Oral squamous cell carcinoma organoid and tumor infiltration T cell immune co-culture system as well as construction method and application thereof

Abstract

The invention relates to an oral squamous cell carcinoma organoid and tumor infiltration T cell co-immune culture system as well as a construction method and application thereof. The construction method comprises the following steps: S1, oral squamous cell carcinoma organoid culture and passage; s2, separating and culturing tumor infiltration T lymphocytes; s3, co-culturing the oral squamous cell carcinoma organoid and the tumor infiltration T lymphocytes. According to the invention, the tumor immune microenvironment is remodeled, and a platform is provided for researching the interaction between tumor cells and immune cells. According to the invention, the morphological structure and pathological characteristics of the primary tissue of the patient are well restored, and the motility rate of the tumor tissue and the TILs is ensured to a great extent. Meanwhile, after the tumor infiltration T cells are cultured and amplified, oral squamous cell carcinoma organoid / TILs co-culture systems with different effect-target ratios are constructed, so that the tumor recognition and killing effect evaluation of the immune cells are realized, and an experimental method / technical path is provided for the evaluation of subsequent immune cell therapy or immunotherapy drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system, and a construction method and application thereof. Background Art

[0002] Oral squamous cell carcinoma (OSC) is a malignant tumor that develops in the oral cavity and is one of the most common malignant tumors of the head and neck. Risk factors for OSC include betel nut chewing, alcohol consumption, smoking, HPV infection, and ultraviolet radiation exposure.

[0003] Despite traditional surgery, cytotoxic chemotherapy, radiotherapy, etc., the median survival of most patients with advanced recurrent / metastatic oral squamous cell carcinoma is low. The prognosis of oral squamous cell carcinoma is closely related to the anatomical location, stage and histological characteristics of the tumor. EGFR is overexpressed in approximately 90% of oral squamous cell carcinomas and is a potential therapeutic target. The anti-EGFR monoclonal antibody cetuximab is the only drug approved for the targeted treatment of advanced recurrent / metastatic oral squamous cell carcinoma. However, due to unclear resistance mechanisms, some patients have a low response rate to cetuximab. In recent years, immunotherapy strategies such as anti-PD-1 therapy-mediated immune checkpoint blockade (ICB) have shown immunomodulatory effects and more durable remissions in patients with oral squamous cell carcinoma. However, only a small number of patients with oral squamous cell carcinoma benefit from anti-PD-1 treatment.

[0004] Currently, patient-derived organoid (PDO) models, as a new type of human-derived model, can well preserve the phenotypes of the original tumor, including histopathological, genetic, and molecular biological characteristics, and can reshape the tumor immune microenvironment, providing a unique opportunity for the evaluation and prediction of therapeutic efficacy and personalized treatment of cancer patients. Tumor-infiltrating lymphocytes (TILs) are a heterogeneous group of lymphocytes with specific anti-tumor effects that exist in the tumor stroma, including T cells and NK cells. The anti-tumor effect produced by TILs cells in tumor immunity is mainly a cellular immune response, and its mechanism of action includes the release of cytotoxins to directly kill tumor cells and regulate the body's immune function. Studying the mechanism of action of TILs and their subpopulations in tumor immunity is of great significance for enhancing the effect of immunotherapy.

[0005] Patent CN111989569A discloses a co-culture of immune cell organoids, which contains diseased immune cells and at least one organoid. The immune cells are isolated and amplified from impure immune samples, and the tumoroids are derived from cultured tumor epithelial cells, such as lung cells, liver cells, breast cells, skin cells, etc. However, the methods and difficulties of constructing co-culture systems are different for different tumor types. However, there are currently few reports on the construction methods of co-culture systems for oral squamous cell carcinoma organoids and TILs cells. Therefore, there is an urgent need for a co-culture system to evaluate the killing ability of TILs cells against oral squamous cell carcinoma in vitro, providing a new platform for screening oral squamous cell carcinoma immunotherapy drugs. Summary of the Invention

[0006] The purpose of the present invention is to overcome the gap in the co-culture of the above two materials, thereby providing an oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system and its construction method and application.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] One of the technical solutions of the present invention is to provide a method for constructing an immune co-culture system of oral squamous cell carcinoma organoids and tumor-infiltrating T cells, comprising the following steps:

[0009] S1. Oral squamous cell carcinoma organoid culture and passaging:

[0010] Oral squamous cell carcinoma tumor tissue was obtained, digested and lysed, resuspended in Matrigel, incubated, and cultured and passaged in oral squamous cell carcinoma organoid culture medium to obtain oral squamous cell carcinoma organoids;

[0011] S2. Isolation and culture of tumor-infiltrating T lymphocytes:

[0012] Tumor-infiltrating T lymphocytes were isolated from oral squamous cell carcinoma tissue, cultured with TILs cell culture medium and CD3 / CD28 cell stimulatory factors, centrifuged, and T cell culture medium containing IL-2 was added to the cell pellet for continued culture and passage.

[0013] S3. Co-culture of oral squamous cell carcinoma organoids with tumor-infiltrating T lymphocytes:

[0014] The oral squamous cell carcinoma organoids cultured in step S1 were digested, mixed with tumor-infiltrating T lymphocytes cultured in step S2, centrifuged, and resuspended in matrigel matrix gel. The cells were incubated and cultured in oral squamous cell carcinoma organoid culture medium and T cell culture medium to obtain an oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system.

[0015] In some embodiments, in step S1, the specific steps of culturing and passaging oral squamous cell carcinoma organoids are:

[0016] Oral squamous cell carcinoma tumor tissue was obtained and digested with tissue digestion solution. After terminating the digestion, the single cell suspension was filtered and centrifuged. Red blood cell lysis solution was added to the cell pellet for lysis and centrifugation. Matrigel matrix gel was added to the cell pellet under ice bath conditions for resuspending and incubation. After solidification, oral squamous cell carcinoma organoid culture medium was added for culture to obtain oral squamous cell carcinoma organoids, and the oral squamous cell carcinoma organoids with good growth status were passaged.

[0017] In some embodiments, in step S1, the tissue digestion solution is composed of collagenase type IV, dispase II, and DNase I at a final concentration of 1 mg / mL;

[0018] The oral squamous cell carcinoma organoid culture medium is prepared by adding a final concentration of 10% fetal bovine serum, 1% penicillin-streptomycin solution, 1% human leukocyte antigen B27, 5mmol / L N-acetylcysteine, 10mmol / L nicotinamide, 5ng / mL EGF protein, 1ng / mL FGF10 protein, and 1% RSPO1 protein to Advanced DMEM culture medium.

[0019] In some embodiments, in step S1, the digestion time is 30±5 min; the diameter of the filtration filter is 70 μm;

[0020] In step S1, matrigel was added and incubated at 37°C for 15 ± 5 min;

[0021] The culture temperature of oral squamous cell carcinoma organoid culture medium was 37°C and the culture time was 5 to 7 days.

[0022] In some embodiments, in step S2, the TILs cell culture medium is a T cell culture medium containing IL-2 (interleukin-2), and the final concentration of IL-2 in the TILs cell culture medium is 2000 IU / mL;

[0023] The volume ratio of TILs cell culture medium to CD3 / CD28 cell stimulatory factors was 100:1.

[0024] In some specific embodiments, in step S2, TILs cell culture medium and CD3 / CD28 cell stimulating factor are added for culturing at a temperature of 37° C. for 48 to 72 hours.

[0025] In some specific embodiments, in step S3, Trypsin-EDTA enzyme is added to the oral squamous cell carcinoma organoids cultured in step S1 for digestion, wherein the mass fraction of the added Trypsin-EDTA enzyme is 0.25%, the digestion temperature is 37° C., and the digestion time is 30±5 min.

[0026] In some embodiments, in step S3, the effector-target ratio of oral squamous cell carcinoma organoids to tumor-infiltrating T lymphocytes is 1:1 to 1:10;

[0027] Add Matrigel to resuspend the cells in an ice bath and incubate at 37°C for 15 ± 5 min.

[0028] The volume ratio of oral squamous cell carcinoma organoid culture medium and T cell culture medium was 1:1, and the culture temperature was 37°C.

[0029] A second technical solution of the present invention is to provide an application of an oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system as described in one of the above technical solutions in identifying a reagent suitable for treating oral squamous cell carcinoma. The oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system is obtained by the construction method described in one of the above technical solutions. A candidate reagent is added to the oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system, and the oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system is detected for one or more changes indicating that the candidate reagent is suitable for treating oral squamous cell carcinoma. If the presence or absence of one or more of the changes in the oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system is detected, the candidate reagent is identified as suitable for treating the oral squamous cell carcinoma.

[0030] The one or more changes are changes in one or more oral squamous cell carcinoma biomarkers, more specifically, the one or more changes are selected from: decreased cell viability, decreased cell proliferation, increased cell death, changes in cell or organoid size, changes in cell motility, changes in the production of cytokines and cytotoxic molecules by co-cultured immune cells, dissociation or destruction of intact / dense epithelial cell layers, and changes in the expression of one or more genes.

[0031] The third technical solution of the present invention is to provide an application of an oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system in testing the efficacy and / or safety of immune cell therapy for the treatment of oral squamous cell carcinoma, and to detect whether there are one or more changes in the oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system, wherein the presence or absence of the one or more changes indicates the efficacy and / or safety of the immune cell therapy.

[0032] The one or more changes are changes in one or more oral squamous cell carcinoma biomarkers, more specifically, the one or more changes are selected from: decreased cell viability, decreased cell proliferation, increased cell death, changes in cell or organoid size, changes in cell motility, changes in the production of cytokines and cytotoxic molecules by co-cultured immune cells, dissociation or destruction of intact / dense epithelial cell layers, and changes in the expression of one or more genes.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The present invention provides an oral squamous cell carcinoma organoid and tumor-infiltrating T cell (TILs) immune co-culture system and its construction method. The construction method includes the cultivation and passaging of oral squamous cell carcinoma organoids, the isolation and cultivation of tumor-infiltrating T lymphocytes, and the immune co-culture of oral squamous cell carcinoma organoids and TILs. The present invention establishes a patient-derived oral squamous cell carcinoma organoid and oral squamous cell carcinoma organoid and TILs immune co-culture model, reshapes the tumor immune microenvironment, and provides a platform for studying the interaction between tumor cells and immune cells.

[0035] (2) The tumor tissue and TILs involved in the present invention are derived from the tumor patient himself, which can better restore the morphological structure and pathological characteristics of the patient's primary tissue, and ensure the viability of the tumor tissue and TILs to a large extent.

[0036] (3) The present invention constructs an oral squamous cell carcinoma organoid / TILs immune co-culture system with different effector-target ratios after culturing and amplifying tumor-infiltrating T cells, thereby realizing the evaluation of the immune cell recognition and killing effect on tumors, and providing an experimental method / technical path for the subsequent evaluation of immune cell therapy or immunotherapy drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the construction process of the oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system.

[0038] Figure 2 Schematic diagram of the growth of oral squamous cell carcinoma organoids and TILs cells after 0, 24, 48, and 72 hours of culture.

[0039] Figure 3 Schematic diagram of the local growth of oral squamous cell carcinoma organoids / TILs after 0, 24h, 48h, and 72h of co-culture.

[0040] Figure 4 Live-dead staining results of oral squamous cell carcinoma organoids / TILs after 72 hours of co-culture.

[0041] Figure 5Results of oral squamous cell carcinoma organoid / TILs co-culture system with different effector-target ratios.

[0042] Figure 6 This is a partial schematic diagram of the growth of oral squamous cell carcinoma organoids / TILs after adding anti-PD1 immune co-culture for 0, 24h, 48h, and 72h. DETAILED DESCRIPTION

[0043] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0044] In the following examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0045] Example 1:

[0046] like Figure 1 As shown, a method for constructing an immune co-culture system of oral squamous cell carcinoma organoids and tumor-infiltrating T cells includes the following steps:

[0047] (1) Culture and passage of oral squamous cell carcinoma organoids

[0048] (1-1) Collect patient-derived oral squamous cell carcinoma tumor tissue under sterile conditions. The sample size should be at least the size of a soybean. Try to obtain tissue from the edge of the tumor (non-fibrotic, non-calcified, non-necrotic tissue). Immerse the tissue in preservation solution and transport it at 4°C. Process it within 48 hours. Place the tissue in a 10 cm culture dish and wash it three times with PBS (Seville, G4202) tissue buffer. Use sterile ophthalmic scissors to cut the tissue into 0.1 mm pieces. 3 size.

[0049] (1-2) Transfer the tissue to a 15 mL centrifuge tube and digest the tissue for half an hour with 5 mL of tissue digestion solution, which contains 1 mg / mL type IV collagenase (Thermo, 17104019), 1 mg / mL dispase II (Sigma, D4693), and 0.1 mg / mL DNase I (Yisheng, 10608ES60).

[0050] (1-3) After the digestion reaches the predetermined time, use a pipette to draw 10 μL of liquid and spread it evenly on a glass slide. Observe the presence of cells under a microscope. If 2-8 cell clumps are observed, terminate the digestion and filter the obtained cell suspension through a 70 μm filter. Centrifuge the filtered single-cell suspension at 1500 rpm for 5 minutes, and discard the supernatant to obtain a cell pellet.

[0051] (1-4) Add 5 mL of red blood cell lysis buffer (Tianjin Haoyang, NH4CL2009) to the cell pellet and incubate for 3-5 minutes to terminate the lysis. Centrifuge again at 1500 rpm for 5 minutes, discard the supernatant to obtain the cell pellet, resuspend the cell pellet, record the cell viability, centrifuge again at 1500 rpm for 5 minutes, and discard the supernatant.

[0052] (1-5) Resuspend the cells in 100% Matrigel on ice, mix the cells and Matrigel evenly, and transfer 10 μL of the cell-matrigel mixture to the center of a 48-well plate, avoiding bubbles. Invert the plate and incubate at 37°C for 15 min until the Matrigel solidifies.

[0053] (1-6) Add 200 μL of oral squamous cell carcinoma organoid culture medium to each well and culture at 37°C. After about 5-7 days, single cells will grow into organoids. Observe the growth status of the organoids under a microscope, and passage and freeze the organoids with good growth status. The oral squamous cell carcinoma organoid culture medium is prepared by adding a final concentration of 10% fetal bovine serum, 1% penicillin-streptomycin solution, 1% human leukocyte antigen B27, 5mmol / L N-acetylcysteine, 10mmol / L nicotinamide, 5ng / mL EGF protein, 1ng / mL FGF10 protein, and 1% RSPO1 protein to Advanced DMEM medium.

[0054] (2) Isolation and culture of TILs cells

[0055] (2-1) Aseptically collect patient-derived oral squamous cell carcinoma tumor tissue. The sample size should be at least the size of a soybean. Try to obtain tissue from the edge of the tumor (non-fibrotic, non-calcified, and non-necrotic tissue). Immerse the tissue in preservation solution and transport it at 4°C. Process it within 48 hours. Place the tissue in a 10 cm culture dish, wash it with PBS tissue buffer, and mince the tissue into 0.1-0.3 mm pieces using sterile ophthalmic scissors. 3 size;

[0056] (2-2) Place the minced tissue into a 90 mm culture dish in a 12-well plate, add 2 mL of T cell culture medium (Stemcell, 10981) containing 2000 IU / mL IL-2 (Stemcell, 78036.3) as TILs cell culture medium, then add 20 uL of LCD3 / CD28 cell stimulator (Stemcell, 10991) and culture at 37°C for 48-72 hours. Observe the cells in the suspension.

[0057] (2-3) Filter and centrifuge to discard the supernatant to obtain a cell pellet, place the cell pellet in a 12-well plate, add 2 mL of the above-mentioned TILs cell culture medium, observe the growth status of the TILs cells under a microscope, and passage and freeze the TILs cells with good growth status.

[0058] (3) Immune co-culture of oral squamous cell carcinoma organoids / TILs

[0059] (3-1) Tumor organoids with good growth status were digested with 0.25% Trypsin-EDTA digestion solution and incubated at 37°C for 30 min. After digestion, double the volume of oral squamous cell carcinoma organoid culture medium buffer was added to terminate the digestion. The organoids were transferred to a centrifuge tube and counted.

[0060] Immune cell processing: Use a 1 mL pipette to gently pipette TILs cells to resuspend them, transfer them to a centrifuge tube, pipette to mix, and count;

[0061] According to the counting results, TILs cells and oral squamous cell carcinoma organoids were mixed in a certain ratio and pipetted to mix evenly. After centrifugation at 1200 rpm at 4°C for 5 min, the supernatant was removed and the cell pellet was placed on ice. Dissolved matrigel was added and pipetted repeatedly to mix evenly. Then, 10 μL / well of the cell pellet was inoculated into the center of a well of a 48-well plate and incubated at 37°C for 15 min.

[0062] After incubation, the oral squamous cell carcinoma organoid culture medium and T cell culture medium were mixed in a 1:1 volume ratio and placed in a 37-degree constant temperature incubator to obtain an oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system.

[0063] The effector-target ratios of the above co-cultured organoids and TILs cells were 1:1, 1:3, 1:5, and 1:10, respectively.

[0064] like Figure 2 As shown, the figures show the growth of oral squamous cell carcinoma organoids (PDO) and TILs cells in 96-well plates at 0h, 24h, 48h, and 72h after culture. The increase in the number of oral squamous cell carcinoma organoids and TILs cells can be observed under a light microscope.

[0065] like Figure 3 As shown, after co-culture of oral squamous cell carcinoma organoids and TILs cells (target ratio of 1:5) for 0h, 24h, 48h, and 72h, the killing of oral squamous cell carcinoma organoids by TILs cells can be observed under a light microscope. The surface of oral squamous cell carcinoma organoids changes from smooth to rough, and the lysis of some oral squamous cell carcinoma organoid spheres can be observed.

[0066] like Figure 4As shown, after co-culture of oral squamous cell carcinoma organoids and TILs cells (target-effect ratio of 1:5), live-dead staining showed that TILs cells killed tumor cells. Red fluorescence showed that after co-culture with TILs cells, some oral squamous cell carcinoma organoids underwent apoptosis and showed a scattered distribution.

[0067] like Figure 5 As shown, oral squamous cell carcinoma organoids and TILs cells at different effector-target ratios (1:1, 1:3, 1:10) were co-cultured for 1 to 3 days. Annexin V staining showed tumor cell apoptosis, and the red fluorescence showed that TILs cells at different effector-target ratios had the ability to kill oral squamous cell carcinoma organoids, including larger and smaller organoids.

[0068] Anti-PD1 was added to the above oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system at a final concentration of 25 μg / mL. Figure 6 As shown in the figure, after adding anti-PD1 drugs, the killing effect was observed after co-culture of oral squamous cell carcinoma organoids and TILs cells (target ratio was 1:5) for 0h, 24h, 48h, and 72h. Under the bright field, it can be seen that as the co-culture time increases, the organoids lyse, and the lysis is obvious at 72h.

[0069] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A method for constructing an immune co-culture system of oral squamous cell carcinoma organoids and tumor-infiltrating T cells, characterized in that: The steps include: S1. Oral squamous cell carcinoma organoid culture and passaging: Oral squamous cell carcinoma tumor tissue was obtained, digested and lysed, resuspended in Matrigel, incubated, and cultured and passaged in oral squamous cell carcinoma organoid culture medium to obtain oral squamous cell carcinoma organoids; S2. Isolation and culture of tumor-infiltrating T lymphocytes: Tumor-infiltrating T lymphocytes were isolated from oral squamous cell carcinoma tissue, cultured with TILs cell culture medium and CD3 / CD28 cell stimulatory factors, centrifuged, and T cell culture medium containing IL-2 was added to the cell pellet for continued culture and passage. S3. Co-culture of oral squamous cell carcinoma organoids with tumor-infiltrating T lymphocytes: The oral squamous cell carcinoma organoids cultured in step S1 were digested, mixed with tumor-infiltrating T lymphocytes cultured in step S2, centrifuged, and resuspended in matrigel matrix gel. The cells were incubated and cultured in oral squamous cell carcinoma organoid culture medium and T cell culture medium to obtain an oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system.

2. The method for constructing an immune co-culture system of oral squamous cell carcinoma organoids and tumor-infiltrating T cells according to claim 1, characterized in that: In step S1, the specific steps for culturing and passage of oral squamous cell carcinoma organoids are as follows: Oral squamous cell carcinoma tumor tissue was obtained and digested with tissue digestion solution. After terminating the digestion, the single-cell suspension was filtered and centrifuged. Red blood cell lysis solution was added to the cell pellet for lysis and centrifugation. Matrigel matrix gel was added to the cell pellet under ice bath conditions for resuspending and incubation. After solidification, oral squamous cell carcinoma organoid culture medium was added for culture to obtain oral squamous cell carcinoma organoids, and the oral squamous cell carcinoma organoids with good growth status were passaged.

3. The method for constructing an immune co-culture system of oral squamous cell carcinoma organoids and tumor-infiltrating T cells according to claim 2, characterized in that: The tissue digestion solution consists of 1 mg / mL type IV collagenase, 1 mg / mL dispase II, and 0.1 mg / mL DNase I enzymes at a final concentration of 1 mg / mL; The oral squamous cell carcinoma organoid culture medium is prepared by adding a final concentration of 10% fetal bovine serum, 1% penicillin-streptomycin solution, 1% human leukocyte antigen B27, 5mmol / L N-acetylcysteine, 10mmol / L nicotinamide, 5ng / mL EGF protein, 1ng / mL FGF10 protein, and 1% RSPO1 protein to Advanced DMEM culture medium.

4. The method for constructing an immune co-culture system of oral squamous cell carcinoma organoids and tumor-infiltrating T cells according to claim 2, characterized in that: In step S1, the digestion time was 30 ± 5 min; the filtration filter diameter was 70 μm; The temperature for incubation after adding matrigel was 37°C and the incubation time was 15 ± 5 min; The culture temperature of oral squamous cell carcinoma organoid culture medium was 37°C and the culture time was 5 to 7 days.

5. The method for constructing an immune co-culture system of oral squamous cell carcinoma organoids and tumor-infiltrating T cells according to claim 1, characterized in that: In step S2, the TILs cell culture medium is a T cell culture medium containing IL-2, and the final concentration of IL-2 in the TILs cell culture medium is 2000 IU / mL; The volume ratio of TILs cell culture medium to CD3 / CD28 cell stimulatory factors was 100:

1.

6. The method for constructing an immune co-culture system of oral squamous cell carcinoma organoids and tumor-infiltrating T cells according to claim 1, characterized in that: In step S2, TILs cell culture medium and CD3 / CD28 cell stimulating factor are added and cultured at a temperature of 37° C. for 48 to 72 hours.

7. The method for constructing an immune co-culture system of oral squamous cell carcinoma organoids and tumor-infiltrating T cells according to claim 1, characterized in that: In step S3, trypsin-EDTA enzyme is added to the oral squamous cell carcinoma organoids cultured in step S1 for digestion, wherein the mass fraction of the added trypsin-EDTA enzyme is 0.25%, the digestion temperature is 37° C., and the digestion time is 30±5 min.

8. The method for constructing an immune co-culture system of oral squamous cell carcinoma organoids and tumor-infiltrating T cells according to claim 1, characterized in that: In step S3, the effector-target ratio of oral squamous cell carcinoma organoids to tumor-infiltrating T lymphocytes was 1:1 to 1:10; Add Matrigel to resuspend the cells in an ice bath and incubate at 37°C for 15 ± 5 min. The volume ratio of oral squamous cell carcinoma organoid culture medium and T cell culture medium was 1:1, and the culture temperature was 37°C.

9. Use of an oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system in identifying a reagent suitable for treating oral squamous cell carcinoma, characterized in that: The oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system is obtained by the construction method of any one of claims 1 to 8, and a candidate agent is added to the oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system, and the oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system is detected to determine whether there is one or more changes indicating that the candidate agent is suitable for treating oral squamous cell carcinoma. If the presence or absence of one or more of the changes in the oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system is detected, the candidate agent is identified as suitable for treating the oral squamous cell carcinoma.

10. Use of an oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system in testing the efficacy and / or safety of immune cell therapy for the treatment of oral squamous cell carcinoma, characterized in that: Detecting whether one or more changes exist in the oral squamous cell carcinoma organoid and tumor-infiltrating T cell immune co-culture system, wherein the presence or absence of the one or more changes indicates the efficacy and / or safety of the immune cell therapy.

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