Culture composition of mesothelioma organoid and culture method of mesothelioma organoid

Abstract

The invention belongs to the field of cell culture, and relates to a mesothelioma organoid culture composition, the mesothelioma organoid culture composition comprises Rspondin-1, Noggin, Y27632, a growth factor and glutamic acid or a derivative thereof, the mesothelioma organoid culture composition is simple in component and reasonable in collocation, the mesothelioma organoid culture composition is combined with a specific culture method, mesothelioma cells from thoracic / peritoneum can be rapidly cultured, and the mesothelioma organoid culture composition is suitable for large-scale production. And mesothelioma organs with physiological characteristics are generated.

Description

Technical Field

[0001] The present invention belongs to the field of cell culture, and relates to a culture combination of mesothelioma organoids and a culture method thereof. Background Art

[0002] Organoids refer to small cell clusters that grow in a specific 3D environment in vitro from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), neonatal or adult stem cells (ASCs), tumor cells, etc., and self-organize and differentiate into functional cell types. Although organoids are not real human organs, they can simulate real organs in structure and function, simulate the in-vivo tissue structure and function to the greatest extent, and can be subcultured stably for a long time.

[0003] Although traditional 2D cell or tissue culture is widely used in various biomedical researches, it is usually considered as a simple physical contact between cells, lacking tissue structure and complexity. Compared with 2D cell culture, organoids have advantages such as a more physiological cell composition and behavior, a more stable genome, being more suitable for biotransfection and high-throughput screening, etc. And compared with animal models, the operation of the organoid model is simpler, the success rate is higher, and it is convenient to study the mechanisms of disease occurrence and development, etc.

[0004] The research focus of organoids is patient-derived organoids (PDOs), which are generally used for disease modeling, research and drug screening of tumor patients, and are generally called tumor organoids. The development of tumor organoids started in 2013 and has been on the rise year by year since then. The existing technology mainly obtains them by culturing patient biopsy, puncture or surgically resected tissues in Matrigel for several weeks. While highly maintaining the heterogeneity of the source tumor and the heterogeneity between patients, the morphology and scale among individuals remain basically uniform, providing a fast and excellent technical platform for fields such as tumor pathogenesis research, drug screening, personalized precision medicine, and regenerative medicine.

[0005] Malignant mesothelioma is usually referred to as mesothelioma. It can originate from four major parts of the body, of which 75% are pleural mesothelioma. Although the incidence is low, its malignancy is high, and the median survival period is only about one year; followed by peritoneal mesothelioma. At present, there is still a lack of effective treatment for mesothelioma, and its pathogenesis and preclinical research on drug development urgently need more realistic in vitro models. Patent CN 113881635 A provides a culture medium and culture method for pleural mesothelioma organoids, but the composition of the culture medium is relatively complex, and the method does not reflect the advantages of application in immunotherapy evaluation. In addition, the culture method of organoids is also an important reason affecting their final morphology. The existing spheroid method, gel drop method, etc., have problems such as flat morphology, low simulation, lack of means for CAR-T efficacy evaluation and detection, and cumbersome operation. Therefore, the development of a culture system that can be used to cultivate stable, real, and efficient thoracic / peritoneal mesothelioma organoids is still a technical problem that needs to be solved in this field. Summary of the invention

[0006] The purpose of the present invention is to provide a culture combination of mesothelioma organoids, which has simple components and good culture effect.

[0007] The specific technical solutions are as follows:

[0008] A culture combination of mesothelioma organoids, comprising Rspondin-1, Noggin, Y27632, growth factors and glutamate or its derivatives.

[0009] In some embodiments, the growth factor is selected from any one of epidermal growth factor (EGF), platelet-derived proliferation factor (PDGF), transforming growth factor (TGFα, TGFβ), fibroblast growth factor (FGF), insulin-like growth factor (IGF), nerve growth factor (NGF), interleukin growth factor (IL), erythropoietin (EPO), and colony stimulating factor (CSF), preferably epidermal growth factor (EGF).

[0010] In some embodiments, the glutamate derivative is selected from any one of L-glutamine and GlutaMAX, preferably GlutaMAX.

[0011] In some specific embodiments, the culture combination includes EGF, Rspondin1, Noggin, GlutaMAX, and Y27632.

[0012] In some embodiments,

[0013] The concentration of Rspondin-1 is 300 - 600 ng / ml, such as 300 ng / ml, 350 ng / ml, 400 ng / ml, 450 ng / ml, 500 ng / ml, 550 ng / ml, 600 ng / ml, and preferably 500 ng / ml.

[0014] The concentration of Noggin is 350 - 650 ng / ml, such as 350 ng / ml, 400 ng / ml, 450 ng / ml, 500 ng / ml, 550 ng / ml, 600 ng / ml, and preferably 500 ng / ml.

[0015] The dosage of Y27632 is 3 - 15 μM, such as 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, and preferably 10 μM.

[0016] The concentration of the growth factor is 100 - 300 ng / ml, such as 100 ng / ml, 150 ng / ml, 200 ng / ml, 250 ng / ml, 300 ng / ml, and preferably 200 ng / ml.

[0017] The dosage of glutamic acid or its derivative is 0.5× - 1.5×, such as 0.5×, 0.6×, 0.7×, 0.8×, 0.9×, 1×, 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, and preferably 1×.

[0018] In some embodiments, the culture combination further contains fetal bovine serum (FBS) or / and penicillin-streptomycin mixture.

[0019] In some specific embodiments, the culture combination includes FBS, EGF, Rspondin1, Noggin, GlutaMAX, Y27632, and penicillin-streptomycin mixture.

[0020] In some embodiments, the dosage of the fetal bovine serum is 3% - 10%, such as 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, and preferably 5%.

[0021] In some specific embodiments, the dosage of the penicillin-streptomycin mixture is 0.5× - 1.5×, such as 0.5×, 0.6×, 0.7×, 0.8×, 0.9×, 1×, 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, and preferably 1×.

[0022] Compared with the prior art, the solution omits various culture components, such as B27 without vitamin A, N2, acetylcysteine, GSK-3, and GSK-3 inhibitors, etc. A significant culture effect can be achieved by using a small number of culture types.

[0023] The present invention also provides a method for culturing mesothelioma organoids, which uses the culture combination as described above.

[0024] In some embodiments, the culturing method includes the following culturing steps:

[0025] 1) Resuspend the mesothelioma cells to be cultured in Matrigel and then drop them onto a peelable material to form gel drops.

[0026] 2) Add a culture medium containing the above-mentioned culture combination into the culture container. After the Matrigel solidifies, dial the gel drops into the culture medium and perform suspension culture to obtain the product.

[0027] In some specific embodiments, each gel drop contains 15 μl of Matrigel, and the gel drop density is 1×10 3 cells / 15 μl of Matrigel - 1×10 5 cells / 15 μl of Matrigel, preferably 1×10 4 cells / 15 μl of Matrigel.

[0028] The method of suspension culture of gel drops adopted by the invention can maintain the 3D structure in the gel drops to the greatest extent. Compared with the existing spheroid method and gel drop method, it can effectively simulate the drug sensitivity reaction of real tumors in vivo, be used to predict the sensitivity of patients to anti-tumor drugs such as CAR-T cell drugs, and solve the defect that there is a difference between the in vitro drug sensitivity detection method and the in vivo sensitivity of mesothelioma. The suspension method is also convenient for collecting organoids and can effectively prevent mechanical damage to organoids during the collection process.

[0029] In some embodiments, the Matrigel is Matrigel matrix glue, which is convenient to prepare and has high batch stability.

[0030] In some embodiments, the peelable material is a film material, preferably a sealing film.

[0031] In some embodiments, the culture medium in step 2) is the basal medium Advanced DMEM / F12.

[0032] In some embodiments, the culture container in step 2) is placed on a shaker for suspension culture, and the physical perfusion method based on the shaker can accelerate nutrient exchange.

[0033] In some specific embodiments, the shaker speed is 10 - 30 rpm, such as 10 rpm, 15 rpm, 20 rpm, 25 rpm, 30 rpm, and preferably 20 rpm.

[0034] In some embodiments, the culture vessel in step 2) is placed in an incubator, and the conditions set for the incubator are 37 °C and 5% CO2.

[0035] In some embodiments, in step 2), 50% of the medium is replaced every 3 days, and the medium contains the above-mentioned culture combination.

[0036] Through the comprehensive optimization of the medium components and the special culture method, it takes only 2 - 3 weeks from the preparation of mesothelioma organoids to their availability for drug testing, greatly shortening the time window for drug testing compared to animal models, and providing a more efficient and reliable means for guiding the selection of clinical treatment for mesothelioma.

[0037] The invention also provides a mesothelioma organoid prepared from the culture combination as described above or the culture method as described above. Compared with the corresponding patient-derived tumor xenograft model (PDX), it has significant advantages such as high preparation success rate, small batch differences, high preparation throughput, short drug efficacy detection time, and good biological simulation.

[0038] The invention also provides the application of the above-mentioned mesothelioma organoids in evaluating the therapeutic effect of anti-tumor drugs.

[0039] In some embodiments, the anti-tumor drugs are selected from cytotoxic drugs, small molecule targeted drugs, antibodies, immune checkpoint inhibitors, hormones, antibody-drug conjugates, biological response modifiers, differentiation inducers, tumor vaccines, CAR-T cell drugs.

[0040] In some specific embodiments, the anti-tumor drug is preferably a CAR-T cell drug.

[0041] Advantages of the present invention:

[0042] 1. The medium can be simultaneously applied to the culture of pleural / peritoneal mesothelioma organoids, and the cultured pleural / peritoneal mesothelioma organoids maintain the morphological structure and genetic characteristics of the primary tissue;

[0043] 2. The medium contains the minimum components required for the culture of pleural / peritoneal mesothelioma organoids and can effectively culture tumor tissues derived from mesothelial cells;

[0044] 3. The invention uses a suspension gel droplet culture for organoids, which can simulate the 3D structure of in vivo tissues to the greatest extent compared with the traditional gel droplet method;

[0045] 4. The organoid culture method used in the invention supports various detection methods for drug sensitivity evaluation, greatly shortening the drug testing time window compared with animal models, and providing a more efficient and reliable means for guiding the clinical treatment selection of mesothelioma. Description of the Drawings

[0046] Figure 1 It is a statistical chart of the viability of tumor cells after the tumor tissue is minced and digested.

[0047] Figure 2 It is a morphological diagram of mesothelioma organoid gel droplets.

[0048] Figure 3 It is a morphological diagram of the growth of pleural mesothelioma organoids.

[0049] Figure 4 It is a morphological diagram of the growth of peritoneal mesothelioma organoids.

[0050] Figure 5 It is a schematic diagram for exploring the culture medium of pleural / peritoneal mesothelioma organoids.

[0051] Figure 6 It is a comparison diagram of the growth of traditional droplet method and the pleural organoids prepared by the present invention.

[0052] Figure 7 It is a comparison diagram of the growth curves of the present invention and spheroid.

[0053] Figure 8 It is a morphological diagram of the growth of pleural mesothelioma organoids after three consecutive passages.

[0054] Figure 9 It is a comparison diagram of the HE staining results of pleural mesothelioma PDX and PDXO.

[0055] Figure 10 It is a comparison diagram of the immunostaining results of the expression levels of pleural mesothelioma characteristic markers in PDX and PDXO.

[0056] Figure 11 It is a diagram of the morphological changes and apoptosis distribution after CAR-T killing pleural mesothelioma organoids analyzed by a 3D fluorescence imager. Detailed Embodiments

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0058] Although the numerical ranges and parameter approximations shown in the broad scope of the present invention, the numerical values shown in the specific embodiments are recorded as accurately as possible. However, any numerical value inherently necessarily contains certain errors, which are caused by the standard deviations existing in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, the range of "300 - 600" should be considered to include any and all sub-ranges between the minimum value of 300 and the maximum value of 600 (including the endpoints); that is, all sub-ranges starting from the minimum value of 300 or greater, such as 300 to 400, and sub-ranges ending at the maximum value of 600 or less, such as 500 to 600. In addition, any reference cited as "incorporated herein" should be understood to be incorporated in its entirety.

[0059] One object of the present invention is to provide a culture combination of mesothelioma organoids, the combination containing the minimum components required for culturing pleural / peritoneal mesothelioma organoids and being capable of effectively culturing tumor tissues derived from mesothelial cells.

[0060] The culture combination of mesothelioma organoids provided by the invention includes Rspondin-1, Noggin, Y27632, growth factors, and glutamate or its derivatives.

[0061] The term "Rspondin-1", also known as "R-Spondin 1", "RSPO1", "Roof-plate-specific Spoddin 1", or cysteine-rich and thrombospondin type 1 domain-containing protein 3, is a ligand of Lgr5 and can activate the Lgr5 receptor, thereby positively promoting the Wnt signaling pathway.

[0062] In some embodiments, the concentration of Rspondin-1 is 300 - 600 ng / ml, such as 300 ng / ml, 350 ng / ml, 400 ng / ml, 450 ng / ml, 500 ng / ml, 550 ng / ml, 600 ng / ml, and preferably 500 ng / ml.

[0063] The term "Noggin" is a secreted homodimeric glycoprotein. The secreted Noggin is close to the cell surface due to binding to heparin-containing proteoglycans and is an antagonist of bone morphogenetic proteins (BMPs).

[0064] In some embodiments, the concentration of Noggin is 350 - 650 ng / ml, such as 350 ng / ml, 400 ng / ml, 450 ng / ml, 500 ng / ml, 550 ng / ml, 600 ng / ml, and preferably 500 ng / ml.

[0065] The term "Y27632" is an orally available, ATP-competitive selective inhibitor of ROCK, and also a highly effective, cell-permeable, reversible, and selective inhibitor of Rho-associated protein kinase.

[0066] In some embodiments, the dosage of Y27632 is 3 - 15 uM, such as 3 uM, 4 uM, 5 uM, 6 uM, 7 uM, 8 uM, 9 uM, 10 uM, 11 uM, 12 uM, 13 uM, 14 uM, 15 uM, preferably 10 uM.

[0067] The term "growth factor" refers to a class of organic substances that are necessary for regulating the normal growth and metabolism of microorganisms but cannot be synthesized by simple carbon and nitrogen sources. In a broad sense, growth factors include, in addition to vitamins, bases, purines, pyrimidines, biotin, nicotinic acid, etc., and sometimes also include the amino acids required by amino acid auxotrophic mutants; while in a narrow sense, growth factors generally only refer to vitamins. A class of polypeptide substances that regulate multiple effects such as cell growth and other cell functions by binding to specific and high-affinity cell membrane receptors. The growth factors described in the invention include, but are not limited to, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor (TGFα, TGFβ), fibroblast growth factor (FGF), insulin-like growth factor (IGF), nerve growth factor (NGF), interleukin growth factor (IL), erythropoietin (EPO), colony-stimulating factor (CSF), preferably epidermal growth factor (EGF).

[0068] In some embodiments, the concentration of the growth factor is 100 - 300 ng / ml, such as 100 ng / ml, 150 ng / ml, 200 ng / ml, 250 ng / ml, 300 ng / ml, preferably 200 ng / ml.

[0069] The glutamate derivative is selected from any one of L-glutamine and GlutaMAX, preferably GlutaMAX.

[0070] The term "GlutaMAX" refers to L-alanyl-L-glutamine (Alanyl-glutamine, Ala-Glu), also known as alanyl glutamine, alanyl glutamic acid dipeptide, which is very stable in aqueous solution and will not spontaneously degrade.

[0071] In some embodiments, the dosage of the glutamate or its derivative is 0.5× - 1.5×, such as 0.5×, 0.6×, 0.7×, 0.8×, 0.9×, 1×, 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, preferably 1×.

[0072] Among them, 0.5× - 1.5× represents the concentration after the original reagent is diluted. Taking the dosage of 1× as an example, for instance, the original concentration of GlutaMAX is 100×, and the dosage means that after GlutaMAX is added to the culture medium, it can be diluted 100 times, and the concentration becomes 1×.

[0073] The culture combination of the present invention may further contain fetal bovine serum (FBS) or / and penicillin-streptomycin mixture (Pen / Strep).

[0074] The dosage of the fetal bovine serum is 3% - 10%, such as 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, and preferably 5%.

[0075] The dosage of the penicillin-streptomycin mixture is 0.5× - 1.5×, such as 0.5×, 0.6×, 0.7×, 0.8×, 0.9×, 1×, 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, and preferably 1×.

[0076] The second object of the present invention is to provide a method for culturing mesothelioma organoids, and the method uses the above-mentioned culture combination to contact with the mesothelioma cells to be cultured.

[0077] The specific culture steps are as follows:

[0078] 1) Resuspend the mesothelioma cells to be cultured in Matrigel and then drop it on the peelable material to form a gel droplet.

[0079] 2) Add the culture medium containing the culture combination into the culture container. After the Matrigel solidifies, dial the gel droplet into the culture medium and perform suspension culture to obtain the product.

[0080] In some specific embodiments, each gel droplet contains 15 μl of Matrigel, and the gel droplet density is 1×10 3 cells / 15 μl of Matrigel - 1×10 5 cells / 15 μl of Matrigel, and preferably 1×10 4 cells / 15 μl of Matrigel.

[0081] In some embodiments, the Matrigel is Matrigel matrix gel.

[0082] In some embodiments, in step 1), the peelable material is a film material, preferably a sealing film. Commonly used materials in the industry such as polyethylene film, polypropylene film, and polyester film can be used.

[0083] In some embodiments, in step 2), the culture medium is the basal medium Advanced DMEM / F12.

[0084] In some embodiments, the culture vessel in step 2) is placed on a shaker for suspension culture, and the shaker speed is 10 - 30 rpm, such as 10 rpm, 15 rpm, 20 rpm, 25 rpm, 30 rpm, preferably 20 rpm.

[0085] In some embodiments, the culture vessel in step 2) is placed in an incubator, and the conditions set for the incubator are 37°C and 5% CO2. Appropriate adjustments can be made according to the actual culture situation.

[0086] In some embodiments, in step 2), 50% of the culture medium is replaced every 3 days, and the culture medium contains the above-mentioned culture combination.

[0087] The mesothelioma cells to be cultured can be from mesothelioma patients, such as pleural mesothelioma patients or peritoneal mesothelioma patients. Methods for obtaining mesothelioma cells include, for example: a pleural mesothelioma cell sample obtained by puncturing a mesothelioma patient, or a peritoneal mesothelioma cell sample obtained by aseptically collecting the patient's ascites.

[0088] The invention also provides a mesothelioma organoid prepared from the culture combination as described above or the culture method as described above.

[0089] The invention also provides the application of the above-mentioned mesothelioma organoids in evaluating the therapeutic effect of anti-tumor drugs.

[0090] The tumor drugs include but are not limited to cytotoxic drugs, small molecule targeted drugs, antibodies, immune checkpoint inhibitors, hormones, antibody-drug conjugates, biological response modifiers, differentiation inducers, tumor vaccines, CAR-T cell drugs. Preferably, they are CAR-T cell drugs.

[0091] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product instructions.

[0092] Example 1 Culture method of pleural mesothelioma organoids

[0093] A culture method of pleural mesothelioma organoids derived from PDX (patient-derived tumor xenograft model) includes the following steps:

[0094] 1) Obtaining pleural mesothelioma cells: Transplanting the puncture sample of a mesothelioma patient subcutaneously into an immunodeficient mouse to construct a PDX model, culturing until it reaches about 1000 mm 3 and then taking it out, removing the surface blood vessels, and cutting it into 1 mm 3Small pieces were added with collagenase / hyaluronidase (#70912, Stemcell) and incubated with shaking at 37°C for 20 - 30 minutes. After digestion, an equal volume of basal medium Advanced DMEM / F12 (containing 5% FBS) was added to terminate the reaction, and the mixture was filtered through a 100μm cell strainer to remove large undigested fragments. Subsequently, the cells were centrifuged at 1500 rpm for 3 minutes. After removing the supernatant, the pellet was resuspended in the basal medium, and the centrifugation was repeated twice to remove debris and enzymes. The tumor cells were counted and the cell viability was statistically analyzed ( Figure 1 );

[0095] 2) Culture of pleural mesothelioma organoids: The pleural mesothelioma cells obtained in step 1) were resuspended in Matrigel matrix gel and dropped at 1×10 4 cells / 15μl on a sterile sealing film ( Figure 2 ); The sealing film was attached to a 6-well plate / culture dish, inverted and placed in a 37°C incubator for 5 minutes to solidify the Matrigel matrix gel. The medium containing the culture combination of the present invention was added to the 6-well plate in advance. The gel drops were transferred into the medium and placed on a shaker (20 rpm) at 37°C in a 5% CO2 incubator for suspension culture. Half of the medium was changed every 3 days to obtain primary pleural mesothelioma organoids (PDXO) ( Figure 3 ).

[0096] Example 2 Culture method of peritoneal mesothelioma organoids

[0097] A culture method of peritoneal mesothelioma organoids derived from ascites, comprising the following steps:

[0098] 1) Obtaining peritoneal mesothelioma cells: The patient's ascites was collected aseptically and aliquoted into 50 ml centrifuge tubes. Subsequently, the cells were centrifuged at 500 g for 5 minutes. After removing the supernatant, the pellet was resuspended with PBS, and the centrifugation was repeated twice to remove cell debris and impurities.

[0099] 2) Culture of peritoneal mesothelioma organoids: The peritoneal mesothelioma cells obtained in step 1) were resuspended in Matrigel matrix gel and dropped at 1×10 4 cells / 15μl on a sterile sealing film; The sealing film was attached to a 6-well plate / culture dish, inverted and placed in a 37°C incubator for 5 minutes to solidify the Matrigel matrix gel. The medium containing the culture combination of the present invention was added to the 6-well plate in advance. The gel drops were transferred into the medium and placed on a shaker (20 rpm) at 37°C in a 5% CO2 incubator for suspension culture. Half of the medium was changed every 3 days to obtain primary peritoneal mesothelioma organoids (PDXO) ( Figure 4 ).

[0100] Example 3 Screening of Components of Mesothelioma Organoid Medium

[0101] The components of the mesothelioma organoid medium are set as shown in Table 3.1. Prepare pleural mesothelioma organoids according to the method described in Example 1, and continuously culture them in an incubator at 37°C and 5% CO2. Perform morphological observation on the 5th day. The results show that as the content of each component increases, the culture effect gradually improves. The effects of Experimental Group 4 and Experimental Group 5 are the best, indicating that they maintain the growth of pleural mesothelioma organoids well( Figure 5 ).

[0102] Table 3.1

[0103]

[0104] Example 4 Effect of Culture Method on the Growth Ability of Pleural Mesothelioma Organoids

[0105] 4.1 Comparison with the Traditional Droplet Method

[0106] Prepare pleural mesothelioma organoids using the traditional droplet method and the method described in Example 1 respectively, and continuously culture them in an incubator at 37°C and 5% CO2. Perform morphological observation and comparison on the 5th and 10th days respectively. The present invention can obtain a more uniform and complete organoid morphology, which is convenient for subsequent drug sensitivity observation( Figure 6 ).

[0107] The traditional droplet method includes the following steps:

[0108] 1) Obtaining pleural mesothelioma cells: the same as step 1) in Example 1;

[0109] 2) Culturing pleural mesothelioma organoids: Resuspend the cell mass precipitate obtained in step 1) with Matrigel matrix gel, and then use a pipette to drop the gel mixed with cells into a 24-well plate, about 20 - 30 μl per drop; Place the 24-well plate with the inoculated gel drops in an incubator at 37°C and 5% CO2 and let it stand for 2 min. After gently shaking the gel drops and there is no obvious flow, carefully invert the plate and wait for it to fully solidify for 30 min; Add 1 ml of the medium containing the culture combination described in the invention to each well in the plate, and then place it in a constant temperature incubator and culture at 37°C and 5% CO2 concentration.

[0110] 4.2 Comparison with the Tumor Cell Spheroids Culture Method

[0111] Prepare pleural mesothelioma cells using the tumor cell spheroids culture method and the method described in Example 1 respectively, and continuously culture them in an incubator at 37°C and 5% CO2. After culturing for the same time, perform cell viability detection. The mesothelioma organoids prepared by the culture method of the present invention can better maintain the proliferation ability of primary cells compared with spheroids( Figure 7 ).

[0112] The method for culturing tumor cell spheroids includes the following steps:

[0113] 1) Obtaining pleural mesothelioma cells: same as step 1) in Example 1;

[0114] 2) Add 100 μl of the culture medium containing the culture combination described in the invention, and then add 100 μl of CellTiter-Glo 3D (#G9681, Promega). According to the operating instructions of the brand manufacturer of the CellTiter-Glo 3D kit, measure and quantify cell viability, and at the same time use an ultra-low attachment 96-well U-bottom plate to make spheroids.

[0115] Example 5 Passage stability of pleural mesothelioma organoids

[0116] After growing for 3 weeks, the pleural mesothelioma organoids were transferred to a 15-ml centrifuge tube, the culture medium was removed, and Matrigel digestive solution (Cell Recovery Solution) was added for digestion at 37 °C for 15 - 30 minutes, with gentle pipetting every 5 minutes during this period. After there were no obvious flocculent substances, the digestion was terminated by adding an equal volume of the basal medium Advanced DMEM / F12. After centrifugation at 300 g for 3 minutes, the supernatant was removed, and 0.25% Trypsin-EDTA was added for digestion for 1 - 2 minutes, and then terminated by adding an equal volume of the basal medium Advanced DMEM / F12 (containing 5% FBS). After centrifugation at 300 g for 3 minutes, the supernatant was removed, and 1 ml of the culture medium containing the culture combination described in the invention was added, followed by cell counting.

[0117] According to the culture method described in Example 1, organoid culture was carried out, and the organoids were passaged continuously three times. Morphological observations were made at 4 days and 12 days of culture, and all organoids maintained good primary cell growth characteristics ( Figure 8 ).

[0118] Example 6 Comparison between pleural mesothelioma organoids and corresponding PDX

[0119] The pleural organoids (PDXO) cultured for three weeks and the corresponding patient-derived tumor xenograft model (PDX) were paraffin-embedded and then stained with HE. The structure and cell characteristics of the pleural mesothelioma organoids maintained a high similarity to the corresponding PDX ( Figure 9 ).

[0120] The PDXOs cultured for three weeks were subjected to immunohistochemical staining with the corresponding PDXs, and the expression levels of pleural mesothelioma characteristic markers Thrombomodulin, CK5+6, Calretinin, and WT-1 on the PDXs and the corresponding PDXOs were compared. The pleural mesothelioma organoids maintained a high similarity with the corresponding PDXs ( Figure 10 ).

[0121] Example 7 Evaluation of the killing efficacy of CAR-T on pleural mesothelioma organoids

[0122] The pleural mesothelioma organoids cultured in suspension in 6-well plates were transferred to 48-well plates, one in each well. MSLN-CAR-T (self-made by the inventors, and the preparation method refers to Chinese Patent CN202111681582.0, Example 1) was added at E:T (Effector cell-MSLN-CAR-T: Target cell-pleural mesothelioma organoid) = 1:1 for co-culture and continuous killing. After 5 days of killing, PI apoptosis staining was performed and photographed using a 3D fluorescence imager. It was found that the structure of the pleural mesothelioma organoids was damaged and the proportion of apoptotic cells increased ( Figure 11 ).

Claims

1. A combination of cultures of mesothelioma organoids, characterized in that, The culture combination includes Rspondin-1, Noggin, Y27632, growth factors, and glutamic acid or its derivatives.

2. The culture combination according to claim 1, characterized in that, The growth factors are selected from any one of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor (TGFα, TGFβ), fibroblast growth factor (FGF), insulin-like growth factor (IGF), nerve growth factor (NGF), interleukin growth factor (IL), erythropoietin (EPO), colony-stimulating factor (CSF), and preferably epidermal growth factor (EGF).

3. The culture combination according to claim 1, wherein The glutamic acid derivatives are selected from any one of L-glutamine and GlutaMAX, and preferably GlutaMAX.

4. The culture combination according to any one of claims 1-3, characterized in that the concentration of Rspondin-1 is 300-600 ng / ml, preferably 500 ng / ml; the concentration of Noggin is 350-650 ng / ml, preferably 500 ng / ml; the dosage of Y27632 is 3-15 uM, preferably 10 uM. the concentration of growth factors is 100-300 ng / ml, preferably 200 ng / ml; the dosage of glutamic acid or its derivatives is 0.5×-1.5×, preferably 1×.

5. The culture combination according to any one of claims 1-4, characterized in that, The culture combination further contains fetal bovine serum (FBS) or / and penicillin-streptomycin mixture.

6. The culture combination according to claim 5, characterized in that the dosage of the fetal bovine serum is 3%-10%, preferably 5%; the dosage of the penicillin-streptomycin mixture is 0.5×-1.5×, preferably 1×.

7. A method for culturing mesothelioma organoids, characterized in that, The method uses the culture combination according to any one of claims 1-6.

8. The culturing method according to claim 7, characterized in that, The culture steps are as follows: 1) Resuspend the mesothelioma cells to be cultured in Matrigel and then drop them on the peelable material to form gel drops; 2) Add the culture medium containing the culture combination according to any one of claims 1-6 into the culture container. After the Matrigel solidifies, dial the gel drops into the culture medium and obtain the product after suspension culture.

9. The culturing method according to claim 8, wherein, The density of the droplet is 1×10 3 cells / 15 μl of Matrigel - 1×10 5 cells / 15 μl of Matrigel, preferably 1×10 4 cells / 15 μl of Matrigel.

10. The cultivation method according to any one of claims 8-9, characterized in that, The method has one or more of the following characteristics: 1) The Matrigel is Matrigel matrix gel; 2) In step 1), the peelable material is a membrane material, preferably a sealing film; 3) In step 2), the culture medium is the basic medium Advanced DMEM / F12; 4) Place the culture container in step 2) on a shaker for suspension culture, and the shaker speed is 10-30 rpm, preferably 20 rpm; 5) In step 2), replace 50% of the culture medium every 3 days.

11. A mesothelioma organoid prepared from the culture combination according to any one of claims 1-6 or the culture method according to any one of claims 7-10.

12. Use of a mesothelioma organoid according to claim 11 in evaluating the therapeutic effect of anti-tumor drugs.

13. The application according to claim 12, characterized in that, The anti-tumor drug is selected from cytotoxic drugs, small molecule targeted drugs, antibodies, immune checkpoint inhibitors, hormones, antibody-drug conjugates, biological response modifiers, differentiation inducers, tumor vaccines, CAR-T cell drugs, preferably CAR-T cell drugs.

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