A method for evaluating the anti-tumor activity of immune cells in vitro and its application

Abstract

The present invention discloses a method for evaluating the anti-tumor activity of immune cells in vitro and its application. The present invention first constructs a co-culture model: (1) peripheral blood mononuclear cells are separated from the peripheral blood of healthy people, and γδT cells are obtained after pre-stimulation and amplification culture; (2) tumor tissue is digested using a tumor tissue dissociation kit Tumor Dissociation Kit, and then the digested tumor cells are cultured for organoids, and tumor organoids of 37μm to 100μm are obtained after screening; (3) Corning 96-well all-white flat-bottom polystyrene microplates are pre-coated with anti-adhesion reagents, and then tumor organoids are plated, counted, and then mixed with γδT cells to form a co-culture system to obtain the co-culture model; finally, the co-culture model is further used to evaluate the anti-tumor activity and evaluate the killing effect of γδT cells on tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biology, and particularly relates to a method for evaluating the anti-tumor activity of immune cells in vitro and an application thereof. Background Art

[0002] Organoids are a type of model that is highly similar to the source tissue or organ in vivo and is based on a 3D in vitro cell culture system. These 3D in vitro culture systems can replicate the complex spatial morphology of differentiated tissues and can show the interactions and spatial position morphology between cells and between cells and their surrounding matrix. They themselves can have similar physiological responses to differentiated tissues and organs in vivo and have extremely high similarity to the source tissues.

[0003] Traditional 2D cell culture models have limited physiological expression, no vascularization, and no immune system. Although they can be screened at high throughput and are very easy to operate, they cannot simulate organ development and human development and disease patterns. Animal models have physiological characteristics, vascularization, and an immune system, but they cannot be screened at high throughput, have limited operation, and the complexity of animal tissues increases the difficulty of research. In contrast, 3D cultured organoids contain a variety of cell types and can form functional "micro-organs", which can be better used to simulate the development process and physiological and pathological states of organ tissues, and therefore have broad application prospects in basic research and clinical diagnosis and treatment.

[0004] Human T cells can be divided into αβT cells and γδT cells according to the different TCR expression chains. The human body's γδT cells can be divided into three subgroups according to the different δ chains, including Vδ1, Vδ2 and Vδ3. Although γδT cells only account for a small part of T lymphocytes (1-10%), they have multiple immune functions, such as defense against viral infection, inflammation regulation, immune homeostasis, helping B cells produce antibodies, and anti-tumor effects. γδT cells do not need MHC molecules to recognize antigens, so they can be used to treat cancer patients through allogeneic transfusion, which is different from αβT cells. The mechanisms of γδT cells killing tumors include secretion of Th1 cytokines, perforins, granzymes, etc., recruitment and activation of antigen-presenting cells, activation of T cells and B cells, etc. Therefore, γδT cells play an important role in the human body's anti-tumor immune response.

[0005] At present, tumor organoids have been used in models of immune cell anti-cancer effectiveness, among which the publicly available immune cells include NK cells, αβT cells, CAR T cells, etc., and they are co-cultured with tumor organoids to explore the anti-cancer effectiveness of immune cells. However, it is difficult to establish a co-culture system of immune cells and tumor organoids, and existing studies have not used tumor organoids to study the anti-cancer effectiveness of γδT cells, and have failed to solve the problem of plating uniformity in the construction of the co-culture system of immune cells and tumor organoids. In addition, technical issues such as the counting method and killing detection method of the co-culture ratio of immune cells and tumor organoids need to be improved. Therefore, it is necessary to provide a more comprehensive, accurate, intuitive and simple method for evaluating the anti-tumor activity of immune cell γδT cells. Summary of the invention

[0006] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for constructing a co-culture model of immune cells and tumor organoids for evaluating anti-tumor activity.

[0007] Another object of the present invention is to provide a method for constructing a co-culture model of immune cells and tumor organoids for evaluating anti-tumor activity.

[0008] Another object of the present invention is to provide a method for evaluating the anti-tumor activity of immune cells in vitro.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A method for constructing a co-culture model of immune cells and tumor organoids for evaluating anti-tumor activity comprises the following steps:

[0011] (1) Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy subjects, and then pre-stimulated and expanded with γδT cells to obtain γδT cells;

[0012] (2) The tumor tissue from the tumor patient is digested using a Tumor Dissociation Kit at 37°C and 180-220 rpm (preferably 200 rpm) for 20-60 minutes, and then the digested tumor cells are passed through a 100 μm cell sieve, and the tumor cells are collected by centrifugation and mixed evenly with matrix gel, and organoid culture medium is added for organoid culture and subculture, and then the size of the tumor organoids is screened using 37 μm and 100 μm cell sieves to obtain humanized tumor organoids of 37 μm to 100 μm;

[0013] (3) Pre-coating a Corning 96-well all-white flat-bottom polystyrene microplate (TC-treated, catalog number: 3917) with an anti-adhesion reagent, then plating the humanized tumor organoids obtained in step (2), counting them, and then mixing them with the γδT cells obtained in step (1) at an effector-target ratio (i.e., the ratio of the number of cells contained in the γδT cells to the number of cells contained in the tumor organoids) of 1 to 10:1 to form a co-culture system, placing the plate in an incubator for culturing, and obtaining the immune cell and tumor organoid co-culture model for evaluating anti-tumor activity.

[0014] The peripheral blood mononuclear cells (PBMC) described in step (1) are separated by conventional methods in the art; preferably, they are prepared by the following method:

[0015] Healthy human peripheral blood was diluted with an equal volume of serum-free RPMI 1640 medium, and then added to human lymphocyte separation solution (Ficoll). The volume ratio of human lymphocyte separation solution (Ficoll) to diluted healthy human peripheral blood was 2:5. Centrifugation was performed, and then the middle white flocculent cell layer was aspirated, and then an equal volume of serum-free RPMI 1640 medium was added. After inversion and thorough mixing, the supernatant was discarded after centrifugation again, and red blood cell lysis solution was added for lysis. After lysis, serum-free RPMI 1640 medium was added to terminate the reaction, and the cells were filtered through a 40μm mesh and centrifuged to discard the supernatant. Finally, serum-free RPMI 1640 medium was added to resuspend the cells to obtain PBMC cells.

[0016] The culture medium used for the γδT cell pre-stimulation culture in step (1) is a γδT cell pre-stimulation culture medium, and its formula is as follows: serum-free RPMI 1640 culture medium (Gibco), 10% by volume of fetal bovine serum (FBS), 1% by volume of penicillin-streptomycin (Gibco), 1200UI / ml Human IL-2, and 50μM zoledronic acid.

[0017] The culture medium used for the expansion culture described in step (1) is a γδT cell culture medium, and its formula is as follows: serum-free RPMI 1640 culture medium (Gibco), 10% by volume of fetal bovine serum (FBS), 1% by volume of penicillin-streptomycin double antibody (Gibco), and 300UI / ml Human IL-2.

[0018] The pre-stimulation culture and expansion culture described in step (1) are preferably cultured in a 24-well plate. The total time of the pre-stimulation culture and expansion culture is 11 to 13 days. When the proportion of Vδ2 in the γδT cells reaches more than 85%, the subsequent γδT cell anti-tumor activity experiment can be performed; wherein the seeding density of the pre-stimulation culture is 3.0×10 6 ~4.0×10 6cells / well, and the seeding density of the expansion culture was 1.2×10 6 ~4.0×10 6 cells / well; more preferably, it is achieved by: firstly performing pre-stimulation culture (Day 0), with a seeding density of 3.0×10 6 ~4.0×10 6 cells / well; then on Day 3, the medium was changed once, and the inoculation density was 3.0×10 6 ~4.0×10 6 cells / well; starting from Day 3, the medium was changed every other day, and the seeding density was 1.0×10 6 ~2.0×10 6 Cells / well were adjusted by changing the medium.

[0019] The tumor described in step (2) is a malignant tumor; preferably breast cancer or lung cancer.

[0020] The Tumor Dissociation Kit described in step (2) is Miltenyi Tumor Dissociation Kit (human, catalog number 130-095-929).

[0021] The digestion solution used in the digestion described in step (2) is preferably prepared by the following method: 325 μl Tumor Dissociation Kit (Miltenyi Biotec, human, catalog number 130-095-929) and 4.7 ml washing solution are mixed evenly to obtain a digestion solution; wherein the washing solution formula is as follows: Advanced DMEM / F12 culture medium (Invitrogen, 12634010), Glutamax additive 1× [Glutamax 100× (Gibco, 35050061) is diluted to 1×], 1M Hepes buffer solution (Gibco, 15630080), penicillin-streptomycin 3× (final concentration 300 U / ml) (Gibco, 15140122), 10 μM Y-27632 (Rock Inhibitor) (Abmole, M1817).

[0022] The digestion time in step (2) is preferably 20 to 40 minutes; more preferably about 30 minutes (when digestion is about 30 minutes, take a small amount of supernatant and observe it under a microscope. If digestion stops when 2 to 10 cells form a cell cluster, the digestion is terminated).

[0023] During the subculture process described in step (2), mechanical blowing is performed before digestion. If mechanical digestion is not possible, a 37 μm cell sieve is used to filter out smaller organoids, and the larger organoids that have not been filtered out are digested with Tryple Express digestion solution. After digestion is completed, the digested organoids are plated together with organoids smaller than 37 μm.

[0024] The 100 μm cell sieve described in step (2) is preferably a 100 μm Reversible Strainer cell sieve; more preferably, it is a 100 μm Reversible Strainer cell sieve (Cat. No. 27270) from Stemcell.

[0025] The 37 μm cell sieve described in step (2) is preferably a 37 μm Reversible Strainer cell sieve; more preferably, it is a 37 μm Reversible Strainer cell sieve (Cat. No. 27215) from Stemcell.

[0026] The organoids described in step (2) are preferably cultured in a 24-well flat-bottom plate.

[0027] The matrix glue described in step (2) is Matrigel matrix glue (Corning).

[0028] The amount of matrix gel used in step (2) can be added according to actual needs; preferably, it is calculated based on 30,000 tumor cells per 30 μl of matrix gel.

[0029] The organoid culture medium described in step (2) can use conventional organoid culture medium; the formula of the organoid culture medium is as follows: Advanced DMEM / F12 culture medium (Invitrogen, 12634010), B27 additive 1× [B27 50× ((Gibco, 17504-044) diluted to 1×], Glutamax additive 1× [Glutamax 100× (Gibco, 35050061) diluted to 1×], 1M Hepes buffer (Gibco, 15630080), penicillin-streptomycin 1× (final concentration 100U / ml) (Gibco, 15140122), 5mM nicotinamide (Sigma, N0636), 1.25mM acetylcysteine ​​(Sigma, A9165), 5μmY-27632 (Rock inhibitor) (Abmole, M1817), 500nM A83-01 (Tocris, 2939), 500nM SB202190 (Sigma, S7067), 5ng / ml fibroblast growth factor 7 (Peprotech, 100-19-1), 20ng / ml fibroblast growth factor 10 (Peprotech, 100-26-25), 5ng / ml epidermal growth factor (Peprotech, AF-100-15), 100ng / ml Noggin (Novoprotein, CB89), 250ng / ml R-spondin-1 (Novoprotein, CX83), 5nM Heregulin β-1 (Peprotech, 100-03), 50μg / ml primary cell antibiotic Primocin (Invivogen, ant-pm-05).

[0030] The anti-adhesion reagent in step (3) is an anti-adhesion rinse solution; preferably, an anti-adhesion rinse solution (Stemcell) is used to pre-coat the cell with an anti-adhesion reagent. 96-well all-white flat-bottom polystyrene microplates prevent human tumor organoids from adhering to the wall.

[0031] In step (3), since tumor organoids are spheres composed of multiple single cells, a new counting method is used for plating: 100 μl of cell suspension is taken from 1.1 ml of cell suspension, and 900 μl of Tryple Express is added to digest it into single cells, and then the number of cells contained in the tumor organoids in the original system is calculated.

[0032] The effector-target ratio in step (3) is preferably 1:1, 5:1 or 10:1; more preferably 10:1.

[0033] The culture described in step (3) is carried out in an incubator at 37°C and 5% CO2.

[0034] The immune cell and tumor organoid co-culture model constructed by any of the above-mentioned methods for constructing an immune cell and tumor organoid co-culture model for evaluating anti-tumor activity.

[0035] A method for evaluating the anti-tumor activity of immune cells in vitro, in addition to the above steps (1) to (3), further comprising at least one of the following steps:

[0036] (4) adding a tracer dye and a fluorescent dye for detecting tumor cell death to the co-culture system in step (3), and then continuously photographing the co-culture system under bright field and fluorescence channels to continuously detect the degree of tumor cell death in the co-culture system, the interaction between γδT cells and tumor organoids, and the killing activity of γδT cells on tumor organoids;

[0037] (5) adding a fluorescent dye for detecting tumor cell death to the co-culture system in step (3), then using a multifunctional microplate reader to detect the relative fluorescence value of the co-culture system (to detect the degree of cell death), and calculating and analyzing the killing activity of γδT cells against tumor organoids;

[0038] (6) Detecting the concentration level of cytotoxicity-related effector factors (killing biomarkers) in the supernatant of the co-culture system in step (3) to comprehensively evaluate the killing activity of γδT cells against tumor organoids.

[0039] The tracking dye described in step (4) is preferably Celltracker red CMTPX dye (Invitrogen, C34552).

[0040] The fluorescent dye described in steps (4) and (5) is preferably Promega Green dye, which is from Promega CellTox Green Cytotoxicity Assay (G8741).

[0041] In step (4), the γδT cells can be first stained with a tracer dye, and then the γδT cells can be co-cultured with tumor organoids. Promega Green Dye reagent is added to the co-culture system to dye the dead cells green. The interaction between the γδT cells and the tumor organoids can be continuously detected by fluorescence imaging and bright field imaging.

[0042] The detection described in step (6) is preferably performed using a CBA multifactor kit (Biolegend, LEGENDplex, Mixand match).

[0043] The effector factor in step (6) includes at least one of IFN-γ, Granzyme B, Perforin and TNF-α.

[0044] Compared with the prior art, the present invention has the following advantages and effects:

[0045] 1. Since tumor organoids have tumor specificity that is highly similar to patient tumor tissues, the method of the present invention does not require the construction of an animal model. It only uses tumor organoids and immune cells to co-culture, thereby accurately evaluating the effectiveness of immune cells in fighting cancer. Compared with traditional animal models, it improves the ease of operation and improves the expression of the model's physiological characteristics compared to 2D cell culture models.

[0046] 2. The present invention provides an experimental research method, comprising co-culturing γδT cells with humanized breast cancer or lung cancer tumor organoids to form a co-culture system, and then continuously monitoring the fluorescent signal value released after the tumor cell death; in addition, continuously analyzing the images of the co-culture system under a microscope bright field and fluorescence, and detecting the expression levels of effector factors related to cytotoxicity in the supernatant of the co-culture system, thereby systematically analyzing and evaluating the anti-tumor activity of γδT cells in killing breast cancer or lung cancer-related tumor organoids in an all-round manner.

[0047] 3. The present invention adds cell death detection fluorescent reagents and tracer dyes to the co-culture system, observes the interaction between immune cells (healthy human γδT cells) and tumor organoids, as well as the cell morphology changes after the tumor organoids are killed under the bright field and fluorescence channel of the microscope, and detects the relative fluorescence value released by the cell death dye by a multifunctional microplate reader, as well as the lethality-related biomarkers in the supernatant of the co-culture system. The killing of tumor organoids by immune cells γδT can be more intuitive, accurate and comprehensive. Therefore, the present invention provides a technical means for evaluating the anti-cancer effectiveness of immune cells γδT that is more universal than personalized precision medicine.

[0048] 4. In the process of culturing humanized tumor organoids, the present invention adopts a new digestion method for digesting cancer tissues. After digestion at 200 rpm in an oscillator at 37°C for 30 minutes using the Tumor Dissociation Kit (Miltenyi Biotec), the cell suspension is taken and the cell aggregates are observed under a microscope to determine the degree of digestion. Compared with the conventional collagenase solution digestion method, it is more conducive to the subsequent culture of tumor organoids.

[0049] 5. In the process of constructing the co-culture system, the present invention uses a 37μm Reversible Strainer cell sieve and a 100μm Reversible Strainer cell sieve to screen the size of tumor organoids, and tumor organoids of similar size between 37μm and 100μm are screened and plated, thereby reducing the differences between wells within the group.

[0050] 6. In the process of constructing the co-culture system, the present invention first pre-coats the cells with an anti-adhesion reagent. 96-well all-white flat-bottom polystyrene microplate to prevent human tumor organoids from adhering to the wall.

[0051] 7. The γδT cell and humanized tumor organoid co-culture model constructed by the present invention is suitable for the evaluation of the anti-tumor activity of immune cells, and can be used to evaluate the direct anti-cancer effectiveness of immune cell γδT, or to evaluate the direct anti-tumor activity and indirect anti-tumor ability mediated by γδT cells.

[0052] 8. The platform provided by the method of the present invention can be used to simply and intuitively evaluate the killing effect of γδT cells on tumors. The effect can be close to the real reaction in the human body, which is of great value for clinical immune cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is the flow cytometry of γδT cells after 11 days of expansion culture.

[0054] Figure 2 The figures are continuous growth images of breast cancer organoid culture under bright field; A shows the growth of two breast cancer organoids observed under bright field (BC10 and BC13, where BC13 shows the growth of the first generation and the second generation after passaging); B shows the corresponding HE and immunohistochemistry images of breast cancer organoids.

[0055] Figure 3 The figures are continuous growth images of lung cancer organoid culture under bright field; A is the growth of two lung cancer organoids observed under bright field (LC5 and LC7, where LC5 shows the growth of the first generation and the second generation after passaging); B is the corresponding HE and immunohistochemistry images of lung cancer organoids.

[0056] Figure 4 This is a graph showing the continuous photography observation results of the γδT cell and breast cancer organoid co-culture killing system under bright field and fluorescence channels.

[0057] Figure 5 This is a graph showing the continuous detection results of Green Dye relative fluorescence values ​​in the co-culture killing system of γδT cells and breast cancer organoids.

[0058] Figure 6This is a graph showing the results of detecting killing biomarkers in the supernatant of the killing system of γδT cells and breast cancer organoids co-cultured.

[0059] Figure 7 This is a picture showing the results of continuous bright-field photography of the co-culture killing system of γδT cells and lung cancer organoids.

[0060] Figure 8 This is a graph showing the continuous detection of the Green Dye relative fluorescence value of the γδT cell and lung cancer organoid co-culture killing system and the supernatant killing biomarker detection results.

[0061] Fig. 9 The figure shows the experimental results of co-culturing lung cancer tumor organoids and γδT cells using three different 96-well plates.

[0062] Fig.10 This is a schematic diagram of the effect of whether an anti-adhesion reagent is used on the plate used in the co-culture experiment of tumor organoids and γδT cells under bright field observation; among them, A means no anti-adhesion reagent is used; B means the anti-adhesion reagent is used.

[0063] Fig.11 Schematic diagram of the effect of using 37μm and 100μm Reversible Strainer cell sieves in the co-culture experiment of tumor organoids and γδT cells under bright field observation; A shows that 37μm and 100μm Reversible Strainer cell sieves were not used; B shows that 37μm and 100μm Reversible Strainer cell sieves were used.

[0064] Fig.12 The digestion status of tumor tissues in the bright field observation group using different digestion methods; A was using 2 mg / ml collagenase IV; B was using Tumor Dissociation Kit (Miltenyi Biopharmaceuticals). DETAILED DESCRIPTION

[0065] The present invention will be described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0066] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0067] As used in this specification, the term "about" typically means ±5% of the stated value, more typically ±4% of the stated value, more typically ±3% of the stated value, more typically ±2% of the stated value, even more typically ±1% of the stated value, and even more typically ±0.5% of the stated value.

[0068] In this specification, certain embodiments may be disclosed in a format of being in a certain range. It should be understood that such description of "being in a certain range" is merely for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0069] The "anti-cancer activity" mentioned in the present invention refers to the killing effect of immune cells directly on cancer cells.

[0070] Example 1 Isolation of peripheral blood mononuclear cells (PBMC)

[0071] 1. Pour the blood of healthy volunteers into a 50ml centrifuge tube, dilute the peripheral blood with an equal volume of serum-free RPMI 1640 medium, and mix thoroughly by inverting;

[0072] 2. Take 4 ml of human lymphocyte separation solution (Ficoll) and add it to a 15 ml conical bottom centrifuge tube. Take 10 ml of diluted peripheral blood along the wall of the test tube and slowly spread it on the surface of the lymphocyte separation solution (4 ml Ficoll + 10 ml of equally diluted blood). First tilt the centrifuge tube, then gradually stand it upright, 25 ° C, 600g, centrifuge for 25 minutes (min), and adjust the speed gear to 6 up and 2 down;

[0073] 3. After centrifugation, use a sterile Pasteur pipette to suck the middle white flocculent cell layer into another sterile centrifuge tube, remove the white film layer and transfer to a 50ml centrifuge tube, add an equal volume of serum-free RPMI 1640 medium, mix thoroughly by inverting, divide into tubes, centrifuge at 1500rpm, centrifuge time for 15min, and adjust the speed gear to 9-9;

[0074] 4. After discarding the supernatant, add an appropriate amount of red blood cell lysis buffer (Tian Gen, RT122-02) to each tube to resuspend the cells, lyse at room temperature for 5 minutes, add 5 times the volume of serum-free RPMI 1640 medium to terminate the reaction, filter with a 40 μm mesh, divide into tubes, centrifuge at 1500 rpm for 7 minutes, and adjust the speed gear to 9 up and 9 down;

[0075] 5. After discarding the supernatant, add an appropriate amount of serum-free RPMI 1640 medium to each tube to resuspend the cells (for example, 5 ml), centrifuge at 1500 rpm for 5 min, and adjust the speed gear to 9 up and 9 down to obtain PBMC cells.

[0076] Example 2 In vitro expansion and culture of γδT cells

[0077] 1. Prepare γδT cell pre-stimulation culture medium (taking the preparation of 50 ml culture medium as an example): serum-free RPMI 1640 medium (Gibco), 10% (v / v) fetal bovine serum (FBS, purchased from AusGeneX, i.e. 5 ml FBS), 1% (v / v) penicillin-streptomycin double antibody (Gibco), 1200 UI / ml Human IL-2 (Beijing Sihuan Biopharmaceuticals) (prepared and used immediately), 50 μM zoledronic acid (Sigma);

[0078] 2. After centrifuging the PBMC cells obtained in Example 1 and discarding the supernatant, the cells at the bottom of the tube were resuspended to 1 ml each with γδT cell pre-stimulation culture medium, and after sufficient and slow mixing, the cells were appropriately diluted and counted using a hemocytometer;

[0079] 3. According to day0:3.0×10 6 ~4.0×10 6 The density of cells / well was used to calculate the number of wells in the seeding plate, and the cells were seeded in a 24-well plate;

[0080] 4. Prepare γδT cell culture medium (taking the preparation of 50 ml culture medium as an example): serum-free RPMI 1640 medium (Gibco), 10% (v / v) FBS (AusGeneX) (i.e. 5 ml FBS), 1% (v / v) penicillin-streptomycin (Gibco), 300 UI / ml Human IL-2 (Beijing Sihuan Biopharmaceuticals) (prepared and used immediately);

[0081] 5. Then, add the prepared γδT cell pre-stimulation culture medium to the 24-well plate inoculated with cells, dilute accordingly, and add 1200UI / ml human IL-2 (final concentration). Inoculate the 24-well plate with a volume of 1ml per well. After inoculation, mix the plate appropriately to make the cells evenly distributed in the wells, and place it in a 37°C incubator for subsequent culture;

[0082] 6. Change the medium once on Day 3, and the inoculation density is 3.0~4.0×10 6 cells / well, and the medium was changed every other day starting from Day 3. On Day 5, Day 7, Day 9, and Day 11, the medium was changed at 1.0-2.0×10 6 The density of cells / well was adjusted by changing the medium. The culture medium used was γδT cell culture medium. The centrifugation conditions for changing the medium were: Day 3, speed 600-800 rpm, centrifugation time 5 min. The centrifugation conditions for changing the medium on subsequent days were: speed 800-1000 rpm, centrifugation time 5 min.

[0083] 7. Perform Vδ2 ratio detection (using flow cytometry) on Day 11 of culture. When the ratio reaches more than 85%, subsequent γδT cell anti-tumor activity experiments can be performed;

[0084] 8. Experimental results: Figure 1 As shown, the Vδ2 ratio was detected on Day 11 and it was found that the ratio could reach 94.2%, indicating that the γδT cells were highly pure after being expanded in vitro by the above culture system and could be used for subsequent γδT cell anti-tumor activity experiments.

[0085] Example 3 Breast cancer / lung cancer organoid culture

[0086] 1. Cancer tissues from breast cancer and lung cancer patients from the First Affiliated Hospital of Jinan University in Guangzhou were placed in centrifuge tubes containing 3 ml of tissue storage solution (Miltenyi Biotec) and 100 μl of penicillin-streptomycin dual antibody, and then placed in a 4°C sample transport box and transferred to the laboratory for the next step of processing (all experiments have been approved by the Ethics Committee of the First Affiliated Hospital of Jinan University, and the informed consent of the participants has been obtained);

[0087] 2. Prepare washing solution, the formula is: Advanced DMEM / F12 medium (Invitrogen, 12634010), Glutamax additive 1× [that is, dilute Glutamax 100× (Gibco, 35050061) to 1×)], 1M Hepes buffer solution (Gibco, 15630080), penicillin-streptomycin 3× (Gibco, 15140122, 10000U / ml) (final concentration is 300U / ml), 10μM Y-27632 (Rock Inhibitor) (Abmole, M1817);

[0088] 3. Prepare two sterile culture dishes with a diameter of 10 cm, add 1 ml of washing solution in the middle, place the cancer tissues in the washing solution, mark the sterile culture dishes, take photos and record;

[0089] 4. Use a scalpel and forceps to remove non-epithelial tissue, keep the cancerous tissue as much as possible, and use ophthalmic scissors to cut the cancerous tissue into 1-2 mm 3 Size, use a disposable cell scraper to gather the tissues in the culture dish together, add appropriate amount of washing solution and PBS solution (Solarbio) to rinse the culture dish, and transfer all tissues to a 15ml centrifuge tube;

[0090] 5. After the tissue suspension in the centrifuge tube is allowed to stand, the layers are separated, the supernatant is removed, and the lower layer of tissue is digested;

[0091] 6. Take 325μl of the prepared Tumor Dissociation Kit (Miltenyi Biotec, human, catalog number 130-095-929), add 4.7ml of washing solution, mix well to obtain digestion solution; then add appropriate amount of digestion solution according to the size of the tissue, put the centrifuge tube into a 37℃ shaker, 200rpm, and digest for 20 to 40 minutes;

[0092] 7. Wait for digestion to last about 30 minutes, take a small amount of supernatant and observe under a microscope. If digestion has reached 2 to 10 cells as a cluster, stop digestion. The total digestion time should not exceed 1 hour.

[0093] 8. When terminating digestion, add more than 1 volume of washing solution to terminate digestion, and then blow evenly with a disposable pipette (note: blow after terminating digestion);

[0094] 9. Pass through a 100μm Reversible Strainer cell sieve (Stemcell). Grind the tissue blocks that cannot pass through with a 10ml syringe pump head;

[0095] 10. The filtered liquid was centrifuged at 350g for 5 min, the sieve was turned over and rinsed with washing liquid, centrifuged at 350g for 5 min, tissue blocks were obtained, and the tissue blocks were frozen (the formula of the freezing solution was FBS: dimethyl sulfoxide (DMSO) = 9:1, v / v);

[0096] 11. After centrifugation, remove the supernatant, add appropriate amount of washing solution to resuspend the cell pellet to 1 ml, and count the cells;

[0097] 12. Place 300 μl of Matrigel matrix gel (Corning) on ​​ice to melt, prepare breast cancer / lung cancer organoid culture medium and restore it to room temperature in advance; the formula of breast cancer / lung cancer organoid culture medium is: Advanced DMEM / F12 medium (Invitrogen, 12634010), B27 additive 1× [dilute B27 50× ((Gibco, 17504-044) to 1×], Glutamax additive 1× [dilute Glutamax 100× (Gibco, 35050061) diluted to 1×], Hepes (1M) buffer (Gibco, 15630080), penicillin-streptomycin 1× (Gibco, 15140122, 10000U / ml) (final concentration is 100U / ml), 5mM nicotinamide (Sigma, N0636), 1.25mM acetylcysteine ​​(Sigma, A9165), 5μmY-27632 (Rock inhibitor) (Abmole, M1817), 500nM A83-01 (Tocris, 2939), 500nM SB 202190 (Sigma, S7067), 5ng / ml fibroblast growth factor 7 (Peprotech, 100-19-1), 20ng / ml fibroblast growth factor 10 (Peprotech, 100-26-25), 5ng / ml epidermal growth factor (Peprotech, AF-100-15), 100ng / ml Noggin (Novoprotein, CB89), 250ng / ml R-spondin-1 (Novoprotein, CX83), 5nM Heregulin β-1 (Peprotech, 100-03), 50μg / ml primary cell antibiotic Primocin (Invivogen, ant-pm-05);

[0098] 13. For scaffolded organoid culture in a 24-well plate, 30,000 cells are needed per well. 300 μl of matrix gel is generally seeded in 10 wells, and a total of 3×10 5cells, take an appropriate amount of cell suspension according to the required number of cells and centrifuge at 350g for 5min;

[0099] 14. After centrifugation, add an appropriate amount of the above-prepared tumor organoid culture medium to resuspend the cell pellet (3×10 5 cells) to 100 μl, add to 300 μl of Matrigel, mix well, and obtain a Matrigel-cell mixture;

[0100] 15. Take out the 24-well plate preheated in the incubator overnight, prepare the seeding plate, seed each well with 40 μl of matrix gel cell mixture, drop it in the center of the well of the 24-well plate to form an arc shape, do not stick to the well wall, and do not generate bubbles, so that the matrix gel accounts for 75% (volume ratio) in each well. Note that the whole process should be operated on ice;

[0101] 16. After seeding, place the plate (24-well plate) upright in a 37°C incubator for one minute;

[0102] 17. Place the plate upside down for 30 minutes to allow the matrix gel to solidify;

[0103] 18. After 30 minutes, add 500 μl of the above-prepared tumor organoid culture medium to each well. When adding the culture medium, do not aim the tip of the pipette at the matrix gel to prevent the matrix gel from being blown apart.

[0104] 19. Mark the plate and place it in an incubator (37°C, 5% CO2) for subsequent organoid culture. Take photos regularly to observe the growth of the organoids. On Day 3, add 500 μl of the above-prepared tumor organoid culture medium to each well. On the next day, discard 500 μl of the old culture medium from each well and add 500 μl of new tumor organoid culture medium.

[0105] 20. Take photos regularly in bright field during the culture process of breast cancer and lung cancer organoids to observe the growth of organoids;

[0106] 21. Experimental results: The continuous growth of two breast cancer organoid cultures (BC10 and BC13) under bright field and their corresponding HE and immunohistochemical results are shown in Figure 2. Figure 2 The continuous growth of lung cancer organoid culture (LC5 and LC7) and their HE and immunohistochemical results are shown in Figure 3 It can be found that under this lung cancer / breast cancer organoid culture system, tumor organoids can be successfully cultured. In addition, tumor organoids can be successfully passaged and cryopreserved, and the corresponding HE and immunohistochemistry results also show the heterogeneity of the tumor, which has a very high similarity with the source tissue. Therefore, it can be explained that the pretreatment method, culture medium and culture method under the lung cancer / breast cancer tumor organoid culture system of the present invention can successfully culture lung cancer / breast cancer tumor organoids.

[0107] Example 4 Passaging of breast cancer / lung cancer tumor organoids

[0108] 1. All subsequent operations in this example were performed on ice, the centrifuge was precooled to 4°C, and the washing solution (same as in Example 3) was precooled in advance;

[0109] 2. Aspirate and discard the culture medium in the organoid culture plate in Example 3;

[0110] 3. Add 500 μl of pre-cooled washing solution to each well, use a pipette tip to mechanically blow the matrix gel in each well, transfer the contents of the well to a 15 ml centrifuge tube, and place the centrifuge tube on ice;

[0111] 4. Centrifuge the tube at 1300 rpm, 5 min, 4°C;

[0112] 5. After centrifugation, remove the supernatant, add 3 ml of pre-cooled washing solution, and pipette up and down 30 times. Take 1 drop of solution and observe under the microscope whether the degree of pipetting reaches the ideal digestion degree. If not, choose Tryple Express enzyme (Gibco, 12605010) for the next step of digestion;

[0113] 6. Filter the organoid solution in step 5 with a 37μm Reversible Strainer (Stemcell, 27215). The sieved suspension contains single cells and smaller organoid clumps. These cells do not need to be digested and are collected in a 15ml centrifuge tube. Then turn the cell sieve over and add washing solution to wash. The washed organoid clumps with a volume larger than 37μm are collected in another 37μm centrifuge tube and centrifuged at 1300rpm, 5min, and 4℃.

[0114] 7. After centrifugation, remove the supernatant and add 1 ml of Tryple Express enzyme (Gibco, 12605010) to the precipitate corresponding to the suspension of larger clumps. Resuspend the cell precipitate by blowing and transfer it to a clean well and place it in a 37°C incubator for digestion. Closely observe the degree of digestion.

[0115] 8.Observe the digestion status in the well for 3 to 5 minutes. If the digestion has not reached the level of 2 to 10 cells forming a cluster, continue digestion. The total digestion time should not exceed 15 minutes.

[0116] 9. If the ideal digestion degree has been reached, transfer the contents of the well to a 15 ml centrifuge tube and gently blow the bottom of the centrifuge tube with a gun tip;

[0117] 10. Add more than 1 volume of washing solution to terminate digestion, mix the precipitate containing single cells and smaller organoid clumps after centrifugation in step 6, centrifuge at 1300 rpm, 5 min, 4°C, and remove the supernatant;

[0118] 11. After centrifugation, add an appropriate amount of tumor organoid culture medium (same as in Example 3) to resuspend the cell pellet to 100 μl, add to 300 μl of matrix gel, mix well, and obtain a matrix gel cell mixture;

[0119] 12. Take out the 24-well plate preheated in the incubator overnight, prepare the seeding plate, seed each well with 40 μl of matrix gel cell mixture, drop it in the center of the well of the 24-well plate to form an arc shape, do not stick to the well wall, and do not generate bubbles, so that the matrix gel in each well accounts for 75%. Note that the whole process should be operated on ice;

[0120] 13. After seeding, place the plate upright in a 37°C incubator for one minute;

[0121] 14. Place the plate upside down for 30 minutes to allow the matrix gel to solidify;

[0122] 15. After 30 minutes, add 500 μl of tumor organoid culture medium to each well. When adding culture medium, do not aim the tip of the pipette at the matrix gel to prevent the matrix gel from being blown apart.

[0123] 16. Mark the plate and place it in an incubator (37°C, 5% CO2) for subsequent organoid culture. Take photos regularly to observe the growth of the organoids. Add 500 μl of tumor organoid culture medium to each well on Day 3. Replace the medium every other day by aspirating 500 μl of the old culture medium from each well and adding 500 μl of new tumor organoid culture medium.

[0124] Example 5 Cryopreservation of breast cancer / lung cancer organoids

[0125] 1. All subsequent operations in this example were performed on ice, the centrifuge was precooled to 4°C, and the washing solution (same as in Example 3) was precooled in advance;

[0126] 2. Aspirate and discard the culture medium in the organoid culture plate in Example 4;

[0127] 3. Add 500 μl of pre-cooled washing solution to each well, use a pipette tip to mechanically blow the matrix gel in each well, transfer the contents of the well to a 15 ml centrifuge tube, and place the centrifuge tube on ice;

[0128] 4. Centrifuge the tube at 1300 rpm, 5 min, 4°C;

[0129] 5. After centrifugation, remove the supernatant, add an appropriate amount of freezing solution (FBS: DMSO = 9:1, v / v), resuspend the cell pellet and transfer it to the cryopreservation tube, mark it, put it into the program freezing box and put it in -80℃, and transfer the cryopreservation tube to liquid nitrogen the next day.

[0130] Example 6 Co-culture experiment of breast cancer / lung cancer organoids and γδT cells

[0131] (I) Organoid plating

[0132] 1. According to the method in Example 4, breast cancer and lung cancer organoids were plated again on the third day after passaging;

[0133] 2. Each well of a 96-well all-white flat-bottom polystyrene microplate (TC-treated, catalog number: 3917) was rinsed with 200 μl of Anti-adherence Rinsing Solution (Stemcell, 07010) in advance to prevent subsequent organoids from adhering to the wall after plating. The 96-well plate was then placed in a 37°C incubator overnight. The next day, an equal volume of PBS solution was used to rinse the wells, ensuring that the liquid in the wells was aspirated and discarded.

[0134] 3. Prepare 1 mg / ml dispase solution: dissolve dispase powder (Sigma-Aldrich, D4693) in washing solution; add 1 mg / ml dispase solution to each well of the tumor organoid plate, mechanically blow with a pipette tip, and place in a 37°C incubator for incubation and digestion for 40 minutes. The purpose of adding dispase is to thoroughly digest the matrix gel to avoid the influence of this component on the subsequent co-culture experiment;

[0135] 4. After digestion, add more than 1 volume of washing solution (same as in Example 3) to terminate digestion, mechanically pipette 10 times, transfer to a centrifuge tube, add appropriate amount of PBS solution to wash each well and transfer the liquid to the centrifuge tube;

[0136] 5. Filter the liquid through a 100 μm Reversible Strainer cell sieve (Stemcell, 27270), and sieve the organoid suspension smaller than 100 μm through a 37 μm Reversible Strainer (Stemcell, 27215); then turn the sieve over and rinse with washing liquid to obtain organoids with a size of 37 μm to 100 μm. These organoids are used for subsequent co-culture experiments with γδT cells;

[0137] 6. Rinse the reverse side of the 100 μm Reversible Strainer (Stemcell, 27270) with washing solution to obtain organoids larger than 100 μm. This part of the organoid solution larger than 100 μm and the single cell or organoid mass solution smaller than 37 μm in step 5 should be centrifuged and frozen or continued to be plated for culture.

[0138] 7. The organoid suspension containing 37 μm to 100 μm obtained in step 5 was centrifuged at 4°C and 1300 rpm for 5 min. After centrifugation, the supernatant was removed and an appropriate amount of γδT cell culture medium (prepared in Example 2) was added to resuspend the suspension to 1.1 ml for counting;

[0139] 8. Take 100 μl of organoid suspension, add 900 μl Tryple Express enzyme (Gibco, 12605010), put it in a 37°C incubator to digest it into single cells, take 10 μl for counting, and then calculate how many cells there are in the original system 1.1 ml;

[0140] 9. In this experiment, the number of wells containing tumors in the co-culture plate is 12, and the required tumor cells are 15,000 cells / well. Two more wells are reserved for consumption loss, and a total of 15,000×14=21×10 4 cells. Since the total system of each co-culture well is 200 μl, half of which is tumor organoid suspension, 12×100=2100 μl γδT cell culture medium is needed to resuspend the corresponding cell amount of tumor organoids.

[0141] (ii) γδT cells stained with Celltracker:

[0142] 1. Equilibrate an unopened 50 μg tracking dye Celltracker Red CMTPX dye (Invitrogen, C34552) to room temperature;

[0143] 2. Add 7.2854 μl DMSO to prepare a 10 mM storage solution;

[0144] 3. Take 1 μl of storage solution and add 9 μl of Advanced DMEM / F12 medium to prepare a 1 mM temporary storage solution;

[0145] 4. Use Advanced DMEM / F12 medium to dilute the 1mM temporary storage solution to 0.5μM working solution. The working solution should be used immediately after preparation and thrown away after use. The storage solution should be stored at -20℃ away from light.

[0146] 5. Preheat the working solution to 37°C and resuspend the γδT cells prepared in Example 2 to 1×10 7cells / ml, stained at 37°C for 45 min;

[0147] 6. Wash the γδT cells twice with PBS solution, centrifuge at 1000 rpm for 5 min each time, and adjust the density to 3×10 6 cells / ml;

[0148] 7. Since the working concentration of the fluorescent dye Promega Green dye (from Promega CellToxGreen Cytotoxicity Assay (G8741)) for detecting cell death is 1:1000 (v / v), the sample addition in this experiment is carried out according to Table 1:

[0149] Table 1

[0150]

[0151] 8. Place the co-culture plate with the added samples in an incubator (37°C, 5% CO2) for culture, take photos regularly under bright field and fluorescence channels to observe the interaction between γδT cells and tumor organoids, and use a multifunctional microplate reader to detect the relative fluorescence value of Green Dye;

[0152] 9. At the end of the experiment, the co-culture plate was centrifuged (500 g, 5 min), and the supernatant of each group was aspirated into an EP tube and stored in a -80°C refrigerator as the test sample, which was subsequently tested using a CBA multifactor kit (Biolegend, LEGENDplex, Mix and match).

[0153] (III) Detection of killing biomarkers in the supernatant of the co-culture system using the CBA multi-factor kit

[0154] 1. The CBA multi-factor kit (Biolegend, LEGENDplex, Mix and match) needs to be equilibrated to room temperature before use. The components of the kit are: ① lyophilized standard; ② lyophilized matrix; ③ capture microspheres; ④ detection antibody; ⑤ PE-labeled streptavidin; ⑥ detection buffer; ⑦ washing solution; ⑧ instrument calibration microspheres;

[0155] 2. Thaw the samples on ice according to the instructions;

[0156] 3. After the kit is equilibrated to room temperature, take out the capture microspheres and vortex at maximum speed for more than 1 minute;

[0157] 4. Add 250 μl of detection buffer to the freeze-dried standard bottle, invert several times to mix thoroughly, let stand for 10 minutes, transfer the dissolved standard to an EP tube (the remaining standard in the experiment needs to be packaged and stored in a -80 degree refrigerator and used within one month), marked as C7;

[0158] 5. Take 7 new EP tubes, label them as C6 / C5 / C4 / C3 / C2 / C1 / C0, and add 75 μl of detection buffer to each tube;

[0159] 6. Take 25 μl from C7 to C6, mix by pipetting, and perform 4-fold gradient dilution in sequence until C1 (C0 is the detection buffer, and the concentration of the standard is 0 pg / ml);

[0160] 7. Add 25 μl of detection buffer to all wells of the V-bottom plate provided with the kit;

[0161] 8. Add 25 μl of the corresponding standard to the standard wells;

[0162] 9. Add 25 μl of the corresponding sample to the sample well;

[0163] 10. Vortex the capture microspheres for 30 seconds before adding them, and add 25 μl to each well;

[0164] 11. Put on the sealing film, shake on the shaker at 800 rpm / min, shake at room temperature and avoid light for 2 hours;

[0165] 12. Centrifuge the V-bottom plate at 250g for 5 minutes.

[0166] 13. After centrifugation, turn the plate over immediately, gently shake off the liquid, and tap it gently on a clean paper towel. After centrifugation, blue microspheres will be precipitated at the bottom of the wells;

[0167] 14. Add 200 μl 1× wash buffer to each well and incubate for 1 minute;

[0168] 15. Centrifuge the plate at 250g for 5 minutes;

[0169] 16. After centrifugation, immediately turn the plate over, gently shake off the liquid, tap it gently on a clean paper towel, add 25μl of detection antibody to each well, and stick the sealing film;

[0170] 17. Incubate at room temperature in the dark at 800 rpm / min;

[0171] 18. Add 25 μl of PE-labeled detection antibody to each well and affix the sealing film;

[0172] 19. Incubate at room temperature in the dark at 800 rpm / min;

[0173] 20. Centrifuge the plate at 250g for 5 minutes;

[0174] 21. Gently shake off the liquid and tap it lightly on a clean paper towel;

[0175] 22. Add 200 μl 1× wash buffer to each well and incubate for 1 minute;

[0176] 23. Centrifuge the plate at 250g for 5 minutes, gently shake off the liquid, and tap it gently on a clean paper towel;

[0177] 24. Add 150 μl of 1× washing solution to each well, resuspend the microspheres with a gun and transfer them to the flow tube / EP tube, prepare for the machine, perform flow analysis, and count the relevant killing biomarkers, such as IFN-γ, Granzyme B, Perforin, TNF-α; set the experiment to repeat three times;

[0178] 25. Experimental results:

[0179] Figure 4 The results of continuous time photography of bright field and fluorescence channels in the co-culture system of γδT cells and breast cancer organoids with an effector-target ratio of 10:1. Figure 4 From the bright field photos, we can see that over time, breast cancer organoids are surrounded by co-cultured γδT cells. The fluorescence channel photos show that γδT cells are stained with red tracer dyes and dead cells are stained with green fluorescence (Green Dye). It can be seen that over time, γδT cells marked with red tracer dyes gradually migrate and surround breast cancer tumor organoids, and the phenomenon that more and more dead cells are stained with green fluorescence also indicates that γδT cells effectively kill tumor organoids in this co-culture system.

[0180] Figure 5 The figure shows the changes in the relative fluorescence value of Green Dye at different time points in the co-culture system of γδT cells and breast cancer organoids with different effector-target ratios. Figure 5 It can be found that with the passage of time, the relative fluorescence values ​​of each effector-target ratio continued to increase (the relative fluorescence value is for Green Dye); after 24 hours of co-culture, the effector-target ratios of 5:1 and 10:1 had higher relative fluorescence values ​​than the control group (0:1), which was statistically significant.

[0181] Figure 6The concentration change diagram of the related killing biomarkers (IFN-γ, Granzyme B, Perforin, TNF-α) in the supernatant at the end time in the co-culture system of γδT cells and breast cancer organoids with different effector-target ratios detected by the CBA method. Figure 6 It can be found that after 24 hours of co-culture, the relevant killing biomarkers contained in the supernatant of the co-culture system with different effector-target ratios increased to varying degrees. For example, IFN-γ increased to varying degrees when the co-culture effector-target ratio was 1:1, 5:1 and 10:1, Granzyme B and Perforin increased significantly when the co-culture effector-target ratio was 5:1 and 10:1, while TNF-α contained in the co-culture supernatant with different effector-target ratios did not change significantly.

[0182] comprehensive Figure 4 , Figure 5 and Figure 6 It can be shown that after 24 h of co-culture, γδT cells with effector-target ratios of 1:1, 5:1, and 10:1 can effectively kill breast cancer tumor organoids.

[0183] Figure 7 The results of the co-culture of γδT cells and lung cancer organoids for 4h and 16h in a co-culture system with an effector-target ratio of 10:1 were photographed. Figure 7 Bright field photos show that over time, γδT cells gradually migrate and surround lung cancer organoids.

[0184] Figure 8 The relative fluorescence value of Green Dye in the co-culture system of γδT cells and lung cancer organoids changes at different time points, and the concentration changes of related killing biomarkers (IFN-γ, Granzyme B, Perforin, TNF-α) in the supernatant of the co-culture system at the end time are detected by CBA method. Figure 8 It can be seen that over time, the relative fluorescence value of the co-culture system with an effector-target ratio of 10:1 gradually increased compared with the control group (the relative fluorescence value is for Green Dye), and the related killing biomarkers such as IFN-γ, Granzyme B and Peforin in the supernatant of this co-culture system increased significantly compared with the control group, while TNF-α did not change significantly.

[0185] Combination Figure 7 and Figure 8 It can be seen that after 24 h of co-culture, γδT cells with an effector-target ratio of 10:1 can effectively kill lung cancer tumor organoids.

[0186] Comparative Example 1

[0187] The method is the same as Example 6, except that the 96-well plates used in the co-culture of lung cancer tumor organoids and γδT cells are different. Three groups of experiments were designed: BD 96-well U-bottom Plate (Cat. No. 353077), 96-well clear round bottom ultra-low attachment microplate (Ultra-low Attachement Plate) (Cat. No. 7007) and The co-culture experiment was completed using a 96-well all-white flat-bottom polystyrene microplate (Flat Bottom Plate) (TC-treated, catalog number: 3917). All three plates were treated with Anti-adherence Rinsing solution (Stemcell) with an effector-target ratio of E:T = 10:1. After the plate was inoculated, it was placed in an incubator for subsequent culture (37°C, 5% CO2). Bright-field photography was performed under a microscope at fixed time points to observe the distribution and aggregation of the co-culture system in different plates. The experiment was repeated three times.

[0188] The results are as follows Fig. 9 As shown by Fig. 9 The experimental results show that when the BD96-well U-shaped plate (U Bottom Plate) was used for co-culture experiments, due to the influence of gravity, the lung cancer tumor organoids and γδT cells were concentrated in the middle of the bottom of the well, but the interaction of γδT cells on tumor organoids could not be clearly and intuitively observed; In the co-culture experiment using a 96-well transparent round-bottom ultra-low attachment plate, it was found that the tumor organoids were concentrated in the center of the bottom of the well, although the concentration was not as good as that of the U-shaped plate. In addition, the γδT cells were evenly distributed around the well, but this would result in the γδT cells being unable to act evenly on the tumor organoids, which also affected the observation effect. The method of the present invention uses In the co-culture experiment of 96-well flat-bottom polystyrene microplates, it can be seen that tumor organoids and γδT cells are evenly distributed in various areas of the wells. As the co-culture experiment progresses, γδT cells gradually migrate to the periphery of tumor organoids and interact with each other. The 96-well all-white flat-bottom polystyrene microplate is conducive to cell interaction, achieving the best co-culture effect of tumor organoids and γδT, and also achieving better observation effects.

[0189] Comparative Example 2

[0190] The method is the same as in Example 6, except that during the construction of the immune cell and breast cancer organoid co-culture system, no anti-adhesion reagent Anti-adhesion Rinsing Solution (Stemcell) was added for pre-coating. 96-well all-white flat-bottom polystyrene microplate (TC-treated, catalog number: 3917), instead, an equal volume of PBS buffer was added to each well; wherein, the effector-target ratio was E:T = 10:1. The experiment was set up for three replicates.

[0191] The results are as follows Fig.10 Shown by: Fig.10 It can be seen that the plates used in the co-culture experiment of breast cancer tumor organoids and γδT cells will adhere to the wall of the tumor organoids when no anti-adhesion reagent is used. Adhesion makes the breast cancer tumor organoids become adherent cells ( Fig.10 A); however, the use of anti-adhesion reagents can effectively avoid the adhesion of tumor organoids and maintain their 3D in vitro growth model ( Fig.10 B).

[0192] Comparative Example 3

[0193] The method is the same as Example 6, except that in the process of constructing the co-culture system of immune cells and lung cancer organoids, the 37 μm Reversible Strainer cell sieve and the 100 μm Reversible Strainer cell sieve are not used to screen the size of tumor organoids; wherein, the effector-target ratio is E:T=10:1. The experiment is repeated three times.

[0194] The results are as follows Fig.11 As shown by Fig.11 It can be seen that in the co-culture experiment of lung cancer tumor organoids and γδT cells, if the 37μm and 100μm Reversible Strainers are not used for sieving and plating, the sizes of the obtained lung cancer tumor organoids vary greatly, increasing the differences between wells within the group and increasing the error of the actual test results ( Fig.11 A); however, in the present invention, after using 37 μm and 100 μm Reversible Strainers for screening and plating, tumor organoids of similar size between 37 μm and 100 μm were screened and plated, and the volume of the obtained tumor organoids was not much different, which effectively reduced the difference between wells within the group and between groups ( Fig.11 B).

[0195] Comparative Example 4

[0196] The method is the same as in Example 3, except that during the culture of tumor organoids, the Tumor Dissociation kit (Miltenyi Biotechnology) was replaced with a traditional digestion method (reference: Bhatia S, Kramer M, Russo S, et al. Patient-derived triple-negative breast cancer organoids provide robust model systems that recapitulate tumor intrinsic characteristics [J]. Cancer Res, 2022, 82 (7): 1174-1192.) to digest the primary cancer tissue, that is, collagenase IV was added to the primary tumor tissue at a final concentration of 2 mg / ml, and the primary tumor tissue was gently shaken at 37 ° C for 45 to 90 minutes. During the culture of the organoids, photos were taken regularly in the bright field to observe the growth of the organoids, which was the same as the steps in Example 3. Three replicates were set.

[0197] The results are as follows Fig.12 As shown, Fig.12 The figure shows the process flow of breast cancer organoid culture pretreatment. Fig.12 It can be seen that the traditional digestion method for conventional organoid culture was used to digest primary breast cancer tissue. It was found that the degree of digestion was not easy to control under bright field observation, and the tissue was easily over-digested, resulting in excessive single cells, which was not conducive to the subsequent culture of breast cancer tumor organoids ( Fig.12 A); the present invention adopts a new digestion method, that is, using Tumor Dissociation Kit (Miltenyi Biotec) in an oscillator at 37°C and 200 rpm for about 30 minutes, taking the cell suspension and observing the cell clumps under a microscope to determine the degree of digestion, which can obtain a good digestion effect. Digestion produces more cell clumps rather than single cells, which is beneficial to the subsequent culture of tumor organoids ( Fig.12 B).

[0198] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for constructing a co-culture model of immune cells and tumor organoids for evaluating anti-tumor activity, characterized in that: The steps include: (1) Peripheral blood mononuclear cells are isolated from the peripheral blood of healthy subjects, and then pre-stimulated and expanded with γδT cells to obtain γδT cells; (2) The tumor tissue from the tumor patient was digested using a Tumor Dissociation Kit at 37°C and 180-220 rpm for 20-60 minutes, and then the digested tumor cells were passed through a 100 μm cell sieve, and the tumor cells were collected by centrifugation and mixed evenly with matrix gel, and organoid culture medium was added for organoid culture and subculture, and then the size of the tumor organoids was screened using 37 μm and 100 μm cell sieves to obtain humanized tumor organoids of 37 μm to 100 μm; (3) pre-coating a Corning 96-well all-white flat-bottom polystyrene microplate with an anti-adhesion reagent, then plating the humanized tumor organoids obtained in step (2), counting them, and then mixing them with the γδT cells obtained in step (1) at an effector-target ratio of 1 to 10:1 to form a co-culture system, placing them in an incubator for culturing, and obtaining the immune cell and tumor organoid co-culture model for evaluating anti-tumor activity; The culture medium used for the γδT cell pre-stimulation culture in step (1) is a γδT cell pre-stimulation culture medium, and its formula is as follows: serum-free RPMI 1640 culture medium, 10% fetal bovine serum by volume, 1% penicillin-streptomycin double antibody by volume, 1200UI / ml Human IL-2, 50μM zoledronic acid; The culture medium used for the expansion culture in step (1) is a γδT cell culture medium, and its formula is as follows: serum-free RPMI1640 culture medium, 10% by volume of fetal bovine serum, 1% by volume of penicillin-streptomycin double antibody, and 300UI / ml Human IL-2; The digestion solution used in the digestion described in step (2) is prepared by the following method: 325 μl TumorDissociation Kit and 4.7 ml washing solution are mixed evenly to obtain a digestion solution; wherein the washing solution has the following formula: Advanced DMEM / F12 culture medium, 1× Glutamax additive, 1M Hepes buffer solution, 3× penicillin-streptomycin, 10 μM Y-27632; The formula of the organoid culture medium described in step (2) is as follows: Advanced DMEM / F12 medium, B27 supplement 1×, Glutamax supplement 1×, 1M Hepes buffer, penicillin-streptomycin 1×, 5mM nicotinamide, 1.25mM acetylcysteine, 5μm Y-27632, 500nM A83-01, 500nM SB 202190, 5ng / ml fibroblast growth factor 7, 20ng / ml fibroblast growth factor 10, 5ng / ml epidermal growth factor, 100ng / ml Noggin, 250ng / ml R-spondin-1, 5nM Heregulin β-1, 50μg / ml primary cell antibiotic Primocin; The 100 μm cell sieve described in step (2) is a 100 μm Reversible Strainer cell sieve; The 37 μm cell sieve described in step (2) is a 37 μm Reversible Strainer cell sieve; The anti-adhesion reagent described in step (3) is Anti-adherence Rinsing Solution.

2. The method according to claim 1, characterized in that: The effector-target ratio in step (3) is 1:1, 5:1 or 10:

1.

3. The method according to claim 1, characterized in that: The pre-stimulation culture and expansion culture in step (1) are cultured in a 24-well plate, and the total time of the pre-stimulation culture and expansion culture is 11 to 13 days; wherein the seeding density of the pre-stimulation culture is 3.0×10 6 ~4.0×10 6 cells / well, and the seeding density of the expansion culture was 1.0×10 6 ~4.0×10 6 cells / well.

4. The method according to claim 1, characterized in that: The matrix glue described in step (2) is Matrigel matrix glue.

5. The method according to claim 1, characterized in that: The tumor described in step (2) is a malignant tumor; The digestion time in step (2) is 20 to 40 minutes; The culture described in step (3) is carried out in an incubator at 37°C and 5% CO2.

6. The method according to claim 5, characterized in that: The tumor in step (2) is breast cancer or lung cancer; The digestion time in step (2) is 30 minutes.

7. The immune cell and tumor organoid co-culture model constructed by the method for constructing an immune cell and tumor organoid co-culture model for evaluating anti-tumor activity as described in any one of claims 1 to 6.

8. A method for evaluating the anti-tumor activity of immune cells in vitro for non-disease diagnosis and treatment purposes, characterized in that: The method comprises steps (1) to (3) as described in any one of claims 1 to 6, and at least one of the following steps: (4) adding a tracer dye and a fluorescent dye for detecting tumor cell death to the co-culture system in step (3), and then continuously photographing the co-culture system under bright field and fluorescence channels to continuously detect the degree of tumor cell death in the co-culture system, the interaction between γδT cells and tumor organoids, and the killing activity of γδT cells on tumor organoids; (5) adding a fluorescent dye for detecting tumor cell death to the co-culture system in step (3), then using a multifunctional microplate reader to detect the relative fluorescence value of the co-culture system, and calculating and analyzing the killing activity of γδT cells against tumor organoids; (6) detecting the concentration level of effector factors related to cytotoxicity in the supernatant of the co-culture system in step (3) to comprehensively evaluate the killing activity of γδT cells against tumor organoids; The fluorescent dye described in steps (4) and (5) is Promega Green dye.

9. The method according to claim 8, characterized in that: The tracking dye described in step (4) is Celltracker red CMTPX dye; The detection described in step (6) is performed using a CBA multifactor kit; The effector factor in step (6) includes at least one of IFN-γ, Granzyme B, Perforin and TNF-α.

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