A tumor organoid-immune cell co-culture method, co-culture system and use thereof

Abstract

The application relates to a tumor organoid-immune cell co-culture method, a co-culture system and application thereof. The method comprises the following steps: S1. adding pre-cooled mixed matrix material into a culture container, and forming a matrix layer after solidification; wherein the mixed matrix material comprises Matrigel matrix glue and gelatin hydrogel in a volume ratio of 1: (0.1-10); S2. inoculating tumor organoid suspension obtained through pretreatment onto the surface of the matrix layer, so that the tumor organoid is attached to the matrix layer, and dropping immune cell suspension obtained through pretreatment onto the surface of the tumor organoid attached to the matrix layer; and S3. adding co-culture medium into the culture container, so that the tumor organoid and the immune cell are immersed in the co-culture medium, and dynamic co-culture is carried out. The method solves the problems of poor co-culture compatibility, poor stability and low repeatability in the prior art, and realizes long-term, efficient and stable co-culture of the tumor organoid and the immune cell.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. Specifically, this invention relates to a method for co-culturing tumor organoids and immune cells, a co-culture system based on this method, and its applications. Background Technology

[0002] Tumor organoids are three-dimensional structures formed by culturing tumor tissue or tumor cells in vitro. They can highly mimic the tissue structure, cellular heterogeneity, and biological functions of tumors in vivo and have been widely used in tumor disease modeling, drug screening, and evaluation of immunotherapy efficacy. Tumor immune co-culture technology involves co-culturing tumor organoids with immune cells (such as T cells, NK cells, and macrophages) to construct an in vitro tumor immune microenvironment. This is a key technological platform for studying the interaction between tumors and the immune system and screening immunotherapeutic drugs (such as PD-1 / PD-L1 inhibitors and CAR-T cells).

[0003] Currently, methods for co-culturing tumor organoids and immune cells are mainly divided into two categories: direct co-culture and indirect co-culture. Direct co-culture involves mixing tumor organoids and immune cells in the same culture system, allowing direct contact between cells. Indirect co-culture, on the other hand, uses devices such as Transwell chambers to physically separate the two, allowing only soluble factors (such as cytokines, nutrients, and metabolites) to communicate. However, the existing co-culture methods mostly use culture systems modified from conventional two-dimensional cell culture systems or single organoid culture systems, without systematic and targeted improvements to address the differentiated needs of co-culturing tumor organoids and immune cells. This results in significant deficiencies in simulating the in vivo tumor immune microenvironment.

[0004] Through analysis, the inventors discovered that the root cause of the defects in the prior art lies in its failure to fully consider the differentiated growth characteristics of tumor organoids and immune cells, specifically manifested in the following three aspects:

[0005] First, the matrix materials have poor adaptability and cannot simultaneously meet the needs of three-dimensional support and immune cell function. Specifically, tumor organoids need to grow in a three-dimensional matrix to maintain their three-dimensional structure and biological function. However, while the Matrigel matrix gel commonly used in existing technologies can provide the basement membrane components required for organoid growth, it is difficult to maintain a stable three-dimensional structure during long-term co-culture. At the same time, immune cells (such as T cells) need to migrate, infiltrate, and recognize target cells in a matrix with a certain mechanical stiffness. The Matrigel system alone cannot simultaneously ensure the structural maintenance of organoids and the functional performance of immune cells.

[0006] Second, the culture medium composition is poorly designed, making it difficult to simultaneously meet the physiological needs of both organoid growth and immune cell activation. Specifically, tumor organoids and immune cells have different requirements for nutritional factors and signaling molecules. Existing co-culture methods mostly employ strategies of using a single culture medium or simply mixing two types of culture media without systematic screening and optimization. This results in the co-culture system failing to simultaneously meet the physiological needs of both cell types, manifesting as problems such as limited organoid growth, decreased immune cell activity, and shortened co-culture cycles.

[0007] Third, the lack of dynamic control over culture conditions makes it impossible to accurately simulate the in vivo microenvironment. Specifically, the in vivo tumor immune microenvironment is characterized by dynamic changes, while existing co-culture methods mostly employ static culture modes, resulting in the inability to simulate the dynamic changes in nutrient concentrations and metabolite levels within the culture system. Furthermore, existing technologies lack systematic optimization and standardized design for key parameters such as inoculation ratio, media change timing, and culture cycle, leading to poor reproducibility between different batches of experiments and low co-culture stability, making it difficult to meet the data reliability requirements of drug screening and mechanism studies.

[0008] Therefore, developing a co-culture method that can simultaneously achieve three-dimensional growth of tumor organoids and maintenance of immune cell function, accurately simulate the dynamic microenvironment in vivo, and possess good stability and reproducibility has become an urgent technical problem to be solved in this field. Summary of the Invention

[0009] To address the above problems, the purpose of this invention is to provide a method for co-culturing tumor organoids and immune cells, a co-culture system based on this method, and its applications. The co-culture method of this invention solves the problems of poor compatibility, inaccurate microenvironment simulation, poor stability, and low reproducibility in existing technologies. It achieves long-term, efficient, and stable co-culture of tumor organoids and immune cells, constructing a tumor immune microenvironment that more closely resembles the actual in vivo state, thus improving the reliability and in vitro-in vivo consistency of experimental results.

[0010] In this invention, the term "pre-cooling" refers to cooling to the ice bath temperature (0-4°C).

[0011] In this invention, the term "Matrigel matrix" should be broadly understood to refer to a class of basement membrane matrix gels extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma or other natural / recombinant sources. These gels contain characteristic basement membrane components such as laminin, type IV collagen, nestin, and heparan sulfate proteoglycans, as well as natural bioactive factors. They can self-assemble under physiological temperature conditions to form a biologically active three-dimensional hydrogel matrix, used to mimic the structure and function of the in vivo cell basement membrane to support cell adhesion, proliferation, differentiation, and three-dimensional morphogenesis. This type of matrix gel includes, but is not limited to, Matrigel. TMGeltrex TM Cultrex TM Commercial products and their functional equivalents. As a specific example, Corning's low growth factor (GFR) and phenol red-free Matrigel matrix adhesive (e.g., catalog number 356231) can be used.

[0012] In this invention, the term "gelatin hydrogel" refers to a polymeric material with a hydrophilic three-dimensional network structure formed on a gelatin base. This material can retain a large amount of water in a swollen state without dissolving, while preserving the inherent biological properties and good biocompatibility of gelatin. The gelatin can be a commercially available product, such as gelatin products with catalog numbers G1890 and G9391 from Sigma-Aldrich; the PhotoGel® series from Advanced BioMatrix; methacryloyl gelatin (GelMA) with models G7002 and fluorescently labeled G7011 from Solarbio; the PhotoGel® series methacryloyl gelatin from Advanced BioMatrix; and functional equivalents prepared in the laboratory based on the principles.

[0013] In this invention, the term "CHIR-99021" is a small molecule GSK-3 inhibitor with the chemical name 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarboxynitrile and the CAS number 252917-06-9.

[0014] The above-mentioned objective of the present invention is achieved by providing the following technical solution:

[0015] In a first aspect, the present invention provides a method for co-culturing tumor organoids and immune cells, comprising the following steps:

[0016] S1. Add pre-cooled mixed matrix material to a culture container, and form a matrix layer after solidification; wherein the mixed matrix material comprises Matrigel matrix glue and gelatin hydrogel in a volume ratio of 1:(0.1-10);

[0017] S2. The pretreated tumor organoid suspension is seeded onto the surface of the matrix layer to allow the tumor organoids to adhere to the matrix layer, and the pretreated immune cell suspension is dropped onto the surface of the tumor organoids attached to the matrix layer.

[0018] S3. Add co-culture medium to the culture container, so that the tumor organoids and immune cells are immersed in the co-culture medium for dynamic co-culture.

[0019] According to some embodiments of the present invention, in step S1, the volume ratio of the Matrigel matrix gel to the gelatin hydrogel is 1:(0.5-3), preferably 1:(1-2).

[0020] According to some embodiments of the present invention, the gelatin hydrogel is formed from gelatin and an aqueous solvent; preferably, the concentration of the gelatin hydrogel is 5-15 g / 100 mL, more preferably 7-9 g / 100 mL; preferably, the aqueous solvent is PBS buffer and / or deionized water; more preferably, the PBS buffer comprises: 6-10 g / L sodium chloride, 0.1-0.3 g / L potassium chloride, 1-2 g / L disodium hydrogen phosphate and 0.15-0.35 g / L potassium dihydrogen phosphate; more preferably, the pH of the PBS buffer is 7.2-7.4.

[0021] According to some embodiments of the present invention, in step S1, the thickness of the matrix layer is 200-300 μm, preferably 240-260 μm.

[0022] According to some embodiments of the present invention, in step S1, the curing includes curing in an incubator at 37±1℃ and 5±1% CO2 for 20-30 min.

[0023] According to some embodiments of the present invention, in step S2, the inoculation ratio of the number of tumor organoid cells to the number of immune cells is 1:(5-80), preferably 1:(10-50).

[0024] According to some embodiments of the present invention, in step S2, the pretreatment of the tumor organoids includes: digesting, washing, and centrifuging tumor organoids cultured for 3-5 days with a diameter of 100-300 μm and intact morphology, and then resuspending them in the pre-cooled mixed matrix material to obtain the tumor organoid suspension; preferably, the digestion is performed using TrypLE Express digestive enzyme, more preferably, the tumor organoids are digested using TrypLE Express digestive enzyme to a semi-discrete state in which each cell cluster retains 2-5 cells.

[0025] According to some embodiments of the present invention, the tumor organoids are selected from one or more of lung cancer organoids, gastric cancer organoids, colorectal cancer organoids, breast cancer organoids, and liver cancer organoids.

[0026] According to some embodiments of the present invention, the tumor organoids are derived from primary tumor tissue or tumor cell lines of tumor patients, or are formed by induced differentiation of induced pluripotent stem cells.

[0027] According to some embodiments of the present invention, in step S2, the pretreatment of the immune cells includes: activating and culturing the immune cells in an activation medium, washing, centrifuging, and then adjusting the cell concentration to 1×10⁻⁶. 6 -5×10 6 The immune cell suspension is obtained by culturing cells at a concentration of 10-20 IU / mL, 5-15 ng / mL of interleukin-7 (IL-7), and 0.5-1.5 μg / mL of anti-CD3 / CD28 antibody. Preferably, the activation culture time is 24-48 h.

[0028] According to some embodiments of the present invention, the immune cells are selected from one or more of T cells, NK cells, and macrophages.

[0029] According to some embodiments of the present invention, in step S3, the co-culture medium comprises a basal medium and the following components added to the basal medium: 2-8% by weight Matrigel, 10-20 ng / mL epidermal growth factor (EGF), 5-10 ng / mL fibroblast growth factor-2 (FGF-2), 5-10 ng / mL hepatocyte growth factor (HGF), 1-2 μM CHIR-99021, 10-20 IU / mL interleukin-2 (IL-2), 5-15 ng / mL interleukin-7, 5-10 ng / mL interleukin-15 (IL-15), 0.5-1.5% by volume B27 additive, 0.5-1.5% by volume penicillin-streptomycin bispecific antibody, and 0.1-0.2 mM ascorbic acid.

[0030] Preferably, in step S3, the co-culture medium comprises a basal medium and the following components added to the basal medium: 4-6% by weight Matrigel, 14-16 ng / mL epidermal growth factor, 7-9 ng / mL fibroblast growth factor-2, 7-9 ng / mL hepatocyte growth factor, 1.4-1.6 μM CHIR-99021, 14-16 IU / mL interleukin-2, 9-11 ng / mL interleukin-7, 7-9 ng / mL interleukin-15, 0.9-1.1% by volume B27 additive, 0.9-1.1% by volume penicillin-streptomycin bispecific antibody, and 0.14-0.16 mM ascorbic acid.

[0031] According to some embodiments of the present invention, the basal culture medium is selected from one or more of DMEM / F-12, Advanced DMEM / F-12, RPMI 1640, IMDM and MEM culture media, preferably Advanced DMEM / F-12 culture medium.

[0032] According to some embodiments of the present invention, in step S3, the culture parameters of the dynamic co-culture include: temperature of 37±2℃; CO2 concentration of 5±1%; oxygen concentration of 5-8%; and a culture period of at least 7 days, preferably 14-21 days.

[0033] According to some embodiments of the present invention, in step S3, the dynamic co-culture adopts a gradient perfusion method for circulating the culture medium; preferably, the flow rate of the gradient perfusion is set as follows: 45-55 μL / h in the early stage of co-culture, 65-75 μL / h in the middle stage of co-culture, and 95-105 μL / h in the later stage of co-culture.

[0034] This invention employs a gradient perfusion strategy. In the early stages of co-culture, a low flow rate is used to reduce the damage to cells caused by fluid shear force, protecting newly inoculated organoids and immune cells. In the later stages, as cell density and metabolic demands increase, the perfusion flow rate is gradually increased to dynamically match the needs of nutrient supply and metabolic waste removal. This avoids the decline in cell viability caused by the accumulation of metabolic waste in traditional static culture and also overcomes the problem of excessive dilution of autologous factors that may occur in the early stages of constant flow rate perfusion, providing an optimized hydrodynamic environment for long-term stable co-culture.

[0035] According to some embodiments of the present invention, in step S3, the dynamic co-culture further includes: periodically performing half-volume replacement of the co-culture medium during the co-culture period; preferably, the half-volume replacement is performed on the 2nd, 4th and 6th days of co-culture, and the perfusion flow rate is reduced to 15-25 μL / h during the replacement, while half of the co-culture medium is replaced with fresh medium.

[0036] According to some embodiments of the present invention, in step S3, the liquid level of the co-culture medium is 500-600 μm, preferably 540-560 μm.

[0037] Secondly, the present invention provides a tumor organoid-immune cell co-culture system, which is constructed by the co-culture method described in the first aspect of the present invention.

[0038] Thirdly, the present invention provides the use of a co-culture system constructed by the co-culture method according to the first aspect of the present invention or the co-culture system according to the second aspect of the present invention in the preparation or screening of tumor immunotherapy drugs and in the study of tumor immune mechanisms.

[0039] The present invention has at least the following beneficial effects:

[0040] 1. This invention utilizes a hybrid matrix material composed of Matrigel and gelatin hydrogel, which balances the structural requirements for three-dimensional growth of tumor organoids with the functional requirements for the survival and activation of immune cells, significantly improving the compatibility of co-culturing the two.

[0041] Specifically, in the co-culture system of this invention, lung cancer, colorectal cancer, and breast cancer organoids all showed good growth trends, maintaining a complete spherical structure on day 21. This indicates that the co-culture system of this invention can support the long-term, stable three-dimensional growth of various tumor organoids and has good universality. In contrast, the Matrigel group alone showed blurred edges and irregular shapes from day 10, and by day 21, most organoids could no longer maintain a spherical structure. This indicates that the combination of Matrigel matrix gel and gelatin hydrogel is the key factor in achieving long-term co-culture in this invention.

[0042] Furthermore, activated PBMCs exhibited a clear dynamic infiltration process during co-culture, gradually infiltrating deeper from their attachment to the organoid surface. This result directly demonstrates that immune cells can effectively infiltrate tumor organoids in the co-culture system of this invention, providing a spatial basis for subsequent killing effects and more closely resembling the true state of the tumor immune microenvironment in vivo.

[0043] 2. In addition to using the mixed matrix material of the present invention, the present invention also optimizes the composition of the co-culture medium. Without adding expensive factors such as Wnt3a, Noggin, and R-Spondin1, it can still effectively meet the differentiated needs of the two types of cells and significantly reduce reagent costs.

[0044] 3. This invention systematically optimizes the entire co-culture process, including key steps such as cell pretreatment, inoculation ratio, and medium replacement strategy. It also maintains the physicochemical stability of the culture environment through dynamic parameter control, avoiding the accumulation of metabolic waste and fluctuations in the culture system, and further solves the pain points of poor reproducibility and low stability of existing technologies.

[0045] 4. Experimental results of this invention show that the co-culture method of this invention can extend the co-culture period to 21 days or more, and the morphological integrity rate of tumor organoids is >90% after 7 days of co-culture. In addition, the results of multiple batches of independent replicate experiments show that the relative standard deviation of the morphological integrity rate of tumor organoids is ≤8%, which is significantly better than existing co-culture methods.

[0046] 5. The co-culture method of this invention has broad applicability and can be applied to various tumor organoids such as lung cancer, colorectal cancer, breast cancer, gastric cancer, and liver cancer, as well as various immune cells such as T cells, NK cells, and macrophages, meeting different experimental needs such as tumor immune mechanism research and immunotherapy drug screening. Furthermore, the standardized and simplified operation process of this invention not only reduces waste of consumables due to operational errors but also allows for compatibility with conventional 24-well and 96-well plates and other high-throughput culture containers, enabling single-sample co-culture of multiple samples, significantly improving experimental efficiency, reducing unit sample cost, and facilitating large-scale application in laboratories. Attached Figure Description

[0047] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0048] Figure 1 Bright-field morphology images of lung cancer organoids in the co-culture system of this invention on days 0, 5, 10, 15 and 21, with a scale bar of 100 μm.

[0049] Figure 2 Bright-field morphology images of colorectal cancer organoids in the co-culture system of this invention on days 0, 5, 10, 15 and 21, with scale bars of 100 μm.

[0050] Figure 3 Bright-field morphology images of breast cancer organoids in the co-culture system of this invention on days 0, 5, 10, 15 and 21, with scale bars of 100 μm.

[0051] Figure 4 Bright-field morphology images of lung cancer organoids on days 0, 5, 10, 15, and 21 in a control system using only Matrigel (gelatin-free hydrogel). The scale bar in the images is 100 μm.

[0052] Figure 5 Fluorescence microscopy images of immune cell infiltration during the co-culture of breast cancer organoids and PBMCs; the PI channel (red fluorescence) is used to label dead cells, and the Merge (overlay image) is used to observe the spatial distribution relationship between activated PBMCs and breast cancer organoid cells.

[0053] Figure 6The images show fluorescence microscopy images of immune cell infiltration and killing in colorectal cancer organoids on day 9 of co-culture under different effector-to-target ratios (E:T=5:1, 10:1, 20:1). Bright images represent bright-field cell images, AO channels (green fluorescence) label surviving tumor organoids, PI channels (red fluorescence) label dead cells, and Merge (overlay image) is used to observe the spatial distribution relationship between activated PBMCs and colorectal cancer organoid cells. The scale bar in the images is 300 μm. Detailed Implementation

[0054] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0055] Unless otherwise specified, the methods or conditions used in the following examples were performed according to conventional methods disclosed in the art. Unless otherwise stated, all reagents or instruments used in the following examples are commercially available products.

[0056] It should be noted that in the following examples, "E:T" refers to the effector-to-target ratio. "E" refers to all cells in peripheral blood mononuclear cells (PBMCs). After treatment with the activation culture medium, T cells, NK cells, macrophages, and other subsets in PBMCs are synergistically activated and exert tumor-killing functions. Therefore, in the E:T ratio used to describe the killing effect in the following examples, "E" refers to the total number of PBMC cells; "T" refers to the target cells (tumor organoid cells).

[0057] Method for detecting morphological integrity: After a specified number of days of co-culture, five fields of view were randomly selected from each well under an inverted fluorescence microscope. The total number of organoids and the number of morphologically intact organoids within each field of view were counted. Morphological integrity rate (%) = (Number of morphologically intact organoid cells / Total number of organoid cells) × 100%. "Morphological integrity" is defined as organoids that, when observed under an inverted fluorescence microscope, have clear boundaries, compact structure, smooth surface, no obvious debris attachment, no cyst formation, no necrotic core, and an overall spherical or near-spherical shape.

[0058] Example 1

[0059] 1. Experimental materials and equipment

[0060] Tumor organoids: Corresponding tumor organoids isolated and cultured from primary tumor tissues of patients with lung cancer, colorectal cancer, and breast cancer, respectively, cultured for 7 days, with a diameter of 200-250 μm and intact morphology.

[0061] Immune cells: PBMCs isolated from peripheral blood of healthy individuals using Ficoll density gradient centrifugation.

[0062] Advanced DMEM / F12 medium: purchased from Gibco, catalog number: 12634028 (500 mL / bottle, standard liquid, containing GlutaMAX and HEPES, sodium pyruvate-free).

[0063] Matrigel: Purchased from Corning, catalog number: 356231 (10 mL, low growth factor (GFR), phenol red-free).

[0064] Gelatin: Purchased from Sigma-Aldrich, catalog number: G1890 (Type A, porcine skin source, approximately 300 g Bloom, cell culture grade).

[0065] The PBS buffer contains: 8.0 g / L sodium chloride, 0.2 g / L potassium chloride, 1.44 g / L disodium hydrogen phosphate and 0.24 g / L potassium dihydrogen phosphate.

[0066] Culture container: 24-well cell culture plate.

[0067] Detection equipment: flow cytometer, inverted fluorescence microscope, and ELISA reader, etc.

[0068] 2. Experimental Procedure

[0069] Step 1: Pretreatment of culture containers

[0070] Disinfect the 24-well culture plate with 75% alcohol by volume, irradiate with ultraviolet light for 30 min, soak in PBS buffer pre-warmed to 37℃ for 10 min, discard the PBS, and dry for later use.

[0071] Step 2: Laying the substrate layer

[0072] Gelatin was dissolved in PBS buffer to prepare a gelatin hydrogel with a concentration of 8.3% by weight. Matrigel matrix and gelatin hydrogel were mixed evenly at a volume ratio of 1:1.5 to obtain a mixed matrix material. After pre-cooling, the mixed matrix material was added to a 24-well culture plate with a thickness of 250 μm in each well and placed in a 37℃, 5% CO2 incubator for 25 min to form a matrix layer.

[0073] Matrix layer control group: To verify the advantages of the hybrid matrix layer of the present invention, a matrix layer control group was set up, in which only Matrigel (gelatin-free hydrogel) was used to lay the matrix layer, and all other culture conditions were exactly the same. This was used for the co-culture of lung cancer organoids and immune cells (results are shown in...). Figure 4 ).

[0074] Step 3: Cell Pretreatment

[0075] Tumor organoid pretreatment: Tumor organoids cultured for 7 days with a diameter of 200-250 μm and intact morphology were gently digested with TrypLE Express digestive enzymes until each cell cluster retained 2-5 cells in a semi-discrete state. After washing twice with PBS, centrifuging at 800 rpm for 5 min, and resuspending in pre-cooled mixed matrix material for later use.

[0076] Immunotherapy: PBMCs were activated and cultured for 48 h in an activation medium containing IL-2 (15 IU / mL), IL-7 (10 ng / mL), and anti-CD3 / CD28 antibody (1 μg / mL). After washing once with PBS, the cells were centrifuged at 1000 rpm for 5 min and the cell concentration was adjusted to 3 × 10⁻⁶ cells / mL. 6 Units / mL are available for use.

[0077] Step 4: Cell seeding

[0078] Tumor organoid inoculation: Pretreated lung cancer, colorectal cancer, and breast cancer tumor organoid suspensions were uniformly inoculated onto the matrix layer surface of the corresponding wells at a density of 80 cell clusters / well. The wells were incubated at 37°C and 5% CO2 for 1.5 h to allow the organoids to fully adhere to the matrix layer.

[0079] Immune cell inoculation: Inoculation was performed at E:T (effect-to-target ratio) ratios of 5:1, 10:1, and 20:1. After inoculation, the culture plate was gently shaken to ensure even distribution of PBMCs.

[0080] Immune cell control group: To assess the basal growth status and spontaneous apoptosis level of tumor organoids, a negative control group without PBMCs was set up for each type of tumor organoid (i.e., only the organoid was received, without the addition of immune cells). The results of the negative control group for colorectal cancer organoids are shown below. Figure 6 .

[0081] Step 5: Add to the co-culture system

[0082] Add co-culture medium preheated to 37°C to each well, ensuring the medium completely covers the cells to a height of approximately 550 μm. The co-culture medium is based on Advanced DMEM / F12, supplemented with the following components: 5% (w / w) Matrigel, 15 ng / mL EGF, 8 ng / mL FGF-2, 8 ng / mL HGF, 1.5 μM CHIR-99021, 15 IU / mL IL-2, 10 ng / mL IL-7, 8 ng / mL IL-15, 1% (v / v) B27 supplement (Gibco B27 Supplement (50×), serum-free, catalog number: 17504-044), 1% (v / v) penicillin-streptomycin stock solution (containing 10000 U / mL penicillin and 10 mg / mL streptomycin), and 0.15 mM ascorbic acid.

[0083] Step 6: Dynamic Co-cultivation Regulation

[0084] The culture plate was placed in a dynamic culture environment, and the culture parameters were set as follows: temperature 37℃, CO2 concentration 5%, oxygen concentration 6%. The culture medium was circulated using a gradient perfusion method, and the perfusion flow rate was set as follows: 50 μL / h for co-culture days 1-3, 70 μL / h for days 4-7, and 100 μL / h for days 8-21.

[0085] Step 7: Fluid Change and Maintenance

[0086] Half-volume media replacement was performed on days 2, 4, and 6 of co-culture. During media replacement, the perfusion flow rate was reduced to 20 μL / h, and fresh pre-warmed co-culture medium was slowly replaced. Morphological changes of various tumor organoids and the distribution of PBMCs were observed daily using an inverted fluorescence microscope. The growth of various organoids and the degree of immune cell infiltration were recorded.

[0087] Step 8: Termination of co-culture and detection

[0088] After 21 days of co-culture, the culture was terminated, and various tumor organoids and their corresponding PBMCs were collected. The infiltration of PBMCs into different tumor organoids was observed using an inverted fluorescence microscope.

[0089] Survival and apoptosis of various tumor organoids under different E:T ratios were detected by combining AO / PI fluorescence staining with inverted fluorescence microscopy.

[0090] 3. Experimental Results

[0091] (1) Dynamic growth of different tumor organoids in co-culture system

[0092] To evaluate the support effect of the co-culture system of this invention on different types of tumor organoids, bright-field observations were conducted on lung cancer, colorectal cancer, and breast cancer organoids for 21 consecutive days. Figures 1 to 3 As shown:

[0093] Lung cancer organoids ( Figure 1 On day 0 after inoculation, the organoids appear as small, round cell clusters; on day 5, they begin to proliferate significantly and increase in size; from day 10 to day 15, the number of organoids increases and their shape tends to be regular spherical; on day 21, the diameter of the organoids further increases, the boundaries are clear, and no obvious disintegration is observed.

[0094] Colorectal cancer organoids ( Figure 2 The growth trend was similar to that of lung cancer organoids. After 10 days, a small number of dense areas appeared inside the organoids, but the overall structure remained intact. By day 21, the organoids maintained good morphology, indicating that the co-culture system also supported the long-term growth of colorectal cancer organoids.

[0095] Breast cancer organoids ( Figure 3 The growth trend is similar to that of lung cancer organoids and colorectal cancer organoids. By day 15, the organoids had formed large spherical structures, and by day 21, the overall integrity rate remained above 85%.

[0096] To verify the advantages of the hybrid matrix material of this invention, a control group using only Matrigel matrix adhesive (gelatin-free hydrogel) was set up. The experimental results are as follows: Figure 4 As shown, when lung cancer organoids were cultured in a single Matrigel group, some organoids exhibited blurred edges and irregular shapes by day 10; significant disintegration and fragmentation began to occur on day 15; and by day 21, most organoids could no longer maintain a spherical structure. In contrast, the hybrid matrix material of this invention (… Figure 1 The lung cancer organoids remained morphologically intact throughout the 21-day culture period. This indicates that the hybrid matrix material, a combination of Matrigel and gelatin hydrogel, is a key factor in achieving long-term co-culture in this invention.

[0097] After 7 days of co-culture, the morphological integrity rates of lung cancer, colorectal cancer, and breast cancer organoids in the co-culture system of this invention were 93%, 91%, and 92%, respectively, and remained above 85% after 21 days, while the integrity rate of the Matrigel-only group had dropped below 70% after 7 days.

[0098] These results also demonstrate that the co-culture system of the present invention can support the long-term, stable three-dimensional growth of various tumor organoids and has good universality.

[0099] (2) Results of fluorescent staining

[0100] Taking breast cancer organoids as an example, the infiltration of activated PBMCs at different time points was observed using fluorescence microscopy. Figure 5 (E:T=10:1). On day 0 (immediately after inoculation), activated PBMCs were evenly distributed around the organoids; on day 7, some activated PBMCs were visible adhering to the surface of the organoids; on day 14, activated PBMCs clearly migrated into the interior of the organoids, exhibiting a typical "encapsulation" phenomenon; on day 21, a large number of activated PBMCs had infiltrated deep into the organoids, while the organoid morphology remained intact. These results visually demonstrate that immune cells in the co-culture system of this invention can effectively infiltrate tumor organoids, providing a spatial basis for subsequent killing effects.

[0101] (3) Immune killing effect at different effect-to-target ratios (E:T)

[0102] Taking colorectal cancer organoids as an example, a control group without PBMCs and experimental groups with E:T ratios of 5:1, 10:1, and 20:1 were set up. AO / PI fluorescence staining was performed on day 9 of co-culture. Figure 6 The results show:

[0103] In the control group without PBMCs, tumor organoids showed strong green fluorescence in the AO channel and no obvious red fluorescence signal in the PI channel, indicating that there was almost no death in the tumor organoids.

[0104] When E:T = 5:1, the AO channel (green, surviving cells) predominates, while the PI channel (red, dead cells) shows only sporadic punctate fluorescence.

[0105] When E:T = 10:1, the red fluorescence signal is significantly enhanced and the green fluorescence intensity decreases, indicating an increase in the number of dead organoid cells;

[0106] When E:T = 20:1, red fluorescence covers most of the organoid region, while green fluorescence is limited to a few residual cell clusters, indicating the presence of a large number of dead organoid cells.

[0107] The above results confirm that the immune cells in the co-culture system of the present invention have a dose-dependent killing effect on tumor organoids, and the higher the effect-to-target ratio, the more significant the killing effect.

[0108] (4) Stability results

[0109] In this embodiment, three batches of experiments were repeated, and the RSD of the morphological integrity rate of tumor organoids was ≤8%, indicating that the method of the present invention has excellent stability and repeatability.

[0110] In summary, the tumor organoid immune co-culture method of the present invention successfully achieved long-term stable co-culture of tumor organoids and T cells. The co-culture period can reach 21 days, during which the tumor organoids maintain their morphology, the immune cell infiltration effect is good, and the tumor organoids exhibit a dose-dependent killing effect. This indicates that the method of the present invention can effectively simulate the interaction process between tumors and immune cells in vivo, and the constructed in vitro tumor immune microenvironment closely resembles the real in vivo state, fully achieving the objectives of the present invention.

[0111] The above descriptions are merely several exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any equivalent or similar implementation schemes obtained by those skilled in the art by making some modifications or alterations to the above-disclosed technical content without departing from the scope of the present invention are within the scope of the present invention.

Claims

1. A method for co-culturing tumor organoids and immune cells, comprising the following steps: S1. Add pre-cooled mixed matrix material to a culture container, and form a matrix layer after solidification; wherein the mixed matrix material comprises Matrigel matrix glue and gelatin hydrogel in a volume ratio of 1:(0.1-10); S2. The pretreated tumor organoid suspension is seeded onto the surface of the matrix layer to allow the tumor organoids to adhere to the matrix layer, and the pretreated immune cell suspension is dropped onto the surface of the tumor organoids attached to the matrix layer. S3. Add co-culture medium to the culture container, so that the tumor organoids and immune cells are immersed in the co-culture medium for dynamic co-culture.

2. The method according to claim 1, wherein, In step S1, the volume ratio of the Matrigel matrix gel to the gelatin hydrogel is 1:(0.5-3), preferably 1:(1-2). Preferably, the gelatin hydrogel is formed from gelatin and an aqueous solvent; More preferably, the concentration of the gelatin hydrogel is 5-15 g / 100mL, more preferably 7-9 g / 100mL; More preferably, the aqueous solvent is PBS buffer and / or deionized water; More preferably, the PBS buffer comprises: 6-10 g / L sodium chloride, 0.1-0.3 g / L potassium chloride, 1-2 g / L disodium hydrogen phosphate and 0.15-0.35 g / L potassium dihydrogen phosphate; More preferably, the pH value of the PBS buffer is 7.2-7.4; Preferably, in step S1, the thickness of the matrix layer is 200-300 μm, more preferably 240-260 μm; Preferably, in step S1, the curing includes curing in an incubator at 37±1℃ and 5±1% CO2 for 20-30 min.

3. The method according to claim 1 or 2, wherein, In step S2, the inoculation ratio of the number of tumor organoid cells to the number of immune cells is 1:(5-80), preferably 1:(10-50).

4. The method according to any one of claims 1 to 3, wherein, In step S2, the pretreatment of the tumor organoids includes: digesting, washing, and centrifuging the tumor organoids cultured for 3-5 days, with a diameter of 100-300 μm and intact morphology, and then resuspending them in the pre-cooled mixed matrix material to obtain the tumor organoid suspension; Preferably, the digestion is performed using TrypLE Express digestive enzymes; Preferably, the tumor organoids are selected from one or more of lung cancer organoids, gastric cancer organoids, colorectal cancer organoids, breast cancer organoids, and liver cancer organoids; Preferably, the tumor organoids are derived from primary tumor tissue or tumor cell lines of cancer patients, or are formed by induced differentiation of induced pluripotent stem cells.

5. The method according to any one of claims 1 to 4, wherein, In step S2, the pretreatment of the immune cells includes: activating and culturing the immune cells in an activation medium, washing, centrifuging, and then adjusting the cell concentration to 1×10⁻⁶. 6 -5×10 6 The immune cell suspension was obtained by measuring cells / mL; Preferably, the activation culture medium contains 10-20 IU / mL IL-2, 5-15 ng / mL interleukin-7, and 0.5-1.5 μg / mL anti-CD3 / CD28 antibody; Preferably, the activation culture time is 24-48 h; Preferably, the immune cells are selected from one or more of T cells, NK cells, and macrophages.

6. The method according to any one of claims 1 to 5, wherein, In step S3, the co-culture medium comprises a basal medium and the following components added to the basal medium: 2-8% by weight Matrigel, 10-20 ng / mL epidermal growth factor, 5-10 ng / mL fibroblast growth factor-2, 5-10 ng / mL hepatocyte growth factor, 1-2 μM CHIR-99021, 10-20 IU / mL interleukin-2, 5-15 ng / mL interleukin-7, 5-10 ng / mL interleukin-15, 0.5-1.5% by volume B27 additive, 0.5-1.5% by volume penicillin-streptomycin bispecific antibody, and 0.1-0.2 mM ascorbic acid; Preferably, in step S3, the co-culture medium comprises a basal medium and the following components added to the basal medium: 4-6% by weight of Matrigel, 14-16 ng / mL of epidermal growth factor, 7-9 ng / mL of fibroblast growth factor-2, 7-9 ng / mL of hepatocyte growth factor, 1.4-1.6 μM of CHIR-99021, 14-16 IU / mL of interleukin-2, 9-11 ng / mL of interleukin-7, 7-9 ng / mL of interleukin-15, 0.9-1.1% by volume of B27 additive, 0.9-1.1% by volume of penicillin-streptomycin bispecific antibody, and 0.14-0.16 mM of ascorbic acid; Preferably, the basal culture medium is selected from one or more of DMEM / F-12, Advanced DMEM / F-12, RPMI 1640, IMDM and MEM culture media, with Advanced DMEM / F-12 culture medium being the most preferred.

7. The method according to any one of claims 1 to 6, wherein, In step S3, the cultivation parameters for the dynamic co-culture include: temperature of 37±2℃; CO2 concentration of 5±1%; oxygen concentration of 5-8%; and a cultivation period of at least 7 days, preferably 14-21 days.

8. The method according to any one of claims 1 to 7, wherein, In step S3, the dynamic co-culture employs a gradient perfusion method for culture medium circulation; Preferably, the flow rate of the gradient perfusion is set as follows: 45-55 μL / h in the early stage of co-culture, 65-75 μL / h in the middle stage of co-culture, and 95-105 μL / h in the later stage of co-culture; Preferably, in step S3, the dynamic co-culture further includes: periodically performing half-volume replacement of the co-culture medium during the co-culture period; more preferably, the half-volume replacement is performed on the 2nd, 4th and 6th days of co-culture, and the perfusion flow rate is reduced to 15-25 μL / h during the replacement, while half of the co-culture medium is replaced with fresh medium. Preferably, in step S3, the liquid level of the co-culture medium is 500-600 μm, more preferably 540-560 μm.

9. A tumor organoid-immune cell co-culture system, which is constructed by the co-culture method according to any one of claims 1 to 8.

10. The use of the co-culture system constructed by the co-culture method according to any one of claims 1 to 8 or the co-culture system according to claim 9 in the preparation or screening of tumor immunotherapy drugs and in the study of tumor immune mechanisms.

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