparation of tumor organoids is completed, 2 mL of the gas-liquid interface culture medium 3 is placed in the outer chamber 2 (cell culture dish). The final concentrations of the relevant functional components in the basic culture medium DMEM / F12 are as follows: the concentration of Glutamax additive is 1×, the concentration of HEPES is 10 mmol / L, the concentration of N-acetylcysteine ​​is 1 mmol / L, the concentration of nicotinamide is 10 mmol / L, the concentration of Pen-Strep Glutamine is 1×, the concentration of B27 is 1×, the concentration of p38MAPK inhibitor SB202190 is 10umol / L, the concentration of A8301 is 0.5umol / L, the concentration of R-spondin1 recombinant protein is 0.05-0.25ug / ml, the concentration of recombinant Human Noggin protein is 0.05-0.2ug / ml, the concentration of EGF is 50ng / mL, the concentration of Gastrin I is 10nmol / L, and the concentration of Human The concentration of Wnt-3A is 0.05-0.2ug / ml, and the concentration of IL-2 is 500IU / mL.

[0067] The reagents used in the present invention: recombinant human R-Spodin-1 protein, recombinant human Noggin protein, recombinant human Wnt-3A protein, N-acetylcysteine ​​(NAC), A83-01, Gastrin I were purchased from MCE; Advance DMEM / F12 basal medium, HEPES buffer, GlutaMAXTM, B27 supplement, Pen-StrepGlutamine were purchased from ThermoFisher; Nicotinamide (Nicotinamide), SB 202190 were purchased from Sigma; Human EGF, IL-2 were purchased from Peprotech. The pH buffer is HEPES, Pen-Strep Glutamine is an antibacterial agent, and N-acetylcysteine ​​is an oxidant.

[0068] In the air-liquid interface double-disc culture system, the liquid level of the air-liquid interface culture medium 3 should not exceed the upper tissue gel layer 4. The lower part of the upper tissue gel layer 4 is in contact with the liquid on the surface of the air-liquid interface culture medium 3, and the top is exposed to the air to form a air-liquid interface.

[0069] Then, the cells were cultured at 37°C and 5% CO2, with the culture medium replaced twice a week. The cells were cultured for 28 days and then subcultured. Figure 1 .

[0070] IV. Passaging

[0071] Transfer the tissue gel layer 4 on the collagen gel containing organoids in the inner chamber 1 to a new sterile culture dish (60 mm*15 mm), and cut the tissue gel layer into pieces;

[0072] Add dissociation solution to a sterile culture dish and transfer to a 15 ml centrifuge tube;

[0073] The dissociation solution was incubated at 37°C for 30 minutes with shaking every 10 minutes;

[0074] Low temperature centrifugation: 1500 rpm, 4°C, 5 min, discard the supernatant;

[0075] Add 3 ml of DMEM medium containing 10% serum, blow thoroughly to mix well, centrifuge again at low temperature: 1500 rpm, 4°C, 5 min, discard the supernatant, and repeat this step 3 times;

[0076] A new air-liquid interface collagen gel was re-formed at a ratio of 1:2, and the operation was the same as constructing an air-liquid interface double-disc system to culture head and neck malignant tumor organoids. After 15-28 days of culture, the cells were subcultured and repeated to the fourth generation.

[0077] HE, immunohistochemistry and immunofluorescence (IF) were used to identify the parotid ductal carcinoma organoids and immune cells produced at the air-liquid interface (ALI). The identification method was to embed and section the ductal carcinoma organoid system obtained after culture. The identification results were as follows: Figures 1 to 4 shown. Figure 1 This is the image shown under a 20X inverted microscope on the 19th day of air-liquid interface culture; Figure 2 (a), (b), (c), and (d) are HE staining images; Figure 3 As shown, CK7 is a specific marker for ductal carcinoma (red), Vimentin is a specific marker for fibroblasts (CAF) (yellow), and CD3 is a specific marker for T cells (green). Figure 3 It can be seen that the HE and IF identification results of the ductal carcinoma organoid system cultured in this example both contain T lymphocytes and CAFs; Figure 4 The immunohistochemistry of parotid ductal carcinoma organoids cultured by the air-liquid interface method from the primary to the fourth generation is shown. The ductal carcinoma tumor-specific immune marker protein CK19 is expressed from the primary to the fourth generation, CK7 and EMA are expressed from the primary to the third generation, but no longer expressed in the fourth generation, indicating that the tumor cannot maintain its original characteristics after being cultured to the fourth generation (i.e., the 88th day of culture).

[0078] Finally, it should be noted that the above implementation modes are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned implementation modes, a person of ordinary skill in the art should understand that the technical solutions described in the aforementioned implementation modes can still be modified, or some or all of the technical features therein can be replaced by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the implementation modes of the present invention.

Claims

1. A method for culturing head and neck malignant tumor organoids for reconstructing the tumor microenvironment, characterized in that: The culture method is based on a gas-liquid interface double-dish culture system, which includes an outer chamber and an inner chamber arranged inside the outer chamber, wherein the outer chamber is a sterile cell culture dish, and the inner chamber is an internal insert cell culture dish with a hydrophilic permeable membrane at the bottom, wherein the inner chamber inner diameter is smaller than the outer chamber inner diameter, and the specific steps of the head and neck malignant tumor organoid culture method for reconstructing the tumor microenvironment are as follows: S1, adding the reconstructed collagen solution into the inner chamber, and forming a bottom gel layer without tumor tissue in the inner chamber of the air-liquid interface double-disc culture system after solidification; S2, processing the obtained head and neck malignant tumor tissue, resuspending it with a reconstructed collagen solution, and obtaining a collagen gel containing the tumor tissue; S3, adding collagen gel containing tumor tissue to the bottom gel layer, and obtaining an upper tissue gel layer after solidification; S4. Cultivate tumor organoids by placing an air-liquid interface culture medium in the outer chamber. The liquid level of the culture medium should submerge the bottom gel layer and contact the lower interface of the upper tissue gel layer but should not submerge the upper tissue gel layer to form an air-liquid interface. Cultivate the cells under the conditions of 37°C, 5% carbon dioxide volume concentration, and replace the culture medium twice a week. S5. After culturing for 14-30 days and passaging, a head and neck malignant tumor organoid system is obtained, wherein the head and neck malignant tumor organoid system comprises head and neck malignant tumor organoids, T cells and fibroblasts.

2. The method for culturing head and neck malignant tumor organoids for reconstructing the tumor microenvironment according to claim 1, characterized in that: The preparation method of the reconstructed collagen solution in step S1 is as follows: by mixing the collagen matrix, 10× concentrated sterile culture medium and sterile reconstitution buffer at a volume ratio of 8:1:1 on ice, first mixing the collagen matrix and 10× concentrated sterile culture medium and then adding the sterile reconstitution buffer and mixing again to prevent the generation of bubbles. It needs to be placed on ice before use to prevent the formation of gel.

3. The method for culturing head and neck malignant tumor organoids for reconstructing tumor microenvironment according to claim 2, characterized in that: In the step S1, after adding the reconstructed collagen solution into the inner chamber, the solution is placed at 37° C. for 30 minutes to obtain a bottom gel layer after solidification; in the step S3, after adding the collagen gel containing the tumor tissue, the solution is placed at 37° C. for 30 minutes to obtain an upper tissue gel layer; the volume ratio of the reconstructed collagen solution taken in step S1 and the reconstructed collagen solution resuspended in step S2 is 1:

1.

4. The method for culturing head and neck malignant tumor organoids for reconstructing tumor microenvironment according to claim 1, characterized in that: In step S2, the sources of the head and neck malignant tumor tissues include squamous epithelial malignant tumors and glandular epithelial malignant tumors, and the processing steps include cutting, soaking, shearing, and washing steps.

5. The method for culturing head and neck malignant tumor organoids for reconstructing tumor microenvironment according to claim 4, characterized in that: The pretreatment step is specifically as follows: fresh tumor specimens are cut and immersed in a sample collection solution, which is a DMEM medium containing 20X penicillin-streptomycin-amphotericin solution, and then washed twice with DPBS, and then minced on ice in an Advance DMEM / F12 medium containing Normocin. The minced tumor tissue should be less than 0.3 mm 3 , and then centrifuged at 1500 rpm, 4°C, and 5 min; then ADMEM / F12 containing Normocin was added for resuspending and washing, and centrifuged at 1500 rpm, 4°C, and 5 min again. The washing operation was repeated twice to obtain the treated head and neck malignant tumor tissue.

6. The method for culturing head and neck malignant tumor organoids for reconstructing tumor microenvironment according to claim 1, characterized in that: The bottom of the internal insert cell culture dish is a hydrophilic permeable membrane, and the hydrophilic permeable membrane is a hydrophilic porous PTFE membrane with a pore size of 0.4 μm.

7. The method for culturing head and neck malignant tumor organoids for reconstructing tumor microenvironment according to claim 1, characterized in that: The specific passaging in step S5 is as follows: transfer the tissue gel layer in the inner chamber to a new sterile culture dish, cut the tissue gel layer into pieces, add dissociation solution to the sterile culture dish, transfer to a centrifuge tube, dissociate at 37°C for 30 minutes, shake once every 10 minutes, and then centrifuge at 1500rpm, 4°C, and 5 minutes, and discard the supernatant; add DMEM culture medium containing 10% serum, blow it thoroughly, centrifuge at 1500rpm, 4°C, and 5 minutes again, discard the supernatant, repeat the washing steps 3 times, and then perform 1:2 passaging.

8. The method for culturing head and neck malignant tumor organoids for reconstructing tumor microenvironment according to claim 7, characterized in that: The dissociation solution is prepared with 200 units / ml of type IV collagenase and 10 ug / ml of DNase I in serum-free DMEM as solvent.

9. A head and neck malignant tumor organoid air-liquid interface culture medium based on the head and neck malignant tumor organoid culture method for reconstructing the tumor microenvironment according to claim 1, characterized in that: The composition of the head and neck malignant tumor organoid air-liquid interface culture medium is as follows: Advanced DMEM / F12 basal culture medium, Glutamax additive, HEPES, Pen-StrepGlutamine, nicotinamide, N-Acetylcysteine, B27, p38MAPK inhibitor SB202190, A8301, R-spondin1 recombinant protein, Noggin protein, EGF, Gastrin I, Wnt 3A, IL-2, wherein the concentration of Glutamax additive is 1×, the concentration of HEPES is 10 mmol / L, the concentration of N-acetylcysteine ​​is 1 mmol / L, the concentration of nicotinamide is 10 mmol / L, and the concentration of Pen-Strep The concentration of Glutamine was 1×, the concentration of B27 was 1×, the concentration of p38 MAPK inhibitor SB202190 was 10umol / L, the concentration of A8301 was 0.5umol / L, the concentration of R-spondin1 recombinant protein was 0.05-0.25ug / ml, the concentration of recombinant Human Noggin protein was 0.05-0.2ug / ml, the concentration of EGF was 50ng / mL, the concentration of Gastrin I was 10nmol / L, the concentration of Human Wnt-3A was 0.05-0.2ug / ml, the concentration of IL-2 was 500IU / mL, and the AdvancedDMEM / F12 basal medium was supplemented to the required volume.

10. Use of head and neck malignant tumor organoids prepared by the head and neck malignant tumor organoid culture method for reconstructing the tumor microenvironment as described in claim 1 in screening drugs for treating tumors.

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