Complex tumor organoid co-culture system as well as construction method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses a complex tumor organoid co-culture system and a construction method and application thereof. The construction method comprises the following steps: construction of tumor organs, separation and induced differentiation culture of dendritic cells, fusion of tumor cells and maturely differentiated dendritic cells (DCs) through oncolytic viruses, and addition of immune cells for co-culture. According to the present invention, the patient-derived tumor cells are utilized to construct the tumor organoid, such that the pathological characteristics of the patient can be well restored; the oncolytic virus is used for inducing fusion of the dendritic cells and the primary tumor cells, so that related antigens, specific antigens and new antigens of the tumor cells can be presented more effectively, and the reaction of immune cells can be stimulated more efficiently; the rapid, simple and effective complex tumor organoid co-culture established by the invention can simulate the positioning and interaction relationship between immune cells and tumor cells in an in-vivo tumor microenvironment, and also provides a platform for evaluation of a novel immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a complex tumor organoid co-culture system, its construction method and application. Background Technology

[0002] Malignant tumors have long and seriously impacted the health of the nation, with lung cancer ranking first in both incidence and mortality among malignant tumors in my country. According to estimates by the International Agency for Research on Cancer, in 2020, China had 820,000 new cases of lung cancer and 710,000 deaths, accounting for 17.9% of all new cancer cases and 23.8% of all cancer deaths. Lung adenocarcinoma accounts for approximately 40% of lung cancers and is the most common type of non-small cell lung cancer. It has a high recurrence rate, is prone to metastasis, has a low survival rate, and a poor prognosis, necessitating the development of novel treatment methods, such as immunotherapy.

[0003] Tumor organoid culture is a cutting-edge technology for three-dimensional in vitro tumor culture. It can establish a tumor model with tumor heterogeneity and simulate the tumor microenvironment. Tumor cells form organ-like tissue structures, which can well represent the interactions between cells and between cells and the extracellular matrix. Therefore, compared with traditional tumor cell lines, this model has higher complexity, heterogeneity, clinical relevance, and individual diversity, making it more suitable for establishing organoid sample banks and for the development and evaluation of novel tumor immunotherapies.

[0004] However, existing lung cancer organoid models still face numerous challenges, such as the difficulty in simulating the interaction between immune cells and tumor tissue (including rejection, invasion, and killing) in the in vitro tumor microenvironment. Therefore, a new culture system model is urgently needed to address the difficulties in simulating the tumor microenvironment in vitro and to recreate the survival status and interaction between tumor tissue and immune cells in patients.

[0005] CN 117603912 A discloses a method for constructing a lung cancer organoid-TILs co-culture system, including the isolation of lung cancer cells / tumor-infiltrating T lymphocytes (TILs), the culture of TILs, the construction of lung cancer organoids, and the co-culture of lung cancer organoids / TILs. In this technology, infiltrating T lymphocytes from the patient's own tumor tissue are used as the source of T cells in the co-culture system. However, the degree of infiltration, cell subset composition, quantity, and distribution area of ​​TILs vary in different solid tumors and different clinical stages, making it difficult to apply the same protocol to different patients. Furthermore, the T cells in the patient's own TILs express limited TCRs and contain some exhausted T cells that are unable to function. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of the existing technology by providing a complex tumor organoid co-culture system comprising oncolytic viruses, immune cells, and other cells, as well as its construction method and applications.

[0007] The present invention achieves the above objectives through the following technical solutions:

[0008] In one aspect, the present invention provides a method for constructing a complex tumor organoid co-culture system, comprising the following steps:

[0009] S1. Obtain tumor tissue and culture it in vitro to form tumor organoids;

[0010] S2. After mixing tumor cells and dendritic cells (DCs), centrifuge to obtain cell pellet; resuspend the cell pellet in fusion cell culture medium containing oncolytic virus, and co-culture the mixed cells containing oncolytic virus to obtain fusion cells of dendritic cells (DCs) / tumor cells containing oncolytic virus.

[0011] S3. The tumor organoids described in step S1, the fusion cells, dendritic cells (DCs) described in step S2, and the immune cells are mixed and co-cultured to construct the complex tumor organoid co-culture system.

[0012] As a preferred technical solution of the present invention, the tumor organoids include, but are not limited to, organoids of lung cancer, intestinal cancer, kidney cancer, liver cancer, pancreatic cancer, stomach cancer, and breast cancer; the tumor cells correspond to the tumor organoids used, that is, when the tumor organoid is a lung cancer organoid, the tumor cells are lung cancer cells.

[0013] As a preferred embodiment of the present invention, the tumor organoid culture step S1 includes:

[0014] Obtain tumor tissue; wash the tumor tissue with tissue washing solution; digest the washed tumor tissue with tissue digestion solution; after a predetermined digestion time, stop digestion and filter the obtained cell suspension through a filter screen to collect single cells; count the collected single cells, centrifuge again to collect the cell pellet, and culture it in matrix gel, changing the tumor organoid culture medium regularly.

[0015] Furthermore, the components of the tumor organoid culture medium include: AdvancedDMEM / F12, P / S, Hepes, Glutamax, NAC, B27, SB202190, A83-01, Y27632, Nicotinamide, R-spindin, Noggin, EGF7, and EGF10.

[0016] In some embodiments of the present invention, the dendritic cells (DCs) in step S2 are mature dendritic cells (DCs) obtained by extracting monocytes or bone marrow cells from peripheral blood or bone marrow and inducing differentiation of the monocytes or bone marrow cells in a DCs cell culture medium containing Flt3L. The specific steps include:

[0017] a) Isolate monocytes from peripheral blood and induce their differentiation into dendritic cells (DCs):

[0018] Peripheral blood was obtained; peripheral blood mononuclear cells (PBMCs) were obtained from whole blood using density gradient centrifugation; the mononuclear cells were cultured in DCs culture medium containing Flt3L to induce differentiation.

[0019] b) Obtain bone marrow cells from bone marrow and induce their differentiation into dendritic cells (DCs):

[0020] The bone was transferred to a clean bench and soaked in alcohol. Then, the bone was transferred to a new culture dish containing PBS. Both ends of the bone were cut off. PBS was then drawn up with a syringe, and the needle was inserted into the bone marrow cavity from both ends of the bone to repeatedly flush out the bone marrow into the culture dish until the bone turned completely white. The bone marrow suspension was collected and filtered to remove small fragments and other tissues. After centrifugation, erythrocyte lysis buffer was added, the cells were resuspended, and incubated at room temperature. After adding erythrocyte lysis buffer as a stop solution, the cells were centrifuged again. The cells were washed with cell washing buffer and counted. The cells were cultured in DCs culture medium containing Flt3L to induce differentiation.

[0021] Furthermore, the components of the DCs cell culture medium include: 1640 medium, FBS, P / S, and Flt3L.

[0022] As a preferred technical solution of the present invention, in step S2, tumor cells are fused with mature dendritic cells (DCs) using oncolytic viruses to achieve the presentation of tumor-specific antigens, tumor-associated antigens and their neotumor antigens.

[0023] The specific steps for fusing tumor cells with mature dendritic cells (DCs) using oncolytic viruses include: mixing tumor cells and mature dendritic cells in the required ratio, centrifuging to obtain a cell pellet; preparing a fusion cell culture medium containing oncolytic viruses, resuspending the mixed cell pellet in the fusion cell culture medium containing oncolytic viruses; culturing the mixed cells containing oncolytic viruses in culture dishes coated with matrix gel, and co-culturing for a certain period of time to obtain fusion cells.

[0024] More preferably, before mixing the tumor cells with the dendritic cells, the dendritic cells are first cultured in a culture medium containing LPS for activation treatment.

[0025] Furthermore, in step S2, the mixing ratio of tumor cells to mature dendritic cells (DCs) is 10:1 to 1:10.

[0026] Furthermore, the oncolytic viruses include dozens of oncolytic viruses such as vesicular stomatitis virus (VSVs), herpes simplex virus (HSV), vaccinia virus, adenovirus (AdV), reovirus, and Newcastle disease virus.

[0027] In a preferred embodiment of the present invention, the oncolytic virus used is vesicular stomatitis virus (VSVs).

[0028] Furthermore, the fusion cell culture medium includes commonly used cell culture media, such as 1640 medium containing 10% FBS and 1x P / S.

[0029] A preferred embodiment is: oncolytic viruses (VSVs), 1640 medium, 10% fetal bovine serum (FBS), P / S, Hepes, Polybrene, and Opti-MEM.

[0030] Furthermore, the immune cells added to the co-culture system in step S3 include, but are not limited to, one or more of CD4+, CD8+ T cells, dendritic cells, and macrophages, which in vitro simulate the formation and function of tumor-infiltrating lymphocytes (TILs).

[0031] In some embodiments of the present invention, the immune cells used are CD4+ or CD8+ T cells obtained from peripheral blood or spleen, and the specific steps include:

[0032] a) Obtaining CD4+, CD8+ T cells from peripheral blood:

[0033] Peripheral blood was obtained; peripheral blood mononuclear cells (PBMCs) were obtained from whole blood using density gradient centrifugation; CD4+ and CD8+ T cells were separated by binding with corresponding magnetic beads; the separated CD4+ and CD8+ T cells were cultured in T cell culture medium.

[0034] b) Obtaining CD4+, CD8+ T cells from the spleen (using mice as an example), the steps include:

[0035] Mice (6-10 weeks old) were euthanized, and their spleens were surgically removed and placed in a cleaning solution. The spleens were then transferred to a laminar flow hood and cleaned with the cleaning solution. The spleens were ground with two sterile coverslips to release and collect the spleen cells. The obtained spleen cells were filtered through a sieve to remove small fragments and tissues, and then centrifuged. Red blood cell lysis buffer was added, the cells were resuspended, and incubated at room temperature. The lysis was terminated by adding a stop solution, and the cells were centrifuged again. The isolated CD8+ T cells and / or CD4+ T cells and other immune cells were cultured in T cell culture medium.

[0036] Furthermore, the components of the T cell culture medium include: 1640 medium, FBS, P / S, Glutamax, 2-mercaptoethanol, and IL-2.

[0037] The method for constructing the complex tumor organoid co-culture system further includes the following steps in obtaining the complex tumor organoid co-culture system:

[0038] The tumor organoids were digested into organoid single cells, and then co-cultured with the fusion cells of dendritic cells / tumor cells containing oncolytic viruses, the dendritic cells, and the immune cells in different concentrations of matrix gel to construct a single-cell organoid-complex tumor organoid co-culture system.

[0039] And / or, the tumor organoids are digested into organoid clusters, and co-cultured with the fusion cells of dendritic cells / tumor cells containing oncolytic viruses, dendritic cells, and immune cells in different concentrations of matrix gel to construct an organoid cluster-complex tumor organoid co-culture system;

[0040] The culture medium components used in the co-culture system include Advanced DMEM / F12, P / S, Hepes, Glutamax, NAC, B27, SB202190, A83-01, Y27632, Nicotinamide, R-spindin, Noggin, EGF7, EGF10, and IL-2.

[0041] Secondly, the present invention provides a complex tumor organoid co-culture system comprising oncolytic viruses, immune cells and other cells obtained by the above-described construction method.

[0042] Thirdly, this invention also provides the application of the method for constructing the complex tumor organoid co-culture system. In clinical practice, it can be applied to the screening of tumor drugs and assist in clinical treatment decisions. In biomedical technology, it can provide personalized disease models and provide a high-quality detection platform for novel immunotherapies.

[0043] Compared with the prior art, the beneficial effects of the present invention are:

[0044] This invention provides a complex tumor organoid co-culture system comprising oncolytic viruses, immune cells, and other cells, along with its construction method and applications. On one hand, constructing tumor organoids using patient-derived tumor cells allows for better replication of the patient's pathological characteristics. On the other hand, the fusion of oncolytic virus-induced dendritic cells with primary tumor cells enables more effective presentation of tumor cell-related antigens, specific antigens, and neoantigens, and more efficiently stimulates the response of immune cells (such as CD4+ T cells and CD8+ T cells). Furthermore, this invention utilizes naive T cells and other helper immune cells from peripheral blood or bone marrow to replace the patient's own tumor cell infiltration (TILs). The co-culture of naive T cells and fused cells will generate a broader and more effective tumor cell response (TCR), resulting in better tumor cell killing. The successfully constructed complex tumor organoid co-culture system can simulate the localization and interaction between immune cells and tumor cells in the in vivo tumor microenvironment, providing strong support for the establishment of tumor models and a platform for the evaluation of novel immunotherapies. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the construction method of the complex organoid co-culture system described in this invention.

[0046] Figure 2 Growth of bone marrow-derived dendritic cells on days 2, 3, and 5 in a medium containing GM-CSF and IL4.

[0047] Figure 3 This study investigated the expression of immune-related molecules in bone marrow-derived dendritic cells after differentiation in a medium containing GM-CSF and IL-4 or Flt3L, and after LPS stimulation.

[0048] Figure 4 This study investigated the presentation of oncolytic virus (FITC), MHCII, and its specific antigen (MHCI-SII) in fusion cells of bone marrow-derived dendritic cells differentiated from Flt3L and mouse lung adenocarcinoma cells (LL / C-OVA) over a continuous 5-day co-culture method.

[0049] Figure 5 The expression of specific antigens (MHCI-SII) and MHCII was studied in different ratios (2:1 or 4:1) and under different fusion conditions (with or without the addition of fusion solution + / -FB) of bone marrow-derived dendritic cells differentiated from Flt3L (with or without LPS stimulation) and oncolytic virus-infected LL / C-OVA cells.

[0050] Figure 6 Analysis of CD8+ T cells obtained in Example 4 of this invention. Detailed Implementation

[0051] The technical solution of the present invention will be clearly and completely described below with reference to embodiments and comparative examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0053] Example 1 This example provides a method for extracting and differentiating dendritic cells derived from bone marrow (using mice as an example), including the following steps:

[0054] 1. The mice (6-10 weeks old) were euthanized by cervical dislocation, and all femurs and tibias were surgically removed. The surrounding muscle tissue was removed as cleanly as possible with scissors and forceps.

[0055] 2. Transfer the bone to the laminar flow hood and soak it in 70% alcohol for 1 minute;

[0056] 3. Transfer the bone to another new culture dish containing PBS, cut off both ends of the bone with scissors, draw PBS with a syringe, insert the needle into the bone marrow cavity from both ends of the bone, and repeatedly rinse out the bone marrow into the culture dish until the bone turns completely white;

[0057] 4. Collect bone marrow suspension and filter out small fragments and muscle tissue using a 40μm filter.

[0058] 5. Centrifuge the filtrate at 400×g for 5 min, and discard the supernatant;

[0059] 6. Add 2 mL of erythrocyte lysis buffer, resuspend the cells, and incubate at room temperature for 3-5 min;

[0060] 7. Add 10 mL of PBS to terminate the lysis, then centrifuge at 400 × g for 5 min and discard the supernatant;

[0061] 8. Wash once with PBS and count the samples;

[0062] 9. After counting the mouse bone marrow cells obtained above, the cell concentration was adjusted to 4 x 10⁻³ cells using DC differentiation medium, 1640 medium containing 10% FBS, 1X P / S, and 150 ng / ml Flt3L. 6 / mL;

[0063] 10. Spread 1 mL of cells into each well of a 24-well culture plate and incubate at 37°C in a 5% CO2 incubator. This is day 0 of the culture.

[0064] 11. After culturing for 9 days, collect suspension cells, which are mature dendritic cells derived from bone marrow.

[0065] Results Analysis

[0066] like Figure 2-3 As shown, Figure 2 This shows the growth of cultured dendritic cells on days 2, 3, and 5. Figure 3 The figure shows the expression of XCR1, CD103, MHCI, CD80, MHCII, and CD8a in mature dendritic cells with and without LPS stimulation. As shown in the figure, the expression of each immune-related marker increased in mature dendritic cells under LPS activation.

[0067] Example 2 This example provides a method for co-culturing oncolytic virus-induced dendritic cells with tumor cells, using bone marrow-derived dendritic cells differentiated from Flt3L and mouse lung adenocarcinoma cells (LL / C-OVA) as examples, including the following steps:

[0068] 1. Dendritic cells derived from bone marrow differentiated from Flt3L were obtained according to the method described in Example 1 above;

[0069] 2. Prepare the fusion cell co-culture medium: 1640 complete culture medium containing oncolytic viruses (VSVs) (MOI=0.01), 10% FBS, 1×P / S, 1×Hepes, Polybrene (8μg / mL) and 1×Opti-MEM;

[0070] 3. Cell counting: Bone marrow-derived dendritic cells differentiated from Flt3L and mouse lung adenocarcinoma cells (LL / C-OVA) were mixed evenly at a ratio of 2:1 and 4:1, and centrifuged at 300×g for 5 min.

[0071] 4. Resuspend the cell pellet in fused cell co-culture medium and seed it in 24-well plates coated with matrix gel;

[0072] 5. Place the 24-well plate in a 37°C, 5% CO2 incubator overnight to obtain fusion cells.

[0073] Results Analysis

[0074] like Figure 4As shown, the proportion of presenting cells, VSV infection status (FITC), and expression of MHCII and its MHCI-SIIN (OVA-specific antigen) in fusion cells obtained through co-culture from day 1 to day 5 are displayed. As shown in the figure, the fusion cells exhibited the highest cell presentation proportion and the highest MHCII expression on day 1, and presented the highest amount of specific antigen on day 3.

[0075] Example 3 This example provides a method for fusing tumor cells infected with oncolytic virus with dendritic cells, using bone marrow-derived dendritic cells differentiated from Flt3L and mouse lung adenocarcinoma cells (LL / C-OVA) as examples, including the following steps:

[0076] 1. Dendritic cells derived from bone marrow differentiated from Flt3L were obtained according to the method in Example 1 above, and some of the cells were cultured in a culture medium containing 100 ng / mL LPS for activation;

[0077] 2. Seed 5 million LL / C-OVA cells in a 10cm plate and culture overnight;

[0078] 3. Prepare DMEM complete medium containing oncolytic viruses: VSVs (MOI = 0.01), 10% FBS, 1×P / S and 1×Hepes;

[0079] 4. Replace the normal culture medium of LL / C-OVA cells with DMEM complete medium containing oncolytic virus;

[0080] 5. Place the 10cm plate in a 37℃, 5% CO2 incubator overnight to obtain LL / C-OVA cells infected with oncolytic virus and expressing VSV-G protein;

[0081] 3. Cell counting: Bone marrow-derived dendritic cells differentiated from Flt3L and LL / C-OVA cells infected with oncolytic virus were mixed evenly at a ratio of 2:1 and 4:1, and centrifuged at 300×g for 5 min.

[0082] 4. Resuspend the cell pellet in 1640 complete culture medium and seed it in 24-well plates coated with matrix gel;

[0083] 5. Incubate the 24-well plate overnight at 37°C in a 5% CO2 incubator, with or without adding acidic fusion solution, to obtain fused cells.

[0084] Results Analysis

[0085] like Figure 5As shown, the fusion cells obtained by fusing tumor cells infected with oncolytic viruses with dendritic cells exhibit different cell ratios (DCs:LL / C-OVA = 2:1 or 4:1), different dendritic cell treatment conditions (with or without LPS stimulation, -LPS or +LPS), and different fusion conditions (with or without fusion solution + / -FB) for the presentation of a specific antigen (MHCI-SII) and the expression of MHCII. As shown in the figure, dendritic cells stimulated with LPS exhibited a higher number of cells capable of presenting the specific antigen under different cell ratios and fusion conditions.

[0086] Example 4 This example provides a spleen CD8 + The method for extracting and culturing T cells includes the following steps:

[0087] 1. Mice (6-10 weeks old) were euthanized by cervical dislocation, and the spleen was surgically removed and placed in PBS containing P / S.

[0088] 2. Transfer the spleen to the laminar flow hood and wash it three times with PBS containing P / S;

[0089] 3. Grind the spleen with the ground surfaces of two sterile coverslips to release spleen cells and collect them in a culture dish containing PBS;

[0090] 4. Filter the obtained spleen cells through a 40μm filter to remove small fragments and tissues, place them in a 15mL centrifuge tube, centrifuge at 300×g for 5min, and remove the supernatant;

[0091] 5. Add 2 mL of erythrocyte lysis buffer, resuspend the cells, and incubate at room temperature for 3-5 min;

[0092] 6. Add 10 mL of PBS to terminate the lysis, then centrifuge at 300 × g for 5 min and discard the supernatant;

[0093] 7. MojoSort TM Mouse CD8 T Cell Isolation Kit extracts CD8 + T cells;

[0094] 8. Resuspend the cells in 4 mL of MojoSort buffer and place them in a 5 mL flow cytometry tube;

[0095] 9. Filter cells through a 70 μm filter, centrifuge at 300 × g for 5 min, resuspend in an appropriate amount of MojoSort buffer, count, and adjust the cell concentration to 1 × 10⁻⁶ cells / mL. 8 / mL;

[0096] 10. Transfer 100 μL of cell suspension to a new flow cytometry tube, add 10 μL of Biotin-Antibody Cocktail, mix thoroughly, and incubate on ice for 15 min;

[0097] 11. Add 10 μL of the corresponding magnetic beads to the tube and incubate on ice for 15 min;

[0098] 12. Add 2.5 mL of Mojosort Buffer and place the tube in a magnet for 5 minutes;

[0099] 13. Pour out and collect the desired cells from the liquid;

[0100] 14. The CD8 obtained above + T cells were cultured in 1640 medium containing 10% FBS, 1X P / S, Glutamax, 50 μM 2-mercaptoethanol, and 2000 U / ml IL-2.

[0101] Spleen CD4 + The extraction and culture methods for T cells are the same as described above. CD4+ T cells are obtained by binding with corresponding magnetic beads.

[0102] Results Analysis

[0103] like Figure 6 As shown, the CD8 extracted using this method + 95.94% of the T cells were CD8a positive.

[0104] Example 5 This example provides a method for culturing lung cancer organoids, including the following steps:

[0105] 1. Obtain lung cancer tumor tissue;

[0106] 2. Cleanse the lung cancer tumor tissue with tissue cleaning solution;

[0107] 3. Digest and cleanse lung cancer tumor tissue with tissue digestion solution;

[0108] 4. After the predetermined digestion time, stop digestion and filter the obtained cell suspension through a filter screen to collect the filtered single cells;

[0109] 5. After counting the collected single cells, centrifuge again to collect the cell pellet and culture it in matrix gel, changing the lung cancer organoid culture medium regularly.

[0110] The steps described are standard procedures, and the components of the lung cancer organoid culture medium include: Advanced DMEM / F12, P / S, Hepes, Glutamax, NAC, B27, SB202190, A83-01, Y27632, Nicotinamide, R-spindin, Noggin, EGF7, and EGF10.

[0111] Example 6 This example provides a method for constructing a co-culture system of single-cell organoids and complex tumor organoids, including the following steps:

[0112] The lung cancer organoids obtained in Example 5 were digested into organoid single cells using tissue digestion solution. These single cells were then co-cultured with fusion cells containing oncolytic virus-containing dendritic cells / tumor cells obtained in Example 3, mature dendritic cells obtained in Example 1, CD4+ T cells and CD8+ T cells obtained in Example 4 in Matrigel at different concentrations. After the desired cells differentiated well, the culture medium used in the co-culture system was added to construct a single-cell organoid-complex tumor organoid co-culture system.

[0113] The culture medium components used in the co-culture system include Advanced DMEM / F12, P / S, Hepes, Glutamax, NAC, B27, SB202190, A83-01, Y27632, Nicotinamide, R-spindin, Noggin, EGF7, EGF10, and IL-2.

[0114] Example 7 This example provides a method for constructing an organoid cluster-complex tumor organoid co-culture system, including the following steps:

[0115] The lung cancer organoids obtained in Example 5 were digested into organoid clusters using tissue digestion fluid. These clusters were then co-cultured with the fusion cells containing oncolytic viruses (dendritic cells / tumor cells) obtained in Example 3, the mature dendritic cells obtained in Example 1, the CD4+ T cells and CD8+ T cells obtained in Example 4, and Matrigel at different concentrations. Once the desired cells had differentiated well, the culture medium used in the co-culture system was added to construct the organoid cluster-complex tumor organoid co-culture system.

[0116] The culture medium components used in the co-culture system include Advanced DMEM / F12, P / S, Hepes, Glutamax, NAC, B27, SB202190, A83-01, Y27632, Nicotinamide, R-spindin, Noggin, EGF7, EGF10, and IL-2.

[0117] In summary, this invention provides a complex tumor organoid co-culture system and its construction method comprising oncolytic viruses, immune cells, and other cells. This includes the culture of lung cancer organoids, the construction and culture of fusion cells containing oncolytic viruses and dendritic cells / tumor cells, the culture of immune cells, and the co-culture of complex tumor organoids comprising oncolytic viruses, immune cells, and other cells. This invention activates dendritic cells differentiated from Flt3L cells in a culture medium containing LPS. In the fusion cells, LPS-stimulated dendritic cells exhibit a higher number of cells capable of presenting specific antigens under different cell ratios and fusion conditions. The cell fusion function of oncolytic viruses is used to fuse dendritic cells with tumor cells, thereby maximizing the presentation of tumor-associated antigens, tumor-specific antigens, and neotumor antigens. Simultaneously, the immunostimulatory function of the oncolytic virus itself can further attract and activate immune cells to enter the tumor microenvironment. Furthermore, this invention utilizes naive T cells and other helper immune cells from peripheral blood or bone marrow to replace the patient's own TILs. The co-culture of naive T cells and fusion cells will generate a broader and more effective TCR, and better kill tumor cells. Using patient-derived tumor cells to construct tumor organoids effectively replicates the pathological characteristics of patients. The provided co-culture system can more effectively simulate the localization and interaction between immune cells and tumor cells in the in vivo tumor microenvironment, providing technical support for the establishment of tumor models and a high-quality detection platform for novel immunotherapies.

[0118] Obviously, the above embodiments of the present invention are merely examples to clearly illustrate the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. A method for constructing a complex tumor organoid co-culture system, characterized in that, Includes the following steps: S1. Obtain tumor tissue and culture it in vitro to form tumor organoids; S2. After mixing tumor cells and dendritic cells, centrifuge to obtain cell pellet; resuspend the cell pellet in fusion cell culture medium containing oncolytic virus, and co-culture the mixed cells containing oncolytic virus to obtain fusion cells of dendritic cells / tumor cells containing oncolytic virus; S3. The tumor organoids described in step S1, the fused cells, dendritic cells, and immune cells described in step S2 are mixed and co-cultured to construct the complex tumor organoid co-culture system.

2. The method for constructing a complex tumor organoid co-culture system according to claim 1, characterized in that, The tumor organoids include organoids for lung cancer, colorectal cancer, kidney cancer, liver cancer, pancreatic cancer, stomach cancer, and breast cancer; the tumor cells correspond to the tumor organoids used.

3. The method for constructing a complex tumor organoid co-culture system according to claim 1, characterized in that, The tumor organoid culture step S1 includes: obtaining tumor tissue; washing the tumor tissue with tissue washing solution; digesting the washed tumor tissue with tissue digestion solution; terminating digestion after a predetermined time, and filtering the obtained cell suspension through a filter screen to collect single cells; counting the collected single cells, centrifuging again to collect the cell pellet, and culturing it in matrix gel, with the tumor organoid culture medium being changed periodically; the components of the tumor organoid culture medium include: AdvancedDMEM / F12, P / S, Hepes, Glutamax, NAC, B27, SB202190, A83-01, Y27632, Nicotinamide, R-spindin, Noggin, EGF7, and EGF10.

4. The method for constructing a complex tumor organoid co-culture system according to claim 1, characterized in that, The dendritic cells described in step S2 are mature dendritic cells obtained by extracting monocytes or bone marrow cells from peripheral blood or bone marrow and inducing differentiation of monocytes or bone marrow cells in DC cell culture medium. The components of the DCs cell culture medium include: 1640 medium, FBS, P / S and Flt3L.

5. The method for constructing a complex tumor organoid co-culture system according to claim 1, characterized in that, In step S2, the mixing ratio of tumor cells to dendritic cells is 10:1 to 1:

10.

6. The method for constructing a complex tumor organoid co-culture system according to claim 1, characterized in that, The oncolytic virus mentioned in step S2 includes vesicular stomatitis virus, herpes simplex virus, vaccinia virus, adenovirus, reovirus, and Newcastle disease virus; the fusion cell culture medium containing the oncolytic virus includes: oncolytic virus, 1640 medium, FBS, P / S, Hepes, Polybrene, and Opti-MEM.

7. The method for constructing a complex tumor organoid co-culture system according to claim 1, characterized in that, The immune cells mentioned in step S3 include one or more of CD4+ T cells, CD8+ T cells, dendritic cells, and macrophages.

8. The method for constructing a complex tumor organoid co-culture system according to claim 1, characterized in that, The culture medium components used in the co-culture system described in step S3 include Advanced DMEM / F12, P / S, Hepes, Glutamax, NAC, B27, SB202190, A83-01, Y27632, Nicotinamide, R-spindin, Noggin, EGF7, EGF10, and IL-2.

9. The method for constructing a complex tumor organoid co-culture system according to claim 1, characterized in that, The steps for constructing the complex tumor organoid co-culture system include: The tumor organoids described in step S1 are digested into organoid single cells, which are then co-cultured with the fusion cells, dendritic cells, and immune cells described in step S2 in a matrix gel to construct a single-cell organoid-complex tumor organoid co-culture system. And / or, the tumor organoids described in step S1 are digested into organoid clusters, and co-cultured with the fusion cells, dendritic cells, and immune cells described in step S2 in a matrix gel to construct an organoid cluster-complex tumor organoid co-culture system.

10. The use of the method for constructing a complex tumor organoid co-culture system according to any one of claims 1-9, characterized in that, The applications include screening for oncology drugs, establishing tumor disease models, or providing a detection platform for immunotherapy.

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