Method for constructing malignant pleuroperitoneal fluid-derived gastric cancer cell organoid model

Abstract

The invention provides a method for constructing a malignant pleuroperitoneal fluid-derived gastric cancer cell organoid model. The method comprises malignant pleuroperitoneal fluid sample treatment, gastric cancer cell separation and gastric cancer cell organoid culture. Gastric cancer cells and lymphocytes are separated through layer-by-layer treatment of malignant pleuroperitoneal fluid samples, so that the purity of the gastric cancer cells in the gastric cancer cell organ model can be improved, the proportion of immune cells can be reduced, the authenticity and accuracy of the gastric cancer cell organ model can be enhanced, and the heterogeneity and individualized characteristics of gastric cancer patients can be reflected; therefore, the method has important value for clinical drug screening and precise treatment of gastric cancer patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering technology, and in particular to a method for constructing a gastric cancer cell organoid model derived from malignant pleural effusion and ascites. Background Art

[0002] Gastric cancer is one of the malignant tumors with the highest morbidity and mortality rates worldwide. In vitro gastric cancer cell culture models have played an important role in studying the molecular mechanisms of gastric cancer occurrence and development and in drug screening. However, traditional two-dimensional culture models cannot fully simulate the three-dimensional structure of gastric cancer cells in vivo, limiting their application in preclinical research. Organoids are three-dimensional cell clusters formed by culturing stem cells or tumor cells, and can simulate the structure and function of organs in vitro. The development of organoid technology provides a more accurate experimental model for preclinical research on gastric cancer.

[0003] Pleural effusion and ascites are one of the most common complications in patients with advanced gastric cancer, and usually contain a large number of free gastric cancer cells. These cells are highly invasive and metastatic, and are ideal tools for studying gastric cancer progression and drug resistance. Constructing gastric cancer cells in pleural effusions into organoid models can better reflect the heterogeneity and dynamic changes of tumors in patients. However, existing technologies have many technical difficulties and limitations in the process of constructing organoid models using gastric cancer cells derived from malignant pleural effusions. The typical problem is that cell purification is difficult: malignant pleural effusions contain not only gastric cancer cells, but also a large number of lymphocytes, fibroblasts and impurities. It is a challenge to purify high-purity gastric cancer cells.

[0004] In order to solve this technical problem, many explorations have been made in the prior art. The commonly used cell separation technologies, such as flow cytometry sorting and immunomagnetic bead sorting, are not efficient and easily lead to cell damage and loss. The Chinese invention patent with patent number CN117247905A discloses "a method for constructing a digestive tract tumor cell organoid model derived from pleural effusion and its application". The mixed cells after red blood cell lysis of the cell precipitation of pleural effusion samples are used as primary cells for culture, and then the primary cultured cells are mixed into matrix gel for organoid culture to obtain a primary cell organoid model. However, it failed to solve the problem of gastric cancer cell purification, and still used mixed cells to construct a gastric cancer cell organoid model. The proportion of immune cells such as lymphocytes in the model is relatively high, which makes it difficult to reflect the heterogeneity and personalized characteristics of gastric cancer patients. Summary of the invention

[0005] The present invention is aimed at the technical problem that the purity of gastric cancer cells in the current gastric cancer organoid model is low. The gastric cancer cells are separated through layer-by-layer processing of pleural effusion and ascites samples, thereby improving the purity of gastric cancer cells in the gastric cancer cell organoid model and enhancing the authenticity and accuracy of the gastric cancer cell organoid model.

[0006] In order to achieve the above object, the specific technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides a method for constructing a gastric cancer cell organoid model derived from malignant pleural effusion and ascites, comprising the following steps:

[0008] A. Treatment of pleural and peritoneal effusion samples

[0009] After the pleural and ascites samples are identified to contain gastric cancer cells, centrifugation, cell sediment collection, red blood cell lysis, and removal of red blood cells and white blood cells in the ascites are sequentially performed. The preferred operation of each step is as follows:

[0010] The method for identifying gastric cancer cells in pleural and ascites samples is as follows: transfer freshly isolated pleural and ascites samples into centrifuge tubes, centrifuge at 400 × g for 5 minutes at room temperature, collect the cell pellet in the pleural and ascites, and add 100 U·mL -1 The cell pellet was washed 2-3 times with DPBS buffer containing 100 mg mL-1 penicillin-streptomycin and 100 μg / mL primocin antibiotics and centrifuged, and the fibrin pellet was discarded. Finally, an appropriate amount of cell suspension was pipetted with a pipette and added to a 96-well cell culture plate to observe whether there were gastric cancer cells.

[0011] The steps for lysing red blood cells are as follows: add 3-5 volumes of red blood cell lysis buffer to 1 volume of cell packed volume at room temperature or 4°C, lyse for 1-2 minutes, centrifuge at 200×g for 5 minutes and discard the red supernatant; then use 100U·mL -1 / 100mg·mL -1 The cell pellet after removal of red blood cells was resuspended in DPBS buffer containing penicillin-streptomycin and 100 μg / mL Primocin antibiotics, centrifuged at 200×g for 2-3 minutes and the supernatant was discarded. The pellet was washed 1-2 times with at least 5 volumes of washing solution to obtain the gastric cancer mixed cells after separation and removal of PBMCs and neutrophils.

[0012] B. Isolation of gastric cancer cells

[0013] After the cell precipitate treated in step A is resuspended with DPBS buffer, an excess lymphocyte separation solution is added to the centrifuge tube at a volume ratio of 3:2; then, with the centrifuge tube tilted at 45°, the cell resuspension with the same volume as the cell suspension is slowly added to the centrifuge tube to ensure that the cell resuspension is spread on the upper layer of the lymphocyte separation solution; at room temperature, after centrifugation at 800×g for 25 minutes, obvious cell stratification is observed, the middle white cell layer is the lymphocyte layer, and the bottom layer is the gastric cancer cell precipitation layer, and gastric cancer cells are obtained after separation and extraction; lymphocytes and gastric cancer cells are extracted separately, and the lymphocytes and gastric cancer cells are washed separately with DPBS buffer, and centrifuged at 100×g for 10 minutes at room temperature, and the supernatant is discarded to obtain two types of cells.

[0014] C. Gastric cancer cell organoid culture

[0015] The gastric cancer cell suspension was mixed with the matrix gel incubated in advance on crushed ice at a volume ratio of 1:1, and the mixture was inoculated into a multi-well culture plate in an ice-cold state; after the inoculation, the multi-well cell culture plate was placed upside down in an incubator at 37°C, 5% CO2, and 95% relative saturated humidity to allow the plate to stand for 24 hours. After the organoid gel droplets solidified, the organoid culture medium (800 μL / well) was added. When adding the culture medium, the pipette tip was kept away from the gel droplets to avoid damaging the matrix gel; DPBS buffer was added to the peripheral wells to reduce the evaporation of the organoid culture medium;

[0016] Place the multi-well cell culture plate upright in the above incubator for culture, and replace the organoid culture medium every 3-4 days; after the organoid culture is completed, digest and separate to obtain a gastric cancer cell organoid model derived from malignant pleural effusion. After the organoid culture is completed, wash each well with the above DPBS buffer 1-2 times, and add 500μL TrypLE TM Express Enzyme (1X) was used to digest the matrix gel. After blowing and breaking the matrix gel droplets, the cell culture plate was placed in the above-mentioned incubator for 10 minutes of static digestion. After the organoids were digested into smaller cell clumps or dispersed gastric cancer cells, the organoid culture medium was added to neutralize the effect of the digestive enzyme; the neutralized cell digestion suspension was added to a sterile centrifuge tube using a pipette, and then centrifuged at 200×g for 5 minutes and the supernatant was discarded. After washing 1-2 times with DPBS buffer, the organoids were coagulated again or frozen at -80℃ using a rapid cell freezing solution and stored in a liquid nitrogen tank.

[0017] The organoid culture medium includes a basal culture medium and basal factors and efficacy factors added thereto.

[0018] The basal culture medium is selected from DMEM / F12 basal culture medium;

[0019] The basic factors include 10% fetal bovine serum, 100 U·mL-1 / 100mg·mL -1 Penicillin-streptomycin and 100 μg / mL Primocin antibiotics;

[0020] Efficacy factors include Wnt pathway agonists, MAPKP38 inhibitors, TGFβ pathway inhibitors, EGF, FGF-10 and Noggin, ROCK inhibitors, Gastrin and Nicotinamide.

[0021] Preferably, in the efficacy factor, the Wnt pathway agonist is composed of 10% R-Spondin 1 and 10% Wnt-3a, the MAPKP38 inhibitor concentration is 3 μM, the TGFβ pathway inhibitor concentration is 0.5-1 μM, the EGF concentration is 5 ng / mL, the FGF-10 concentration is 20 ng / mL and the Noggin concentration is 100 ng·mL -1 , ROCK inhibitor concentration was 10 mM, Gastrin concentration was 10 nM, and Nicotinamide concentration was 10 mM.

[0022] More preferably, the MAPK P38 inhibitor is selected from SB 202190, the TGFβ pathway inhibitor is selected from A83-01, and the ROCK inhibitor is selected from Y27632.

[0023] In a second aspect, the present invention provides a gastric cancer cell organoid model derived from pleural effusion and ascites, which is prepared by any of the methods described above.

[0024] The organoid models obtained by culturing gastric cancer cells after separating lymphocytes were compared with the organoid models obtained by mixed culture of lymphocytes and gastric cancer cells. The results showed that the number of organoid models obtained by simple gastric cancer cell culture was nearly 5 times the number of organoid models obtained by mixed cell culture; there were also significant differences in the purity of gastric cancer cells in the organoid models.

[0025] In a third aspect, the present invention provides the application of the above-constructed gastric cancer cell organoid model derived from pleural effusion, such as for the molecular mechanism of gastric cancer occurrence and development and in vitro drug screening. The gastric cancer cell organoid model with higher gastric cancer cell purity can better simulate the three-dimensional structure of gastric cancer cells in vivo and the gastric cancer microenvironment, which will help improve the accuracy of research.

[0026] The beneficial protection and effects of the present invention are as follows:

[0027] The improved method of the present invention can improve the culture success rate of the gastric cancer cell organoid model derived from pleural effusion and ascites to more than 90%.

[0028] The present invention can improve the purity of gastric cancer cells in the gastric cancer cell organoid model and reduce the proportion of immune cells, which is beneficial to enhance the authenticity and accuracy of the gastric cancer cell organoid model and better reflect the heterogeneity and individual characteristics of gastric cancer patients. Therefore, it has important value for clinical drug screening and precision treatment of gastric cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The first example of the separation of lymphocytes from gastric cancer ascites sediment cells is shown;

[0030] Figure 2 The second example of gastric cancer is shown in which lymphocytes and gastric cancer cells are separated from the pleural effusion sediment cells.

[0031] Figure 3 The figure shows the growth of organoids cultured using a mixed cell sample of lymphocytes and gastric cancer cells and an organoid model cultured using isolated gastric cancer cell samples, as observed under a microscope during the organoid culture stage;

[0032] Figure 4 The comparison of organoid numbers cultured using mixed cell samples of lymphocytes and gastric cancer cells and using isolated gastric cancer cell samples is shown;

[0033] Figure 5 Two organoid models cultured with gastric cancer cells isolated from gastric ascites and pleural effusion were shown for pathological HE and IHC (CK7 and CDX2) staining identification, showing gastric cancer cells isolated from ascites and pleural effusion. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described in detail and completely in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention.

[0035] The reagents and raw materials used in the present invention are all commercially available or can be prepared according to literature methods. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers.

[0036] 1. Reagents and consumables

[0037] (1) Consumables: 50 mL centrifuge tubes, 15 mL centrifuge tubes, 24-well cell culture plates, 100 mm cell culture dishes, 200 μL pipette tips, and 1000 μL pipette tips;

[0038] (2) Reagents: DPBS balanced salt buffer, TrypLE TM Express Enzyme (1X), rapid cell freezing solution, lymphocyte separation solution, DMEM / F12 basal medium, 10% fetal bovine serum, Wnt pathway agonists (R-Spondin1 (10% concentration) and Wnt-3a (10% concentration)), MAPKP38 inhibitor (SB 202190) at a concentration of 3 μM, TGFβ pathway inhibitor (A83-01) at a concentration of 0.5-1 μM, EGF (5 ng / mL), FGF-10 (20 ng / mL), Noggin (100 ng·mL-1), ROCK inhibitor (Y27632) at a concentration of 10 mM, small molecule compounds (Gastrin (10 nM), Nicotinamide (10 mM), Y27632 (10 mM)), penicillin-streptomycin (100 U·mL-1), -1 / 100mg·mL -1 ) and Primocin antibiotic (100 μg / mL).

[0039] 2. Construction of Gastric Cancer Cell Organoids

[0040] 2.1 Pretreatment of gastric cancer pleural effusion and ascites samples

[0041] (1) Treatment of pleural and ascites fluid: 300 mL of fresh pleural and ascites fluid samples from gastric cancer patients were transferred into six 50 mL centrifuge tubes under sterile conditions and centrifuged at 400 × g for 5 min at room temperature to collect the cell precipitate in the pleural and ascites fluid. -1 / 100mg·mL -1 Wash the cell pellet 2-3 times with DPBS buffer containing penicillin-streptomycin and 100 μg / mL Primocin antibiotics and centrifuge. During this period, the fibrin pellet can be aspirated and discarded. Finally, use a pipette to aspirate an appropriate amount of cell suspension and add it to a 96-well cell culture plate to observe whether there are gastric cancer cells. If there are gastric cancer cells, proceed to the next experimental operation.

[0042] (2) Lysis of red blood cells: Usually, the cell sediment of the pleural and ascites of gastric cancer patients contains a large number of red blood cells and other cellular components. It is necessary to add red blood cell lysis solution (add 3-5 times the volume of red blood cell lysis solution to 1 volume of cell packed volume), gently blow and mix, and lyse for 1-2 minutes. This experimental operation can be performed at room temperature or 4°C. Then, centrifuge at 200×g for 5 minutes and discard the red supernatant. The centrifugation effect is better at 4°C. If the red blood cell lysis is found to be incomplete, this step can be repeated once. Usually, a very small amount of red blood cells will not affect subsequent operations and organoid culture. Use a 100U·mL -1 / 100mg·mL-1 Resuspend the cell pellet after removing red blood cells in DPBS buffer containing penicillin-streptomycin and 100μg / mL Primocin antibiotics, centrifuge at 200×g for 2-3 minutes and discard the supernatant. Centrifugation at 4℃ is better, and wash 1-2 times in total. The amount of washing buffer should usually be at least 5 times the volume of the cell pellet.

[0043] (3) Separating lymphocytes and retaining the underlying gastric cancer cells:

[0044] A. Resuspend the cell pellet using the above DPBS buffer and make the volume of the cell resuspension reach 2mL;

[0045] B. Use a pipette to draw 3 mL of lymphocyte separation solution and add it into a 15 mL sterile centrifuge tube;

[0046] C. Tilt the 15 mL centrifuge tube at a 45° angle and use a pipette to slowly add 2 mL of cell resuspension into the centrifuge tube along the tube wall, ensuring that the cell resuspension is spread evenly on the upper layer of the lymphocyte separation solution;

[0047] D. Centrifuge with a swinging bucket centrifuge. Place the 15 mL centrifuge tube in the swinging bucket adapter. Reduce the centrifuge acceleration and deceleration gear (3-5 is appropriate). Centrifuge at 800 × g for 25 minutes at room temperature.

[0048] E. After centrifugation, gently take the 15mL centrifuge tube and place it on the tube rack. You can observe obvious cell stratification: the middle white cell layer is the lymphocyte layer, and the bottom layer is the gastric cancer cell sedimentation layer (see Figure 1 and Figure 2 );

[0049] F. Aspirate the liquid in the upper layer and carefully pipette the lymphocyte layer in the middle into another clean 15mL sterile centrifuge tube. Resuspend the gastric cancer cell pellet in the bottom layer and pipette it into a new clean 15mL sterile centrifuge tube.

[0050] G. Wash the lymphocytes and gastric cancer cells respectively with 8 mL of the above DPBS buffer, centrifuge at 100 × g for 10 minutes at room temperature, and discard the supernatant;

[0051] H. Resuspend the lymphocytes with the above-mentioned DPBS buffer and plant the resuspended lymphocytes into a 100 mm cell culture dish. Place the dish in an incubator at 37°C, 5% CO2, and 95% relative saturated humidity for 1-2 hours. Pipette the suspended cells and transfer them back to a new 100 mm cell culture dish to further improve the purity of the lymphocytes for future research or freeze them at -80°C and store them in a liquid nitrogen tank.

[0052] 2.2 Organoid Culture

[0053] The two methods of gastric cancer cell sedimentation culture after separation of lymphocytes and mixed culture of lymphocytes and gastric cancer cells (hereinafter referred to as mixed cells) were compared:

[0054] (1) Organoid gel droplet seeding: Take an appropriate amount of gastric cancer cell pellet or mixed cells, resuspend the cells in liquid matrix gel incubated on crushed ice, ensure that the volume ratio of matrix gel to cell suspension is 1:1, use a pre-cooled 200 μL pipette tip to draw 50 μL of the matrix gel cell mixture and inoculate it into the middle of the well of a 24-well cell culture plate, and avoid the droplet contacting the well wall to cause the water droplet to be destroyed and the generation of bubbles during the seeding process;

[0055] (2) Solidification and culture of organoid gel droplets: After the plate is seeded, place the 24-well cell culture plate upside down in an incubator at 37°C, 5% CO2, and 95% relative saturated humidity for 10 minutes. After the organoid gel droplets solidify, take out the 24-well cell culture plate and use a pipette to add 800 μL / well of organoid culture medium. Adding 1 mL of DPBS buffer to the peripheral wells of the 24-well cell culture plate can effectively reduce the evaporation of the organoid culture medium. Place the 24-well cell culture plate upright in the above incubator for culture. Replace the organoid culture medium every 3-4 days. When adding the culture medium, keep the pipette tip away from the gel droplets to avoid damaging the matrix gel.

[0056] An inverted microscope was used to regularly observe and compare the growth status of organoids in the gastric cancer cell pellet culture group and the mixed cell culture group. Figure 3 and Figure 4 As shown, with the increase of culture date, the organoid model gradually took shape, but the proportion of gastric cancer cells in the organoid model of the mixed cell group was significantly lower than that in the organoid model of the single gastric cancer cell group, and the number of organoids formed was also much lower than that in the single gastric cancer cell group.

[0057] (3) Organoid passaging and cryopreservation: Use a pipette to discard the organoid culture medium in the 24-well cell culture plate, wash each well with the above-mentioned DPBS buffer 1-2 times, and add 500 μL TrypLE TMExpress Enzyme (1×) digests the matrix gel, and after using a pipette to blow and destroy the matrix gel droplets, place the 24-well cell culture plate in the above-mentioned incubator and let it digest for 10 minutes. After the organoids are digested into smaller cell clumps or dispersed gastric cancer cells, add organoid culture medium to neutralize the effect of the digestive enzyme. Use a pipette to add the neutralized cell digestion suspension to a 15 mL sterile centrifuge tube, then centrifuge at 200×g for 5 minutes and discard the supernatant. After washing 1-2 times with the above-mentioned DPBS buffer, plate the organoid gel droplets again (for specific operation steps, refer to step 2.2 (1)) or use the rapid cell freezing solution to freeze at -80℃ and place in a liquid nitrogen tank for storage.

[0058] (4) Pathological identification of gastric cancer organoids: Organoids were cultured using agar paraffin-embedded gastric cancer cell pellets. Organoid paraffin sections were stained with HE and IHC for gastric adenocarcinoma cell markers CDX2 and CK7. Pathological results showed that the cultured organoids had pathological characteristics of gastric cancer cells ( Figure 5 ), proving that gastric cancer organoids were successfully cultured and that this organoid model can be used for subsequent in vitro tumor molecular biology studies and in vivo PDOX studies.

[0059] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A method for constructing a gastric cancer cell organoid model derived from malignant pleural effusion and ascites, characterized in that: The steps include: A. Treatment of pleural and peritoneal effusion samples After the presence of gastric cancer cells was identified in the pleural and ascites samples, they were centrifuged, cell pellets were collected, and red blood cells were lysed to separate and remove PBMCs and neutrophils in the pleural and ascites samples; B. Isolation of gastric cancer cells After the cell precipitate treated in step A is resuspended, lymphocyte separation solution and cell resuspension solution are added in sequence. After low-speed horizontal centrifugation, obvious cell stratification is observed. The middle white cell layer is the lymphocyte layer, and the bottom layer is the gastric cancer cell precipitate layer. After separation and extraction, gastric cancer cells are obtained; C. Gastric cancer cell organoid culture The gastric cancer cell suspension was mixed with the matrix gel incubated in advance on crushed ice at a volume ratio of 1:1, and inoculated into the multi-well culture plate in an ice-cold state; after the inoculation, the multi-well cell culture plate was placed upside down in an incubator at 37°C, 5% CO2, and 95% relative saturated humidity to stand, and after the organoid gel droplets solidified, the organoid culture medium was added, and DPBS buffer was added to the surrounding wells to reduce the evaporation of the organoid culture medium; The multi-well cell culture plate was placed upright in the above-mentioned incubator for culture, and the organoid culture medium was replaced every 3-4 days. After the organoid culture was completed, digestion and separation were performed to obtain a gastric cancer cell organoid model derived from pleural effusion. The organoid culture medium includes a basal culture medium and basal factors and efficacy factors added thereto, wherein the efficacy factors include Wnt pathway agonists, MAPK P38 inhibitors, TGFβ pathway inhibitors, EGF, FGF-10 and Noggin, ROCK inhibitors, Gastrin and Nicotinamide.

2. The method for constructing a gastric cancer cell organoid model derived from malignant pleural effusion according to claim 1, characterized in that: in, In step A, the method for identifying gastric cancer cells in pleural effusion samples is as follows: transfer the freshly isolated pleural effusion samples to a centrifuge tube, centrifuge at 400 × g for 5 minutes at room temperature, collect the cell pellet in the pleural effusion, and add 100 U·mL -1 / 100mg·mL -1 The cell pellet was washed 2-3 times with DPBS buffer containing penicillin-streptomycin and 100 μg / mL Primocin antibiotics and centrifuged, and the fibrin pellet was aspirated and discarded. Finally, an appropriate amount of cell suspension was aspirated with a pipette and added to a 96-well cell culture plate to observe whether there were gastric cancer cells.

3. The method for constructing a gastric cancer cell organoid model derived from malignant pleural effusion according to claim 1, characterized in that: in, The steps for lysing red blood cells are as follows: add 3-5 volumes of red blood cell lysis buffer to 1 volume of cell packed volume at room temperature or 4°C, lyse for 1-2 minutes, centrifuge at 200×g for 5 minutes and discard the red supernatant; then use 100U·mL -1 / 100mg·mL -1 The cell pellet after red blood cell removal was resuspended in DPBS buffer containing penicillin-streptomycin and 100 μg / mL Primocin antibiotics, centrifuged at 200×g for 2-3 minutes and the supernatant was discarded. The pellet was washed 1-2 times with at least 5 volumes of washing solution to obtain gastric cancer mixed cells after red blood cells and white blood cells were removed.

4. The method for constructing a gastric cancer cell organoid model derived from malignant pleural effusion according to claim 1, characterized in that: in, In step B, after resuspending the cell pellet with DPBS buffer, an excess of lymphocyte separation solution was added to the centrifuge tube at a volume ratio of 3:2; and then, with the centrifuge tube tilted at 45°, the cell resuspension solution with the same volume as the cell suspension was slowly added to the centrifuge tube to ensure that the cell resuspension solution was spread on the upper layer of the lymphocyte separation solution. At room temperature, after centrifugation at 800×g for 25 minutes, obvious cell stratification was observed. The middle white cell layer was the lymphocyte layer, and the bottom layer was the gastric cancer cell sedimentation layer. Lymphocytes and gastric cancer cells were extracted separately, and washed with DPBS buffer respectively. At room temperature, the mixture was centrifuged at 100×g for 10 minutes, and the supernatant was discarded to obtain two types of cells.

5. The method for constructing a gastric cancer cell organoid model derived from malignant pleural effusion according to claim 1, Features: in, In step C, when inoculating into a multi-well culture plate, the matrix gel cell mixture is inoculated into the middle of the well of the multi-well cell culture plate to prevent the droplet from contacting the well wall to cause the water droplet to be destroyed and bubbles to be generated during the inoculation process; The amount of organoid culture medium added was 800 μL / well. When adding the culture medium, the pipette tip was kept away from the gel droplet to avoid damaging the matrix gel; After the organoid culture is completed, each well is washed 1-2 times with the above DPBS buffer and 500 μL TripLE is added to each well. TM Express Enzyme (1X) was used to digest the matrix gel. After blowing and destroying the matrix gel droplets, the cell culture plate was placed in the above-mentioned incubator for 10 minutes of static digestion. After the organoids were digested into smaller cell clumps or dispersed gastric cancer cells, the organoid culture medium was added to neutralize the effect of the digestive enzyme; the neutralized cell digestion suspension was added to a sterile centrifuge tube using a pipette, and then centrifuged at 200×g for 5 minutes and the supernatant was discarded. After washing 1-2 times with DPBS buffer, the organoids were coagulated again or frozen at -80℃ using a rapid cell freezing solution and stored in a liquid nitrogen tank.

6. The method for constructing a gastric cancer cell organoid model derived from malignant pleural effusion according to claim 1, characterized in that: in, The basal culture medium is selected from DMEM / F12 basal culture medium; The basic factors include 10% fetal bovine serum, 100 U·mL -1 / 100mg·mL -1 Penicillin-streptomycin and 100 μg / mL Primocin antibiotics; Among the efficacy factors, the Wnt pathway agonist is composed of 10% R-Spondin 1 and 10% Wnt-3a, the MAPK P38 inhibitor concentration is 3 μM, the TGFβ pathway inhibitor concentration is 0.5-1 μM, the EGF concentration is 5 ng / mL, the FGF-10 concentration is 20 ng / mL and the Noggin concentration is 100 ng·mL -1 , ROCK inhibitor concentration was 10 mM, Gastrin concentration was 10 nM, and Nicotinamide concentration was 10 mM.

7. The method for constructing a gastric cancer cell organoid model derived from malignant pleural effusion according to claim 6, characterized in that: in, The MAPK P38 inhibitor is selected from SB 202190, the TGFβ pathway inhibitor is selected from A83-01, and the ROCK inhibitor is selected from Y27632.

8. A gastric cancer cell organoid model derived from malignant pleural effusion and ascites, characterized in that: The method is prepared by any one of claims 1 to 7.

9. Use of the gastric cancer cell organoid model derived from malignant pleural effusion and ascites as described in claim 8 in in vitro drug screening for gastric cancer.

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