Construction method and application of gastric cancer treatment effect evaluation model of CAR-T cells

Abstract

The invention discloses a construction method and application of a gastric cancer treatment effect evaluation model of CAR-T cells, and the constructed gastric cancer treatment effect evaluation model of the CAR-T cells comprises a CAR-T cell and gastric cancer organ co-culture killer dynamic detection system and a CAR-T cell, intestine organ and lung organ co-culture safety evaluation system. Based on a CAR-T cell and gastric cancer organoid co-culture lethality dynamic detection system, the process that CAR-T cells act on gastric cancer organoid can be dynamically observed through a living cell imaging method, then the action mechanism of the CAR-T cells for treating solid tumors is studied, and meanwhile, based on a CAR-T cell, intestine organoid and lung organoid co-culture safety evaluation system, the CAR-T cells and the gastric cancer organoid co-culture lethality dynamic detection system can be used for evaluating the CAR-T cells and the gastric cancer organoid co-culture lethality. Compared with the prior art, the preclinical model has the advantages that the effect of the CAR-T cells on gastric cancer para-tissues and other tissue-derived organs can be detected, and safety evaluation is carried out, so that the preclinical model for more comprehensively detecting the gastric cancer treatment effect of the CAR-T cells is obtained, and the research on CAR-T cell targets is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for constructing and applying a CAR-T cell gastric cancer treatment effect evaluation model. Background Art

[0002] For the treatment of gastric cancer, traditional treatments such as radiotherapy and chemotherapy are often accompanied by severe side effects and disease recurrence. Therefore, more personalized treatment strategies are urgently needed. Harnessing the great potential of the immune system appears to be a new hope for improving treatment outcomes for gastric cancer patients.

[0003] Immunotherapy using chimeric antigen receptor engineered T cells (CAR-T cells) is effective in the treatment of leukemia. For solid tumors such as gastric cancer, the dense structure of solid tissues will lead to poor CAR-T cell treatment effects. The organoid model simulates the dense structure of solid tumors very well. Although there are a few studies using tumor organoids to study the killing function of CAR-T cells, there is currently no CAR-T cell killing function test for gastric cancer. In addition, antibodies overexpressed in tumor tissues may also be expressed in normal tissues, which may cause serious side effects. Existing studies have verified the killing ability of CAR-T cells and ignored the safety testing of CAR-T cells. Therefore, how to build a more comprehensive preclinical model for detecting the effect of CAR-T cells in the treatment of gastric cancer is a technical problem that technicians in this field need to solve. Summary of the invention

[0004] The present invention provides a method for constructing a CAR-T cell gastric cancer treatment effect evaluation model and its application, so as to simultaneously detect the lethality and safety of CAR-T cells, thereby obtaining a more comprehensive preclinical model for detecting the effect of CAR-T cells in treating gastric cancer.

[0005] One aspect of the present invention provides a method for constructing a CAR-T cell gastric cancer treatment effect evaluation model, wherein the CAR-T cell gastric cancer treatment effect evaluation model includes a CAR-T cell and gastric cancer organoid co-culture lethality dynamic detection system, and a CAR-T cell and intestinal organoid and lung organoid co-culture safety evaluation system; The CAR-T cell and gastric cancer organoid co-culture lethality dynamic detection system is obtained by the following steps: Step S11, culturing the gastric cancer organoids into a three-dimensional structure in Matrigel and gastric cancer organoid culture medium; Step S12, separating the gastric cancer organoids from the gel using a phosphate buffer, and staining them with a Cell mask dye to make the gastric cancer organoids have red fluorescence; Step S13, staining the CAR-T cells with DIO dye to make them have green fluorescence; Step S14, mixing the stained gastric cancer organoids with the CAR-T cells to obtain a mixed cell resuspension; Step S15, culturing the mixed cell resuspension with a co-culture medium to obtain a dynamic detection system for the lethality of CAR-T cells and gastric cancer organoids co-cultured; The CAR-T cell and intestinal organoid and lung organoid co-culture safety assessment system is obtained by the following steps: Step S21, mixing the intestinal organoids with the CAR-T cells to obtain a mixed cell resuspension a, mixing the lung organoids with the CAR-T cells to obtain a mixed cell resuspension b, mixing the intestinal organoids with the control T cells to obtain a mixed cell resuspension c, and mixing the lung organoids with the control T cells to obtain a mixed cell resuspension d; Step S22, using the co-culture medium a to culture the mixed cell resuspension a and the mixed cell resuspension c, respectively, and using the co-culture medium b to culture the mixed cell resuspension b and the mixed cell resuspension d, respectively, to obtain a safety assessment system for the co-culture of CAR-T cells with intestinal organoids and lung organoids.

[0006] Another aspect of the present invention provides an application of a gastric cancer treatment effect evaluation model of CAR-T cells obtained by the above-mentioned construction method, wherein the dynamic detection system for the co-culture of CAR-T cells and gastric cancer organoids is used to detect the lethality of CAR-T cells, and the safety evaluation system for the co-culture of CAR-T cells with intestinal organoids and lung organoids is used to evaluate the safety of CAR-T cells.

[0007] Specifically, when detecting the lethality of CAR-T cells, a live cell imaging microscope is used to shoot the dynamic detection system of the co-culture lethality of CAR-T cells and gastric cancer organoids for a preset time to observe the mode of action of CAR-T cells on gastric cancer organoids; When evaluating the safety of CAR-T cells, the cultured mixed cell resuspension a, mixed cell resuspension b, mixed cell resuspension c, and mixed cell resuspension d were first stained with PI and photographed under a microscope. Then, the CAR-T cells and intestinal organoid co-cultures were separated and washed from the gel with phosphate buffer, the CAR-T cells and lung organoid co-cultures were separated and washed from the gel, the control T cells and intestinal organoid co-cultures were separated and washed from the gel, and the control T cells and lung organoid co-cultures were separated and washed from the gel. TryplE was used to digest each washed co-culture into single cells, and then Epcam antibodies were used to mark the single cells. PI and Epcam double-positive cells were detected by flow cytometry, and the killing of intestinal organoids and lung organoids by CAR-T cells and control T cells was compared to evaluate the safety of CAR-T cells.

[0008] Beneficial effects: The gastric cancer treatment effect evaluation model constructed by the present invention includes a dynamic detection system for the lethality of co-culture of CAR-T cells and gastric cancer organoids, and a safety evaluation system for co-culture of CAR-T cells with intestinal organoids and lung organoids. Based on the dynamic detection system for the lethality of co-culture of CAR-T cells and gastric cancer organoids, the live cell imaging method can be used to dynamically observe the process of CAR-T cells acting on gastric cancer organoids, and then study the mechanism of action of CAR-T cells in treating solid tumors. At the same time, based on the safety evaluation system for co-culture of CAR-T cells with intestinal organoids and lung organoids, the effects of CAR-T cells on gastric cancer adjacent tissues and organoids from other tissues can be detected for safety evaluation. Therefore, the present invention obtains a more comprehensive preclinical model for detecting the effect of CAR-T cells in treating gastric cancer, which is beneficial to the study of CAR-T cell targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a picture taken using a live cell imaging microscope to detect the dynamic lethality of CAR-T cells and gastric cancer organoids co-cultured. Figure 2 These are bright field images of PI staining of intestinal organoids and normal lung organoids after co-culture with control T cells and CAR-T cells for one day; Figure 3 These are the flow cytometry images of PI and Epcam co-staining of intestinal organoids and normal lung organoids after co-culture with control T cells and CAR-T cells for one day. DETAILED DESCRIPTION

[0010] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0011] An embodiment of the present invention provides a method for constructing a gastric cancer treatment effect evaluation model of CAR-T cells, wherein the gastric cancer treatment effect evaluation model of CAR-T cells includes a dynamic detection system for the lethality of co-culture of CAR-T cells and gastric cancer organoids, and a safety evaluation system for co-culture of CAR-T cells with intestinal organoids and lung organoids; The CAR-T cell and gastric cancer organoid co-culture lethality dynamic detection system is obtained through steps S11-S15: Step S11, culturing the gastric cancer organoids into a three-dimensional structure in matrix gel and gastric cancer organoid culture medium to simulate the dense structure of solid tumors, wherein the components and contents of the gastric cancer organoid culture medium are as follows: Advanced DMEM / F12, 1X; penicillin-streptomycin mixture, 1X; B27, 1X; GluMAX supplement, 1mM; acetylcysteine, 1X; HEPES Buffer, 1X; N2, 1X; B27, 200ng / mL; Wnt3a, 1μg / mL; R-spondin 1, 100ng / mL; Noggin, 10nM; gastrin protein Gastrin, 10nM; EGF, 50ng / mL; fibroblast growth factor FGF-10, 100ng / mL; TGF-β type I receptor inhibitor A83-01, 0.5μM; nicotinamide, 10Mm; Y-27632, 10μM.

[0012] Step S12, separating the gastric cancer organoids from the gel using phosphate buffer, and staining them with Cell mask dye to make the gastric cancer organoids have red fluorescence.

[0013] Step S13, staining the CAR-T cells with DIO dye to make them have green fluorescence.

[0014] Step S14, mixing the stained gastric cancer organoids and CAR-T cells in a ratio of 1:2 to obtain a mixed cell resuspension.

[0015] Step S15, the matrix gel and the mixed cell resuspension are mixed at a ratio of 1:19, and then inoculated into a glass-bottomed culture dish after blowing on ice, and placed in a 37°C incubator. After the gel solidifies, the co-culture medium is added to obtain a CAR-T cell and gastric cancer organoid co-culture lethality dynamic detection system, wherein the components and contents of the co-culture medium are: Advanced DMEM / F12, 1X; penicillin-streptomycin mixture, 1X; B27, 1X; GluMAX supplement, 1mM; acetylcysteine, 1X; HEPESBuffer, 1X; N2, 1X; B27, 200ng / mL; Wnt3a, 1μg / mL; R-spondin 1, 100ng / mL; Noggin, 10nM; gastrin protein Gastrin, 10nM; EGF, 50ng / mL; fibroblast growth factor FGF-10, 100ng / mL; TGF-β Type I receptor inhibitor A83-01, 0.5 μM; nicotinamide, 10 Mm; Y-27632, 10 μM; IL-2, 200 U / mL.

[0016] The CAR-T cell and intestinal organoid and lung organoid co-culture safety assessment system is obtained by following the steps S21-S22: Step S21, mixing the intestinal organoids with the CAR-T cells to obtain a mixed cell resuspension a, mixing the lung organoids with the CAR-T cells to obtain a mixed cell resuspension b, mixing the intestinal organoids with the control T cells to obtain a mixed cell resuspension c, and mixing the lung organoids with the control T cells to obtain a mixed cell resuspension d.

[0017] Specifically, step S21 includes steps S211-S213: Step S211, culturing normal intestinal organoids into a three-dimensional structure in Matrigel and intestinal organoid culture medium, and culturing normal lung organoids into a three-dimensional structure in Matrigel and lung organoid culture medium. Among them, the components and contents of intestinal organoid culture medium are: Advanced DMEM / F12, 1X; EGF, 50ng / mL; IGF 1, 100ng / mL; Noggin, 100ng / mL; A83-01, 500nM; Gastrin, 10 nM; FGF2, 50 ng / mL; Wnt-3A serum-free conditioned medium, 50%; B27 supplement, 1X; N-Acetylcysteine, 1mM; Nicotinamide, 5mM; GlutaMax, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; Y-27632, 5μM; R-spondin 1, 500ng / mL.

[0018] The composition and content of the lung organoid culture medium are as follows: Advanced DMEM / F12, 1X; R-Spondin1, 500ng / mL; FGF 7, 25ng / mL; FGF 10, 100ng / mL; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL.

[0019] Step S212, using phosphate buffer to separate the intestinal organoids and lung organoids from the gel.

[0020] Step S213, the intestinal organoids are mixed with CAR-T cells in a ratio of 1:2 to obtain a mixed cell resuspension a, the lung organoids are mixed with CAR-T cells in a ratio of 1:2 to obtain a mixed cell resuspension b, the intestinal organoids are mixed with control T cells in a ratio of 1:2 to obtain a mixed cell resuspension c, and the lung organoids are mixed with control T cells in a ratio of 1:2 to obtain a mixed cell resuspension d.

[0021] Step S22, the matrix gel and the mixed cell resuspension a are mixed at a ratio of 1:19, and then the mixture is pipetted on ice and inoculated into a glass-bottomed culture dish, which is placed in a 37°C incubator. After the gel solidifies, the co-culture medium a is added; the matrix gel and the mixed cell resuspension b are mixed at a ratio of 1:19, and then the mixture is pipetted on ice and inoculated into a glass-bottomed culture dish, which is placed in a 37°C incubator. After the gel solidifies, the co-culture medium b is added; the matrix gel and the mixed cell resuspension c are mixed at a ratio of 1:19, and then the mixture is pipetted on ice and inoculated into a glass-bottomed culture dish, which is placed in a 37°C incubator. After the gel solidifies, the co-culture medium b is added; The culture dish was placed in a 37°C incubator, and co-culture medium a was added after the gel solidified; the matrix gel and mixed cell resuspension d were mixed in a ratio of 1:19, pipetted on ice and inoculated into a glass-bottomed culture dish, placed in a 37°C incubator, and co-culture medium b was added after the gel solidified; after culturing the mixed cell resuspension a, mixed cell resuspension b, mixed cell resuspension c, and mixed cell resuspension d for 24 hours, a safety evaluation system for co-culture of CAR-T cells with intestinal organoids and lung organoids was obtained.

[0022] Among them, the components and contents of the co-culture medium a are: Advanced DMEM / F12, 1X; EGF, 50ng / mL; IGF 1, 100ng / mL; Noggin, 100ng / mL; A83-01, 500nM; Gastrin, 10 nM; FGF2, 50ng / mL; Wnt-3A serum-free conditioned medium, 50%; B27 supplement, 1X; N-Acetylcysteine, 1mM; Nicotinamide, 5mM; GlutaMax, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; Y-27632, 5μM; R-spondin 1, 500ng / mL; IL-2, 200U / mL.

[0023] The composition and content of the co-culture culture medium b are: Advanced DMEM / F12, 1X; R-Spondin1, 500ng / mL; FGF 7, 25ng / mL; FGF 10, 100ng / mL; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; IL-2, 200U / mL.

[0024] An embodiment of the present invention also provides an application of a gastric cancer treatment effect evaluation model of CAR-T cells obtained by the above-mentioned construction method, wherein the dynamic detection system for the co-culture of CAR-T cells and gastric cancer organoids is used to detect the lethality of CAR-T cells, and the safety evaluation system for the co-culture of CAR-T cells with intestinal organoids and lung organoids is used to evaluate the safety of CAR-T cells.

[0025] Specifically, when testing the cytotoxicity of CAR-T cells, a live cell imaging microscope was used to film the cytotoxicity dynamic detection system of CAR-T cells and gastric cancer organoids for 20 hours to observe the action mode of CAR-T cells on gastric cancer organoids. Please refer to Figure 1 , and save the pictures taken at 0H (H means hour), 3.5H, 4.5H, 5.5H, 6.5H, 7.5H, 8.5H, 9.5H, 10.5H, 11.5H, 12.5H, 13.5H, 14.5H, 15.5H, 16.5H, 17.5H, 18.5H, 19.5H respectively, from Figure 1 It can be seen that CAR-T cells act on tumor organoids not through the simple secretion of factors. During the action process, CAR-T cells produce filaments that entangle gastric cancer organoids, causing them to decompose and die. Based on this result, it is conducive to further studying the mechanism of action of CAR-T cells in treating solid tumors.

[0026] When evaluating the safety of CAR-T cells, the cultured mixed cell resuspension a, mixed cell resuspension b, mixed cell resuspension c, and mixed cell resuspension d were first stained with PI and photographed under a microscope. Then, the CAR-T cells and intestinal organoid co-cultures were separated and washed from the gel with phosphate buffer, the CAR-T cells and lung organoid co-cultures were separated and washed from the gel, the control T cells and intestinal organoid co-cultures were separated and washed from the gel, and the control T cells and lung organoid co-cultures were separated and washed from the gel. TryplE was used to digest each washed co-culture into single cells, and then Epcam antibodies were used to mark the single cells. PI and Epcam double-positive cells were detected by flow cytometry, and the killing of intestinal organoids and lung organoids by CAR-T cells and control T cells was compared to evaluate the safety of CAR-T cells.

[0027] Figure 2 The PI-stained bright field images of intestinal organoids and normal lung organoids after one day of co-culture with control T cells and CAR-T cells are shown. Figure 2 It can be seen that no matter whether it is normal lung organoids or normal intestinal organoids, some cells can be stained with PI after co-culture with CAR-T cells or control T cells, proving that cell apoptosis exists. However, simple PI staining cannot determine whether it is T cells or organoids that are apoptotic, so Epcam antibodies are further used to mark epithelial cells.

[0028] Figure 3 The PI and Epcam co-staining flow cytometry images of intestinal organoids and normal lung organoids after one day of co-culture with control T cells and CAR-T cells were shown. Figure 3 It can be seen that the proportion of apoptotic cells in normal intestinal organoids after co-culture with control T cells was 51.57%, and the proportion of apoptotic cells after co-culture with CAR-T cells was 50.42%. There was no significant difference in the proportion of apoptotic cells between the two. The proportion of apoptotic cells in normal lung organoids after co-culture with control T cells was 15.5%, and the proportion of apoptotic cells after co-culture with CAR-T cells was 16.39%. There was no significant difference in the proportion of apoptotic cells between the two.

[0029] Based on the above results, it can be concluded that CAR-T cells have a significant killing effect on gastric cancer organoids, but a weak killing effect on normal intestinal and lung organoids, with little impact on surrounding normal tissues, and are safe. Therefore, while performing dynamic detection of CAR-T cell killing, the present invention can detect the effect of CAR-T cells on gastric cancer adjacent tissues and other tissue-derived organoids to conduct safety assessments.

[0030] In summary, the method for constructing and applying the gastric cancer treatment effect evaluation model of CAR-T cells provided by the present invention has the following beneficial effects: The gastric cancer treatment effect evaluation model constructed by the present invention includes a dynamic detection system for the lethality of co-culture of CAR-T cells and gastric cancer organoids, and a safety evaluation system for co-culture of CAR-T cells with intestinal organoids and lung organoids. Based on the dynamic detection system for the lethality of co-culture of CAR-T cells and gastric cancer organoids, the live cell imaging method can be used to dynamically observe the process of CAR-T cells acting on gastric cancer organoids, and then study the mechanism of action of CAR-T cells in treating solid tumors. At the same time, based on the safety evaluation system for co-culture of CAR-T cells with intestinal organoids and lung organoids, the effects of CAR-T cells on gastric cancer adjacent tissues and organoids from other tissues can be detected for safety evaluation. Therefore, the present invention obtains a more comprehensive preclinical model for detecting the effect of CAR-T cells in treating gastric cancer, which is beneficial to the study of CAR-T cell targets.

[0031] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for constructing a gastric cancer treatment effect evaluation model using CAR-T cells, characterized in that: The gastric cancer treatment effect evaluation model of the CAR-T cells includes a dynamic detection system for the lethality of co-culture of CAR-T cells and gastric cancer organoids, and a safety evaluation system for co-culture of CAR-T cells with intestinal organoids and lung organoids; The CAR-T cell and gastric cancer organoid co-culture lethality dynamic detection system is obtained by the following steps: Step S11, culturing the gastric cancer organoids into a three-dimensional structure in Matrigel and gastric cancer organoid culture medium; Step S12, separating the gastric cancer organoids from the gel using a phosphate buffer, and staining them with a Cell mask dye to make the gastric cancer organoids have red fluorescence; Step S13, staining the CAR-T cells with DIO dye to make them have green fluorescence; Step S14, mixing the stained gastric cancer organoids with the CAR-T cells to obtain a mixed cell resuspension; Step S15, culturing the mixed cell resuspension with a co-culture medium to obtain a dynamic detection system for the lethality of CAR-T cells and gastric cancer organoids co-cultured; The CAR-T cell and intestinal organoid and lung organoid co-culture safety assessment system is obtained by the following steps: Step S21, mixing the intestinal organoids with the CAR-T cells to obtain a mixed cell resuspension a, mixing the lung organoids with the CAR-T cells to obtain a mixed cell resuspension b, mixing the intestinal organoids with the control T cells to obtain a mixed cell resuspension c, and mixing the lung organoids with the control T cells to obtain a mixed cell resuspension d; Step S22, using the co-culture medium a to culture the mixed cell resuspension a and the mixed cell resuspension c, respectively, and using the co-culture medium b to culture the mixed cell resuspension b and the mixed cell resuspension d, respectively, to obtain a safety assessment system for the co-culture of CAR-T cells with intestinal organoids and lung organoids.

2. The method for constructing a gastric cancer treatment effect evaluation model of CAR-T cells according to claim 1, characterized in that: Step S21 specifically includes: Step S211, culturing normal intestinal organoids into a three-dimensional structure in Matrigel and intestinal organoid culture medium, and culturing normal lung organoids into a three-dimensional structure in Matrigel and lung organoid culture medium; Step S212, separating the intestinal organoids and the lung organoids from the gel respectively using phosphate buffer; Step S213, the intestinal organoids are mixed with CAR-T cells in a ratio of 1:2 to obtain a mixed cell resuspension a, the lung organoids are mixed with CAR-T cells in a ratio of 1:2 to obtain a mixed cell resuspension b, the intestinal organoids are mixed with control T cells in a ratio of 1:2 to obtain a mixed cell resuspension c, and the lung organoids are mixed with control T cells in a ratio of 1:2 to obtain a mixed cell resuspension d.

3. The method for constructing a gastric cancer treatment effect evaluation model of CAR-T cells according to claim 1, characterized in that: Step S22 specifically includes: The matrix gel and the mixed cell resuspension a were mixed at a ratio of 1:19, pipetted on ice and inoculated into a glass-bottomed culture dish, placed in a 37°C incubator, and the co-culture medium a was added after the gel solidified; the matrix gel and the mixed cell resuspension b were mixed at a ratio of 1:19, pipetted on ice and inoculated into a glass-bottomed culture dish, placed in a 37°C incubator, and the co-culture medium b was added after the gel solidified; the matrix gel and the mixed cell resuspension c were mixed at a ratio of 1:19, pipetted on ice and inoculated into a glass-bottomed culture dish The mixture was placed in a 37°C incubator, and co-culture medium a was added after the gel solidified; the matrix gel and mixed cell resuspension d were mixed in a ratio of 1:19, pipetted on ice and inoculated into a glass-bottomed culture dish, and placed in a 37°C incubator, and co-culture medium b was added after the gel solidified; after culturing the mixed cell resuspensions a, b, c, and d for 24 hours, a safety evaluation system for the co-culture of CAR-T cells with intestinal organoids and lung organoids was obtained.

4. The method for constructing a gastric cancer treatment effect evaluation model of CAR-T cells according to claim 1, characterized in that: In step S11, the components and contents of the gastric cancer organoid culture medium are: Advanced DMEM / F12, 1X; penicillin-streptomycin mixture, 1X; B27, 1X; GluMAX supplement, 1mM; acetylcysteine, 1X; HEPES Buffer, 1X; N2, 1X; B27, 200ng / mL; Wnt3a, 1μg / mL; R-spondin 1, 100ng / mL; Noggin, 10nM; Gastrin protein Gastrin, 10nM; EGF, 50ng / mL; fibroblast growth factor FGF-10, 100ng / mL; TGF-β type I receptor inhibitor A83-01, 0.5μM; nicotinamide, 10Mm; Y-27632, 10μM.

5. The method for constructing a gastric cancer treatment effect evaluation model of CAR-T cells according to claim 1, characterized in that: In step S15, the composition and content of the co-culture medium are: Advanced DMEM / F12, 1X; penicillin-streptomycin mixture, 1X; B27, 1X; GluMAX supplement, 1mM; acetylcysteine, 1X; HEPES Buffer, 1X; N2, 1X; B27, 200ng / mL; Wnt3a, 1μg / mL; R-spondin 1, 100ng / mL; Noggin, 10nM; gastrin protein Gastrin, 10nM; EGF, 50ng / mL; fibroblast growth factor FGF-10, 100ng / mL; TGF-β type I receptor inhibitor A83-01, 0.5μM; nicotinamide, 10Mm; Y-27632, 10μM; IL-2, 200U / mL.

6. The method for constructing a gastric cancer treatment effect evaluation model of CAR-T cells according to claim 2, characterized in that: In step S211, the composition and content of the intestinal organoid culture medium are: Advanced DMEM / F12, 1X; EGF, 50ng / mL; IGF 1, 100ng / mL; Noggin, 100ng / mL; A83-01, 500nM; Gastrin, 10 nM; FGF2, 50ng / mL; Wnt-3A serum-free conditioned medium, 50%; B27 supplement, 1X; N-Acetylcysteine, 1mM; Nicotinamide, 5mM; GlutaMax, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; Y-27632, 5μM; R-spondin 1, 500ng / mL; In step S211, the composition and content of the lung organoid culture medium are: Advanced DMEM / F12, 1X; R-Spondin1, 500ng / mL; FGF 7, 25ng / mL; FGF 10, 100ng / mL; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL.

7. The method for constructing a gastric cancer treatment effect evaluation model of CAR-T cells according to claim 1, characterized in that: In step S22, the composition and content of the co-culture medium a are as follows: Advanced DMEM / F12, 1X; EGF, 50ng / mL; IGF 1, 100ng / mL; Noggin, 100ng / mL; A83-01, 500nM; Gastrin, 10 nM; FGF2, 50ng / mL; Wnt-3A serum-free conditioned medium, 50%; B27 supplement, 1X; N-Acetylcysteine, 1mM; Nicotinamide, 5mM; GlutaMax, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; Y-27632, 5μM; R-spondin 1, 500ng / mL; IL-2, 200U / mL; In step S22, the composition and content of the co-culture culture medium b are: Advanced DMEM / F12, 1X; R-Spondin1, 500ng / mL; FGF 7, 25ng / mL; FGF 10, 100ng / mL; Noggin, 100ng / mL; A83-01, 500nM; Y-27632, 5μM; SB202190, 500μM; B27 supplement, 1X; N-Acetylcysteine, 1.25mM; Nicotinamide, 5mM; GlutaMax, 1X; HEPES, 10mM; Penicillin / Streptomycin, 100μg / mL; IL-2, 200U / mL.

8. Application of the gastric cancer treatment effect evaluation model of CAR-T cells obtained by the construction method according to any one of claims 1 to 7, characterized in that: The dynamic detection system for the co-culture of CAR-T cells and gastric cancer organoids is used to detect the lethality of CAR-T cells, and the safety evaluation system for the co-culture of CAR-T cells with intestinal organoids and lung organoids is used to evaluate the safety of CAR-T cells.

9. The use according to claim 8, characterized in that: When testing the lethality of CAR-T cells, a live cell imaging microscope was used to shoot the dynamic detection system of the co-cultured lethality of CAR-T cells and gastric cancer organoids for a preset time to observe the mode of action of CAR-T cells on gastric cancer organoids; When evaluating the safety of CAR-T cells, the cultured mixed cell resuspension a, mixed cell resuspension b, mixed cell resuspension c, and mixed cell resuspension d were first stained with PI and photographed under a microscope. Then, the CAR-T cells and intestinal organoid co-cultures were separated and washed from the gel with phosphate buffer, the CAR-T cells and lung organoid co-cultures were separated and washed from the gel, the control T cells and intestinal organoid co-cultures were separated and washed from the gel, and the control T cells and lung organoid co-cultures were separated and washed from the gel. TryplE was used to digest each washed co-culture into single cells, and then Epcam antibodies were used to mark the single cells. PI and Epcam double-positive cells were detected by flow cytometry, and the killing of intestinal organoids and lung organoids by CAR-T cells and control T cells was compared to evaluate the safety of CAR-T cells.

Images