A culture medium for constructing an in vitro 3D micro-tumor model of thymic tumors and its method and application

Abstract

The present invention discloses a culture medium for constructing an in vitro 3D micro-tumor model of thymic tumors and its method and application. The present invention relates to the field of biotechnology, and provides a culture medium for constructing an in vitro 3D micro-tumor model of thymic tumors, which is composed of double anti-P / S, HEPES, GlutaMax, MEM non-essential amino acid solution, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein MSP, human recombinant protein HGF, human recombinant protein IL-2, human recombinant protein IL-15, human recombinant protein Noggin, human recombinant protein IGF, forskolin, A83-01, SB202190, B27, ITS-X, Y-27632 and basal culture medium. The in vitro 3D micro-tumor model of thymic tumors constructed using the culture medium of the present invention can accurately reflect the various characteristics of the original lesions of patients, and is a good scientific research experimental model and preclinical experimental model in the field of accurate diagnosis and treatment of tumors.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a culture medium for constructing an in vitro 3D micro-tumor model of a thymic tumor, and a method and application thereof. Background Art

[0002] As the place where T cells mature, the thymus plays a core role in adaptive immunity. It is also a key site for the production of pathogenic antibodies, which in turn causes the occurrence of autoimmune diseases. Thymic tumors are a relatively rare cancer, accounting for 0.2%-1.5% of all malignant tumors, and they mainly occur in the mediastinum. According to the WHO histological classification of thymic tumors, they can be roughly divided into type A, type AB, type B1, type B2, type B3 thymomas and type C thymic carcinomas.

[0003] For patients with early thymic tumors, surgery is the main treatment method; however, for patients with advanced or recurrent thymic tumors, systemic treatment is usually used. According to the NCCN medication guidelines, the first-line treatment for thymoma is cisplatin, doxorubicin and cyclophosphamide, and the first-line treatment for thymic carcinoma is carboplatin or paclitaxel. Correspondingly, after treatment with the above drugs, the objective response rate of thymoma is about 50%, while the objective response rate of thymic carcinoma is only 21.7%. This shows that the clinical effectiveness of the current empirical treatment regimen is low and needs to be further improved and improved.

[0004] In recent years, the emergence of various patient "drug surrogate" models has gradually made personalized and precise tumor treatment possible. For example, cancer cells are obtained directly from patient tumor samples, and their sensitivity to drugs can predict the clinical objective remission rate of colorectal cancer, prostate cancer, and breast cancer. This technology has been applied to thymic biology research, including the use of artificial thymic organs to study T cell development and therapeutic engineered T cells. However, due to the rarity of thymic tumor diseases and the complexity of pathological mechanisms, the development of new therapies is limited. Moreover, compared with other solid tumors, the development of in vitro humanized models of thymic tumors is slow, and related literature and patents are rarely reported.

[0005] At present, the mainstream tumor drug sensitivity detection technology on the market is based on patient-derived tumor xenograft models (PDX) and patient-derived tumor organoid models (PDO). However, PDX has a long modeling cycle (2-4 months), a low success rate (50%-80%), and high costs (50,000-100,000 yuan). In addition, due to the presence of heterologous cell infiltration, it interferes with immune-related research. The PDO model lacks an immune microenvironment, relies on matrix gel culture, has poor repeatability, requires a large number of samples, has limited throughput of test solutions (<10 types), and has a poor correspondence with clinical practice (about 80%). At the same time, due to the presence of tumor matrix components (the culture medium is not a fully liquid environment), it brings inconvenience to the relevant research on tumor metabolism and secretion.

[0006] The existing technology for culturing thymic tumor organoids has the following disadvantages: (1) Large sample volume requirement: thymic tumor organoids generally require a large sample (>1g) after surgery for culture; (2) Long detection time: thymic tumor organoids usually take 7-10 days to form, and drug sensitivity testing is completed after about 4 weeks; (3) Low culture success rate: According to data reported in the literature, the overall culture success rate of thymic tumor organoids is about 76.2% (16 / 21); (4) High construction cost: The cost of thymic tumor organoids that rely on matrix gel as raw material for culture is more than 10,000 yuan per case; (5) Lack of immune microenvironment: Thymic tumor organoids are formed based on the induced differentiation mechanism of tumor stem cells, and their main components are tumor epithelial cells, and they contain almost no other cell types; (6) Poor clinical consistency: According to data reported in the literature, the specificity of drug sensitivity test results of thymic tumor organoids is 60% (3 / 5), with a large proportion of false negatives. Reference "Wu YH, Chao HS, Chiang CL, et al. Personalizedcancer avatars for patients with thymic malignancies: A pilot study with circulating tumor cell-derived organoids. Thorac Cancer. 2023;14(25):2591-2600. doi:10.1111 / 1759-7714.15039".

[0007] In view of the current clinical application limitations of PDX and PDO models, it is urgent to develop an anthropomorphic model that can overcome the above defects and fully represent the biological characteristics of the patient's original tumor tissue, so as to promote the development of basic research and translational applications related to thymic tumors. Summary of the invention

[0008] In order to solve the problem that existing tumor models such as PDX / PDO are limited in clinical research, the present invention provides a culture medium and a method for constructing an in vitro 3D micro-tumor model of thymic tumor, and on this basis tests its application effect in the precise treatment and diagnosis of tumors.

[0009] In a first aspect, the present invention claims a culture medium for constructing an in vitro 3D micro-tumor model of thymic tumor.

[0010] The culture medium for constructing an in vitro 3D micro-tumor model of thymic tumor claimed in the present invention consists of double-antibody P / S (penicillin-streptomycin), HEPES, GlutaMax, MEM non-essential amino acid solution, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein MSP, human recombinant protein HGF, human recombinant protein IL-2, human recombinant protein IL-15, human recombinant protein Noggin, human recombinant protein IGF, forskolin (an adenylate cyclase agonist), A83-01, SB202190, B27, ITS-X (Insulin, Transferrin, Selenium, Ethanolamine Solution), Y-27632 and basal culture medium.

[0011] In the culture medium, the final concentration of HEPES is 8-12 mM (such as 10 mM); the final concentration of GlutaMax is 0.8-1.2% (such as 1%) by volume; the final concentration of the MEM non-essential amino acid solution is 0.8-1.2% (such as 1%) by volume; the final concentration of the human recombinant protein EGF is 10-100 ng / mL (such as 50 ng / mL); the final concentration of the human recombinant protein bFGF is 10-50 ng / mL (such as 20 ng / mL); the final concentration of the human recombinant protein MSP is 5-25 ng / mL (such as 20 ng / mL); the final concentration of the human recombinant protein HGF is 10-100 ng / mL (such as 20 ng / mL); the final concentration of the human recombinant protein IL-2 is 10-100 ng / mL (such as 20 ng / mL); The final concentration of the recombinant protein IL-15 is 10-100 ng / mL (such as 20 ng / mL); the concentration of the human recombinant protein Noggin is 100-200 ng / mL (such as 100 ng / mL); the final concentration of the human recombinant protein IGF is 10-40 ng / mL (such as 20 ng / mL); the final concentration of Forskolin is 2-10 μM (such as 5 μM); the concentration of A83-01 is 0.25-1.25 μM (such as 1 μM); the concentration of SB202190 is 5-10 μM (such as 10 μM); the final concentration of B27 is 1.5-2.5% (such as 2%) by volume; the final concentration of ITS-X is 0.8-1.2% (such as 1%) by volume; and the final concentration of Y-27632 is 5-20 μM (such as 10 μM).

[0012] Furthermore, the solvent of the MEM non-essential amino acid solution is water, and the solutes and concentrations are as follows: glycine 10mM; L-alanine 10mM; L-asparagine 10mM; L-aspartic acid 10mM; L-glutamate 10mM; L-proline 10mM; L-serine 10mM. The GlutaMax is an advanced cell culture additive that can directly replace L-glutamine in cell culture medium. The GlutaMax is "GlutaMAX™ Supplement" (such as Gibco#35050061, or other products with the same composition). The ingredient of the "GlutaMAX™ Supplement" is L-alanyl-L-glutamine, which is a substitute for L-glutamine, with a concentration of 200nM, and the solvent is 0.85% NaCl solution. The A83-01 is "3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide" (such as Tocris#2939, or other products with the same composition). The SB202190 is "4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole" (such as Sigma#S7067, or other products with the same composition). The B27 is "B-27™ Supplement (50X), minus vitamin A" (such as Gibco#12587010, or other products with the same composition).The “B-27™ Supplement (50X), minus vitamin A” contains biotin, DL-α-tocopherol acetate, DL-α-tocopherol, BSA (fatty acidfree fraction V), catalase, human recombinant insulin, human transferrin, superoxide dismutase, corticosterone, D-galactose, ethanolamine hydrochloride, glutathione (reduced), L-carnitine hydrochloride, linoleic acid, linolenic acid, progesterone, putrescine2HCl, sodium selenite, triiodothyronine (T3 (triodo-I-thyronine)). The solvent of the ITS-X is EBSS solution (Earle's balanced salt solution), and the solutes and concentrations are as follows: insulin 1g / L; transferrin 0.55g / L; sodium selenite 0.00067g / L; ethanolamine 0.2g / L. The Y-27632 is "Y-27632 dihydrochloride (an ATP-competitive ROCK-I and ROCK-II inhibitor, Ki 220nM and 300nM, respectively)" (such as MCE#129830-38-2, or other products with the same composition).

[0013] Furthermore, the basic culture medium may be Advanced DMEM / F12 culture medium.

[0014] Furthermore, in the culture medium, the final concentration of penicillin in the dual-antibody P / S may be 100-200 U / mL (such as 100 U / mL); the final concentration of streptomycin in the dual-antibody P / S may be 100-200 μg / mL (such as 100 μg / mL).

[0015] Furthermore, the culture medium may exist in two forms:

[0016] First, the culture medium is a solution formed by mixing the double-antibody P / S, the HEPES, the GlutaMax, the MEM non-essential amino acid solution, the human recombinant protein EGF, the human recombinant protein bFGF, the human recombinant protein MSP, the human recombinant protein HGF, the human recombinant protein IL-2, the human recombinant protein IL-15, the human recombinant protein Noggin, the human recombinant protein IGF, Forskolin, the A83-01, the SB202190, the B27, the ITS-X, the Y-27632 and the Advanced DMEM / F12 culture medium.

[0017] After the culture medium is prepared, it needs to be filtered and sterilized using a 0.22 μM syringe filter (Millipore SLGP033RS) and can be stored at 4° C. for two weeks.

[0018] Secondly, each component in the culture medium exists independently and is prepared according to a formula when used.

[0019] Furthermore, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein MSP, human recombinant protein HGF, human recombinant protein IL-2, human recombinant protein IL-15, human recombinant protein Noggin, human recombinant protein IGF, Forskolin, SB202190 and Y-27632 can exist in the form of stock solution (mother solution) (can be stored for a long time at -80°C); specifically, it can be 1000 times stock solution (mother solution). Among them, A83-01 can exist in the form of stock solution (mother solution) (can be stored for a long time at -20°C), specifically, it can be 100000 times stock solution (mother solution).

[0020] The 1000× human recombinant protein EGF stock solution is composed of human recombinant protein EGF, BSA and PBS, wherein the final concentration of the human recombinant protein EGF is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0021] The 1000× human recombinant protein bFGF stock solution is composed of human recombinant protein bFGF, BSA and PBS, wherein the final concentration of the human recombinant protein bFGF is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0022] The 1000× human recombinant protein MSP stock solution consists of human recombinant protein MSP, BSA and PBS, wherein the final concentration of the human recombinant protein MSP is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0023] The 1000× human recombinant protein HGF stock solution consists of human recombinant protein HGF, BSA and PBS, wherein the final concentration of the human recombinant protein HGF is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0024] The 1000× human recombinant protein IL-2 stock solution consists of human recombinant protein IL-2, BSA and PBS, wherein the final concentration of the human recombinant protein IL-2 is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0025] The 1000× human recombinant protein IL-15 stock solution consists of human recombinant protein IL-15, BSA and PBS, wherein the final concentration of the human recombinant protein IL-15 is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0026] The 1000× human recombinant protein Noggin stock solution consists of human recombinant protein Noggin, BSA and PBS, wherein the final concentration of the human recombinant protein Noggin is 100 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0027] The 1000× human recombinant protein IGF stock solution is composed of human recombinant protein IGF, BSA and PBS, wherein the final concentration of the human recombinant protein IGF is 40 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0028] Among the above eight 1000-fold stock solutions, the BSA can exist in the form of a 100-fold stock solution (mother solution) (prepared and used immediately), which is specifically composed of BSA and PBS, wherein the final concentration of BSA (Sigma#A1933) is 0.1 g / mL, and the remainder is PBS.

[0029] In addition, 1000×Forskolin consists of Forskolin and DMSO, wherein the final concentration of Forskolin is 10 mM and the remainder is DMSO.

[0030] The 1000×SB202190 stock solution consists of SB202190 and DMSO, wherein the final concentration of the SB202190 is 10 mM and the remainder is DMSO.

[0031] 1000×Y-27632 is composed of Y-27632 and ultrapure water, wherein the final concentration of Y-27632 is 10 mM, and the remainder is ultrapure water.

[0032] In addition, the 100000×A83-01 stock solution consisted of A83-01 and DMSO, wherein the concentration of A83-01 was 25 mM and the remainder was DMSO.

[0033] In a second aspect, the present invention claims a kit of reagents for culturing an in vitro 3D micro-tumor model of thymic tumor.

[0034] The set of reagents for culturing an in vitro 3D micro-tumor model of thymic tumors claimed in the present invention is composed of the culture medium described in the first aspect above and all or part of the following: sample dissociation solution, sample preservation solution, sample cleaning solution, cell digestion solution, digestion termination solution and cell freezing solution.

[0035] The sample preservation solution can be used for temporary preservation of the sample after it is removed from the body, and can maintain the activity of cells in the sample for a short period of time after the sample is removed from the body. The sample cleaning solution can be used for cleaning and disinfecting the sample. The sample dissociation solution can be used for dissociation of the sample.

[0036] Furthermore, the sample dissociation solution consists of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of collagenase I is 150-250U / mL (such as 200U / mL); the final concentration of collagenase II is 150-250U / mL (such as 200U / mL); the final concentration of collagenase IV is 150-250U / mL (such as 200U / mL); and the remainder is PBS.

[0037] The unit U of collagenase (the collagenase I or the collagenase II or the collagenase IV) is defined by the enzymatic activity of the protease: at 37°C and pH 7.5, 1 U of protease is used to treat the collagenase (the collagenase I or the collagenase II or the collagenase IV) for 5 hours to release 1 μmol of L-leucine.

[0038] Furthermore, the sample preservation solution is composed of fetal bovine serum, double-antibody P / S, HEPES and HBSS; wherein the final concentration of the fetal bovine serum is 1-5% (such as 2%) by volume; the final concentration of penicillin in the double-antibody P / S is 100-200U / mL (such as 100U / mL), and the final concentration of streptomycin in the double-antibody P / S is 100-200μg / mL (such as 100μg / mL); the final concentration of HEPES is 8-12mM (such as 10mM); and the remainder is HBSS.

[0039] Furthermore, the sample cleaning solution consists of dual-antibody P / S and PBS; wherein the final concentration of penicillin in the dual-antibody P / S is 100-200U / mL (such as 100U / mL); the final concentration of streptomycin in the dual-antibody P / S is 100-200μg / mL (such as 100μg / mL); and the remainder is PBS.

[0040] Furthermore, the cell digestion solution has the following composition: every 10 mL of the cell digestion solution contains 4-6 mL (eg, 5 mL) of Accutase, EDTA with a final concentration of 5 mM, 1.5-2.5 mL (eg, 2 mL) of TrypLE Express, and the remainder is PBS.

[0041] Wherein, the Accutase is "StemPro™ Accutase™ Cell Dissociation Reagent" (such as Gibco#A11105-01, or other products with the same composition). The Accutase is a single-component enzyme that is dissolved in D-PBS, 0.5mM EDTA solution. The TrypLE Express is "TrypLE™ Express Enzyme (1X), no phenol red" (such as Gibco#12604013, or other products with the same composition). The "TrypLE™ Express Enzyme (1X), no phenol red" contains 200mg / L KCl, 200mg / L KH2PO4, 8000mg / L NaCl, 2160mg / L Na2HPO4·7H2O, 457.6mg / L EDTA; and also contains recombinant protease.

[0042] Furthermore, the digestion stop solution is composed of fetal bovine serum, double-antibody P / S and DMEM culture medium; wherein the final concentration of the fetal bovine serum is 8-12% (10%) by volume; the final concentration of penicillin in the double-antibody P / S is 100-200U / mL (such as 100U / mL); the final concentration of streptomycin in the double-antibody P / S is 100-200μg / mL (such as 100μg / mL); and the remainder is DMEM culture medium.

[0043] Furthermore, the cell freezing solution consists of Advanced DMEM / F12 culture medium, DMSO and 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM / F12 culture medium, the DMSO and the 1% methylcellulose solution is 20:2:(0.8-1.2), such as 20:2:1; and the 1% methylcellulose solution is a methylcellulose aqueous solution with a concentration of 1 g / 100 ml.

[0044] In a third aspect, the present invention claims the use of the culture medium described in the first aspect or the set of reagents described in the second aspect in constructing an in vitro 3D micro-tumor model of thymic tumor.

[0045] The thymic tumor may be type A thymoma, type AB thymoma, type B1 thymoma, type B2 thymoma, type B3 thymoma or type C thymic carcinoma.

[0046] In a fourth aspect, the present invention claims a method for constructing an in vitro 3D micro-tumor model of thymic tumor.

[0047] The method for constructing an in vitro 3D micro-tumor model of thymic tumor claimed in the present invention may include the following steps:

[0048] (a1) dissociating thymic tumor solid tumor tissue using the sample dissociation solution described in the second aspect above;

[0049] (a2) Using the culture medium described in the first aspect above to suspend and culture the single cells dissociated in step (a1) to form cell clusters, thereby obtaining an in vitro 3D micro-tumor model of thymic tumor.

[0050] In step (a1), the thymic tumor solid tumor tissue can be dissociated using the sample dissociation solution according to a method comprising the following steps: using 1 mL of the sample dissociation solution to no more than 0.5 mg of tissue, the minced thymic tumor solid tumor tissue is subjected to sample dissociation using the sample dissociation solution at 37° C. for a dissociation time of 15 minutes to 2 hours (e.g., 1 hour).

[0051] In step (a2), the cells dissociated from (a1) can be suspended and cultured using the culture medium according to a method comprising the following steps: using a cell culture container with a low adsorption surface, suspending and culture the cells dissociated from (a1) using the culture medium, and culturing the cells under conditions of 37° C. and 5% CO2.

[0052] The initial seeding density can be 10 5 Pieces / cm 2 The bottom area of ​​the container, taking a six-well plate as an example, is 10 per well. 6 The cells were plated at a density of 10 cells.

[0053] Furthermore, the culturing time in step (a2) is 3-5 days.

[0054] Furthermore, before step (a1), the following steps may be included for pre-dissociation treatment of the thymic tumor solid tumor tissue: washing the surface of the thymic tumor solid tumor tissue sample with 70-75% by volume ethanol for 10-30 seconds; washing the thymic tumor solid tumor tissue sample with the sample washing solution described in the second aspect above for 5-10 times (e.g., 5 times), and washing the thymic tumor solid tumor tissue sample with a sterile PBS solution for 5-10 times (e.g., 5 times); and then removing impurities, connective tissue, adipose tissue, necrotic tissue and other components that affect micro-tumor culture in the thymic tumor solid tumor tissue sample.

[0055] The step of pre-dissociation treatment of the thymic tumor solid tumor tissue needs to be performed on ice, and the entire operation step needs to be completed within 10 minutes.

[0056] Furthermore, in step (a1), after the thymic tumor solid tumor tissue is dissociated with the sample dissociation solution, the following steps are also included: terminating the dissociation reaction with 8-15 times (such as 10 times) the volume of the digestion stop solution described in the second aspect above, and collecting the cell suspension; filtering the cell suspension with a 100μm or 40μm sterile cell filter to remove tissue debris and adherent cells; centrifuging at 800-1000g (such as 800g) at room temperature for 10-15 minutes (such as 10 minutes), and discarding the supernatant; then resuspending the cells with 3-5 mL (such as 5mL) of sterile PBS; centrifuging again at 800-1000g (such as 800g) at room temperature for 10-15 minutes (such as 10 minutes), and discarding the supernatant; and then resuspending the cell pellet with the culture medium described in the first aspect above.

[0057] The thymic tumor solid tumor tissue sample subjected to the pre-dissociation treatment is isolated from the body within 24 hours, and is kept in the sample preservation solution described in the second aspect above before the pre-dissociation treatment.

[0058] The thymic tumor may be type A thymoma, type AB thymoma, type B1 thymoma, type B2 thymoma, type B3 thymoma or type C thymic carcinoma.

[0059] In a fifth aspect, the present invention claims a method for obtaining primary cells of thymic tumor solid tumors.

[0060] The method for obtaining thymic tumor solid tumor primary cells claimed in the present invention is to separate thymic tumor solid tumor primary cells from an in vitro 3D micro-tumor model of thymic tumor obtained by the method described in the fourth aspect above.

[0061] The method may specifically include the following steps:

[0062] (b1) digesting the thymic tumor micro-tumor model using a cell digestion solution (such as the cell digestion solution described above) to obtain single cells;

[0063] Furthermore, the method further comprises the step of terminating the digestion, such as using the digestion terminating solution as described above to terminate the digestion.

[0064] (b2) sorting out CD326 positive cells from the single cells obtained in step (b1) to obtain the thymic tumor solid tumor primary cells.

[0065] Furthermore, CD326 positive cells can be sorted using CD326 magnetic beads.

[0066] The thymic tumor may be type A thymoma, type AB thymoma, type B1 thymoma, type B2 thymoma, type B3 thymoma or type C thymic carcinoma.

[0067] In a sixth aspect, the present invention claims protection for the use of an in vitro 3D micro-tumor model of thymic tumor obtained by the method described in the fourth aspect above in screening diagnostic and / or therapeutic drugs for thymic tumor.

[0068] In a seventh aspect, the present invention claims protection for the use of an in vitro 3D micro-tumor model of thymic tumor obtained by the method described in the fourth aspect above in in vitro companion diagnosis.

[0069] Beneficial effects of the present invention:

[0070] (1) Small sample volume requirement: culture can be completed on micro-samples such as puncture and biopsy (<100 mg);

[0071] (2) Short detection cycle: The micro-tumor model of thymic tumor can be formed in only 2-3 days, and drug sensitivity testing can be completed within 10 days;

[0072] (3) High culture success rate: The culture medium of the present invention had a culture success rate of 90.7% (88 / 97) for thymic tumor samples, of which 9 failed samples were caused by objective reasons such as poor sampling quality (1 sample was contaminated and 8 samples had no tumor cells);

[0073] (4) Low construction cost: The micro-tumor model of thymic tumor does not rely on matrix gel, and its culture medium contains specific cytokines and is serum-free, with a total cost of only RMB 2,000 per case;

[0074] (5) Preservation of immune microenvironment: The micro-tumor model of thymic tumor is characterized by digesting or separating the patient-derived tumor tissue into a state dominated by single cells, and then suspending and culturing it in a serum-free culture medium containing specific cytokines, so that the single cells self-assemble and aggregate with each other and proliferate to obtain cell clusters with 3D structures. It can retain a variety of cell types in the patient's tumor sample, including tumor epithelial cells, various immune cells (such as B cells, T cells), etc., and can simulate the tumor microenvironment to a high degree. Generally, the proportion of tumor epithelial cells in the micro-tumor model is relatively large, and the presence and proportion of other cell types depends on the specific situation of the patient-derived sample (especially the type and proportion of immune cells), fully restoring the individual differences of tumor patients.

[0075] (6) High clinical consistency: The present invention detects the secretion level of AChR-Ab antibodies in the culture supernatant of micro-tumor models of thymoma-associated Myasthenia Gravis (TAMG) patient samples. The results show that the relative content of antibodies is significantly correlated with disease characteristics.

[0076] In summary, compared with the prior art, the in vitro 3D micro-tumor model of thymic tumor constructed by the present invention can accurately reflect the various characteristics of the patient's original lesion, and is a good scientific research experimental model and preclinical experimental model in the field of precision diagnosis and treatment of tumors. Its construction method is faster, has a shorter detection cycle, lower cost, higher degree of standardization, better operability and repeatability; the thymic tumor micro-tumor model can replace patients for drug testing, and find unconventional second- and third-line drugs or cross-indication drugs for some drug-resistant patients; the raw materials of the thymic tumor micro-tumor model are all provided by domestic reagent manufacturers, which is easy to operate and suitable for large-scale promotion, and will further accelerate the development of related basic research and translational applications in China. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 This is the culture and identification of the in vitro 3D micro-tumor model of thymic tumors. This bright field image depicts the phenotype of thymoma tumor tissue after being cultured into a micro-tumor model. There are solid clusters, solid dense clusters, and grape-like clusters in the micro-tumor model. The cell diameter is 50-300μm, and the number of cells in a single clone is from dozens to hundreds of cells. The scale in the figure is 100μm, 10× bright field field.

[0078] Figure 2 Comparison of the morphological structure consistency between the original tumor tissue and the corresponding PTC micro-tumor model. AB, B2, and B3 thymoma were selected and HE staining was performed to verify that the morphological structure was relatively consistent.

[0079] Figure 3Comparison of the consistency of pathological features between the original tumor tissue and the corresponding PTC micro-tumor model (CK staining). AB, B2, and B3 thymoma types were selected, and CK was stained by IHC to verify that the pathological features were relatively consistent.

[0080] Figure 4 Comparison of the consistency of pathological features between the original tumor tissue and the corresponding PTC micro-tumor model (TdT staining). AB, B2, and B3 thymomas were selected, and IHC staining of TdT was performed to verify that the pathological features were relatively consistent.

[0081] Figure 5 To identify immune cells in the PTC microtumor model, a successfully cultured TAMG PTC microtumor model was digested and single cell suspension was prepared. Then, various immune cells were labeled by flow cytometry to determine the presence and diversity of immune cells in the model.

[0082] Figure 6 Comparison of the distribution of somatic mutation SNVs in tumor tissues from different cases and PTC models. In the figure, "#number" represents the sample number, corresponding to different thymic tumor patients; T represents tumor tissue; PTC is the PTC microtumor model.

[0083] Figure 7 Comparison of chromosome variation—CNV distribution between tumor tissue and corresponding PTC microtumor. Chr: chromosome number. A is the CNV distribution map of tumor tissue; B is the CNV distribution map of the corresponding PTC. Each point in the figure represents a capture interval of the gene, the horizontal axis represents the chromosome position of the gene, and the vertical axis represents the copy number.

[0084] Figure 8 The primary thymic tumor cell line was isolated from the in vitro 3D micro-tumor model of thymic tumor. The scale of the figure is 100μm, 10× bright field field.

[0085] Fig. 9 The results of AChR-Ab in peripheral blood serum. The ELISA method was used to detect the AChR-Ab level in the serum of TAMG patients. A red line was drawn with y=0.536 as the boundary. The level above this line was antibody positive. The horizontal axis 1-29 represents the number of 29 TAMG patients.

[0086] Fig.10The results of AChR-Ab in the culture supernatant of the PTC microtumor model. The ELISA method was used to detect AChR-Ab in the culture supernatant of the PTC model of TAMG patients. A green line was drawn with y=0.322 as the boundary. Above this line, the antibody concentration in the PTC culture supernatant was significant. *Indicates that the patient used glucocorticoids or immunosuppressants. The horizontal axis 1-29 represents the number of 29 TAMG patients. DETAILED DESCRIPTION

[0087] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0088] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0089] Example 1: Preparation of reagents for constructing an in vitro 3D microtumor model of thymic tumor

[0090] 1. Sample preservation solution

[0091] The specific formula of the sample preservation solution (100 mL) is shown in Table 1.

[0092]

[0093] After the sample preservation solution is prepared, it is divided into 15mL centrifuge tubes, 5mL per tube. After division, it can be stored at 4℃ for 1 month.

[0094] 2. Sample cleaning solution

[0095] The specific formula of the sample cleaning solution (100 mL) is shown in Table 2.

[0096]

[0097] The sample cleaning solution must be prepared and used immediately.

[0098] 3. Sample dissociation solution

[0099] The specific formula of the sample dissociation solution (10 mL) is shown in Table 3.

[0100]

[0101] Note: The sample dissociation solution should be prepared and used immediately.

[0102] In Table 3, the preparation of collagenase stock solution is shown in Tables 4 to 6.

[0103]

[0104] After the 10× collagenase I stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored at -20°C for a long time.

[0105]

[0106] After the 10× collagenase II stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored at -20°C for a long time.

[0107]

[0108] After the 10× collagenase IV stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored at -20°C for a long time.

[0109] In Tables 4 to 6, the unit U of collagenase (the collagenase I or the collagenase II or the collagenase IV) is defined by the enzymatic activity of the protease: at 37° C., pH 7.5, 1 U of protease is used to treat the collagenase (the collagenase I or the collagenase II or the collagenase IV) for 5 hours to release 1 μmol of L-leucine.

[0110] 4. Cell digestion fluid

[0111] The specific formula of the cell digestion solution (10 mL) is shown in Table 7.

[0112]

[0113] The cell digestion solution is prepared and used immediately.

[0114] 5. Digestion stop solution

[0115] The specific formula of the digestion stop solution (100 mL) is shown in Table 8.

[0116]

[0117] After the digestion stop solution is prepared, it can be stored at 4°C for one month.

[0118] 6. Culture medium for constructing an in vitro 3D microtumor model of thymic tumors

[0119] The specific formula of the culture medium (100 mL) for constructing an in vitro 3D microtumor model of thymic tumor is shown in Table 9.

[0120]

[0121] After the culture medium (100 mL) for constructing the in vitro 3D micro-tumor model of thymic tumors is prepared, it is sterilized by filtration using a 0.22 μM syringe filter (Millipore SLGP033RS) and can be stored at 4°C for two weeks.

[0122] In Table 9, the preparation of human recombinant protein stock solutions is shown in Tables 11 to 18 (the preparation of BSA stock solution is shown in Table 10), the preparation of Forskolin stock solution is shown in Table 19, the preparation of A83-01 stock solution is shown in Table 20, the preparation of SB202190 stock solution is shown in Table 21, and the preparation of Y-27632 stock solution is shown in Table 22.

[0123]

[0124] 100× BSA solution is prepared and used immediately.

[0125]

[0126] After the 1000× human recombinant protein EGF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0127]

[0128] After the 1000× human recombinant protein bEGF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0129]

[0130] After the 1000× human recombinant protein MSP stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0131]

[0132] After the 1000× human recombinant protein HGF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0133]

[0134] After the 1000× human recombinant protein IL-2 stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0135]

[0136] After the 1000× human recombinant protein IL-15 stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0137]

[0138] After the 1000× human recombinant protein Noggin stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0139]

[0140] After the 1000× human recombinant protein IGF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0141]

[0142] After the 1000× Forskolin stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0143]

[0144] After the 100000×A83-01 stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0145]

[0146] After the 1000×SB202190 stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0147]

[0148] After the 1000×Y-27632 stock solution is prepared, it is packaged in 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0149] 7. Cell Cryopreservation Solution

[0150] The specific formula of the cell freezing solution is shown in Table 23.

[0151]

[0152] Cell freezing solution is prepared and used immediately.

[0153] In Table 23, the preparation of 1% methylcellulose solution is shown in Table 24.

[0154]

[0155] 1% methylcellulose solution can be stored at 4°C for a long time after preparation.

[0156] Example 2: Cultivation and identification of in vitro 3D micro-tumor model of thymic tumor

[0157] The operation process for collecting samples of thymic tumors is as follows: solid tumor resection, puncture, biopsy, etc., all sampling is completed in the operating room to avoid microbial contamination of samples; sampling requirements are as follows: for solid tumor tissue, fresh cancer tissue with rich blood vessels should be taken when cutting (fresh tissue should be cut within 30 minutes of ex vivo), avoiding necrotic areas, fibrotic areas, adipose tissue and tissue burned by electric knife, to maintain cell vitality in the tissue to the greatest extent, and tumor tissue is required to be no less than 50mg (about 5mm×5mm). For puncture samples, more than 2 strips are required, each with a length of ≥1cm.

[0158] The excised solid tumor tissue of thymic tumor should be washed by shaking in physiological saline to remove dirt. After the sample is collected, it should be immediately stored in a 15mL sterile centrifuge tube containing 5ml of sample preservation solution (see Example 1) or stored in a sterile cryopreservation tube, transported at 4°C, and sent for inspection as soon as possible (completed within 24 hours).

[0159] The specific culture process of the in vitro 3D micro-tumor model of thymic tumor is as follows:

[0160] Step 1: Pre-treatment of thymic tumor solid tumor tissue dissociation

[0161] The following operations need to be performed on ice and the entire operation should be completed within 10 minutes.

[0162] 1. After weighing the sample, clean the sample surface with medical alcohol (75% by volume) for 10 to 30 seconds.

[0163] 2. Wash the sample 5 times with sample washing solution (see Example 1), and wash the sample 5 times with sterile PBS solution.

[0164] 3. Use ophthalmic scissors, ophthalmic tweezers, scalpel and other instruments to carefully peel off the fat tissue, connective tissue and necrotic tissue in the sample.

[0165] Step 2: Dissociation of thymic tumor solid tumor tissue samples

[0166] 1. Use ophthalmic scissors to cut the tissue into 0.5mm pieces. 3 Small pieces around.

[0167] 2. Treat the tissue with sample dissociation solution (see Example 1). For tissues with a sample size not exceeding 0.5 mg, use 1 mL of sample dissociation solution. For tissues with a sample size exceeding 0.5 mg, add 0.1 mL of sample dissociation solution for every 0.1 mg increase in tissue weight. The sample dissociation solution treatment condition is 37°C, and the dissociation time is 1 hour. During the dissociation process, observe the dissociation of the sample under a microscope every 15 minutes until most cells are observed to fall off from the tissue.

[0168] 3. Use 10 times the volume of digestion stop solution (see Example 1) to terminate the dissociation reaction. Filter the cell suspension through a 100 μm sterile cell strainer to remove tissue debris and adherent cells, centrifuge at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0169] 4. Resuspend the cells in 5 mL of sterile PBS, centrifuge at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0170] 5. Resuspend the cell pellet in the culture medium of the in vitro 3D micro-tumor model of thymic tumor (see Table 9 in Example 1), count the cells, and determine the cell viability by trypan blue staining. If the viability of the isolated cells is greater than 70%, the cells can be inoculated and cultured.

[0171] Step 3: In vitro 3D micro-tumor model cultivation of thymic tumors

[0172] 1. A low-attachment-surface was used to perform suspension culture of an in vitro 3D micro-tumor model of a thymic tumor. The culture medium used was the culture medium for constructing an in vitro 3D micro-tumor model of a thymic tumor in Table 9 of Example 1 (wherein the final concentration of penicillin in the dual-antibody P / S was 100 U / mL; the final concentration of streptomycin in the dual-antibody P / S was 100 μg / mL; the final concentration of HEPES was 10 mM; the final concentration of GlutaMax was 1% by volume; the final concentration of the MEM non-essential amino acid solution was 1% by volume; the final concentration of the human recombinant protein EGF was 50 ng / mL; the final concentration of the human recombinant protein bFGF was 20 ng / mL; the final concentration of the human recombinant protein The final concentration of white MSP is 20ng / mL; the final concentration of the human recombinant protein HGF is 20ng / mL; the final concentration of the human recombinant protein IL-2 is 20ng / mL; the final concentration of the human recombinant protein IL-15 is 20ng / mL; the concentration of the human recombinant protein Noggin is 100ng / mL; the final concentration of the human recombinant protein IGF is 20ng / mL; the final concentration of Forskolin is 5μM; the concentration of A83-01 is 1μM; the concentration of SB202190 is 10μM; the final concentration of B27 is 2% by volume; the final concentration of ITS-X is 1% by volume; the final concentration of Y-27632 is 10μM). Taking a six-well plate as an example, 10 per well 6 The cells were plated at a density of 100 cells / well, with 2-3 mL of culture medium per well. The inoculated cells were cultured in a cell culture incubator at 37°C and 5% CO2.

[0173] 2. Observe the cell status every day until the cells form clumps with a diameter of about 100 μm. Then replace the culture medium every 2-3 days to maintain the growth status of thymic tumor microtumors.

[0174] The present invention cultured 97 fresh surgical tissue samples in vitro, including 9 cases of type A thymoma, 18 cases of type AB thymoma, 12 cases of type B1 thymoma, 36 cases of type B2 thymoma, 17 cases of type B3 thymoma and 5 cases of type C thymic carcinoma. The overall culture success rate was 90.7% (88 / 97), of which 9 failed cases were caused by objective reasons such as poor sampling quality (1 case of contamination and 8 cases without tumor cells). Bright field view of the in vitro 3D micro-tumor model of thymic tumor Figure 1 As shown, the diameter is 50-300 μm, the number of cells in a single microsphere is from dozens to hundreds of cells, and at least three different types of cell clusters are observed in morphology: including compact solid spheres of different sizes, solid spheres with lumens, and grape-shaped spheres, which fully reflect the individual differences of tumors. It can be seen that the present invention has successfully constructed a stable in vitro 3D micro-tumor model of thymic tumors in a full liquid culture environment.

[0175] Example 3. Scientific research characterization of in vitro 3D microtumor model of thymic tumor

[0176] In order to further determine the consistency between the in vitro 3D micro-tumor model of thymic tumor (hereinafter referred to as PTC micro-tumor) constructed in Example 2 of the present invention and the original tumor tissue in terms of morphological structure, pathological characteristics, genetic information, etc., and to identify the diversity of cellular components in the PTC micro-tumor model, this example conducted a series of evaluation experiments to ensure that the model has consistent characteristics with the original tumor tissue in the in vivo environment, so that the model can be used for subsequent mechanism research and drug screening, etc.

[0177] 1. Evaluate the consistency of morphological structure

[0178] HE staining was performed on the original tumor tissue and the corresponding PTC micro-tumor to evaluate the consistency of the morphological structure between the two. The present invention selected AB, B2, and B3 thymoma as representatives for morphological consistency evaluation. The results showed that the morphological structure of the PTC micro-tumor model had obvious consistency characteristics with the original tumor tissue ( Figure 2 ). Among them, the A-type area of ​​AB-type thymoma is not obvious, and only B-type thymoma components are seen, with neoplastic epithelial cells and scattered immature lymphocytes. Under the microscope, a large number of immature lymphocytes can be seen in B2-type thymoma, among which there are scattered neoplastic epithelial cells. Some epithelial cells are arranged in a reticular or small nest shape, and some epithelial cells have obvious nucleoli. Under the microscope, a large number of neoplastic epithelial cells can be seen in B3-type thymoma, and the cells are arranged in sheets, and nucleoli can be seen in some cells; the number of immature lymphocytes is significantly less than that of B2-type thymoma.

[0179] 2. Evaluating the consistency of pathological features

[0180] On the basis of evaluating the consistency of morphological structure, the present invention further explores the consistency of pathological characteristics between PTC microtumors and original tumor tissues. Therefore, IHC staining was performed on the above 3 samples to evaluate the consistency of pathological characteristics. CK, which is commonly used in clinical practice, was used to evaluate the presence of tumor epithelial cells, and TdT was used to evaluate the presence of immature lymphocytes.

[0181] CK staining ( Figure 3 ) The results showed that the neoplastic epithelial cells of AB and B2 thymomas were scattered or arranged in nests, while the neoplastic epithelial cells of B3 thymoma were distributed in sheets.

[0182] TdT staining results ( Figure 4 ) shows: The immature lymphocytes of AB type thymoma are distributed in sheets with high density. The immature lymphocytes of B2 type thymoma are scattered, with high cell density in some areas. The immature lymphocytes of B3 type thymoma are scattered.

[0183] The above results show that the pathological characteristics of the PTC microtumor model are obviously consistent with those of the original tumor tissue.

[0184] 3. Identification of the diversity characteristics of cell components

[0185] As is well known, immune cells have a short survival period in vitro. Given that thymoma tissue is rich in a large number of immune cells, among which helper T lymphocytes, memory T lymphocytes, regulatory T cells, B cells and memory B cells have been repeatedly found to be involved in disease regulation. The present invention plans to further determine whether the above immune cells exist in the self-assembled PTC micro-tumor model. A successfully cultured micro-tumor model of a thymic tumor was digested to prepare a single cell suspension, and then various types of immune cells were labeled by flow cytometry experiments to determine the presence and diversity of immune cells in the model.

[0186] like Figure 5 As shown, helper T cells (CD3 + CD4 + ), double positive T cells (CD3 + CD4 + CD8 + ), double negative T cells (CD3 + CD4 - CD8 - ), memory T cells (CD3 + CD4 + CD45RA - ), total B cells (CD19 + ), memory B cells (CD19 + CD27 + ), regulatory T cells (CD3+ CD4 + CD25 + CD127 - ) and Th17 cells (CD3 + CD4 + CD45RA - CCR6 + CXCR3 - CCR4 + ) exist in the model in a certain proportion.

[0187] 4. Evaluating the consistency of genetic information

[0188] As described above, the present invention has evaluated the consistency of tissue morphology and pathological characteristics. The present invention will continue to evaluate the consistency of genetic information between the original tumor tissue and the corresponding PTC micro-tumor model at the molecular level.

[0189] 1. Somatic mutations--SNV distribution results

[0190] SNV is a site where a single base on the genome changes and is widely distributed on the genome. The somatic SNV test was performed on paired samples using the software VarScan, and the test results were filtered using the software default parameters to obtain highly reliable somatic SNV results. The results of this study showed that the SNV results of each pair of samples were roughly the same ( Figure 6 Specifically, the relative contents of frameshift mutations and non-frameshift mutations, synonymous SNVs and non-synonymous SNVs, and termination gain and termination loss between the original tumor tissue and the corresponding PTC micro-tumor model showed good consistency.

[0191] 2. Chromosome variation—CNV distribution results

[0192] After detecting single nucleotide polymorphisms, the present invention continues to detect copy number variations on chromosomes. CNV, also known as copy number polymorphism, refers to the variation of DNA fragments from 1KB to several MB in the genome compared to the reference sequence, including complex chromosome structural variations derived from insertions, deletions, amplifications and their mutual combinations. The amount of information contained in CNV is usually several times that of SNVs, which greatly enriches the diversity of genomic genetic variation. The present invention uses the BIC-seq2 method to perform somatic CNV detection on whole genome data. The CNV distribution of whole genome data is shown in Figure 7 The normal population CNV distribution is located between the two blue lines. The results of this patient showed that the red dotted line distribution on the chromosomes of tumor tissue and PTC microtumor showed a relatively obvious consistency.

[0193] 3. Results of mRNA expression correlation analysis

[0194] The present invention continues to perform transcriptome sequencing on the original tumor tissue and the corresponding PTC micro-tumor model to evaluate the similarity of mRNA expression between samples. The gene expression values ​​after analyzing the mRNA sequencing data using the Pearson and Spearman method are calculated to calculate their correlation coefficients. The closer the correlation coefficient is to 1, the higher the similarity of the expression patterns between samples. The sequencing and analysis results show that the correlation coefficient of the mRNA expression values ​​between samples is between 0.75-0.88, and the gene expression has a certain similarity. As shown in Table 25.

[0195]

[0196] Example 4: Establishment of primary cell line of thymic tumor microtumor model

[0197] The cell magnetic bead sorting process mentioned in the following examples uses the Miltenyi CD326 magnetic bead positive selection kit (Miltenyi #130-061-101).

[0198] 1. Collect the thymic tumor micro-tumor model cultured in suspension in Example 2, centrifuge at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0199] 2. Wash the cell pellet once with sterile PBS solution, centrifuge at 800g for 10 minutes at room temperature, and discard the supernatant.

[0200] 3. Reselect the cell pellet with cell digestion solution (see Example 1) and digest at 37°C for 5-30 minutes. During the digestion process, observe the digestion of cell clumps under a microscope every 5 minutes until most cell clumps are digested into single cells.

[0201] 4. Terminate the digestion with 10 times the volume of the digestion stop solution (see Example 1), centrifuge the cell suspension at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0202] 5. The single cell suspension obtained by digestion was sorted with Miltenyi CD326 magnetic bead positive selection kit to obtain CD326 positive tumor cells: resuspend the cell pellet with sorting buffer (formula is Table 26), and the cell amount is less than 10 7 Add 20 μL CD326 magnetic beads to the system and incubate on ice for 30 minutes. Wash the cell pellet with 2 mL sorting buffer. Wash the sorting column with 5 mL sorting buffer and place the sorting column on the magnetic rack. Pass the cell suspension through the cell sorting column and rinse the sorting column three times with 3 mL sorting buffer to elute the CD326 negative cells. Remove the sorting column from the magnetic rack and rinse the sorting column once with 5 mL sorting buffer to elute the CD326 positive cells.

[0203]

[0204] Note: Prepare and use immediately.

[0205] 6. Centrifuge the sorted CD326 positive cells at 800g for 10 minutes at room temperature and discard the supernatant. 5 / 3.5cm cell culture dish for inoculation and adherence culture to obtain thymic tumor primary cell line ( Figure 8 ).

[0206] Example 5: Evaluation of the correlation between antibody secretion levels and disease characteristics in the PTC microtumor model

[0207] After evaluating the consistency characteristics from the perspectives of tissue structure, pathological characteristics, genetic information, etc., the present invention continues to explore the correlation between the AChR-Ab level in the culture supernatant of the PTC microtumor model and disease characteristics. Previous studies have suggested that the tumor tissue of patients with thymoma-associated Myasthenia Gravis (TAMG) can continuously produce AChR-Ab, so the present invention uses ELISA to detect AChR-Ab in the peripheral blood serum and PTC culture supernatant of this type of patients. In order to facilitate the display of experimental results, the present invention logarithmically transforms the AChR-Ab results: y=log2 (x+1) , where x is the original result of AChR-Ab in serum and PTC culture supernatant, and y is the converted analytical value.

[0208] According to the instructions in the test kit, when AChR-Ab ≥ 0.45nmol / L, it is considered to be antibody positive. Therefore, after conversion according to the above formula, y = 0.536 is the converted antibody positive critical value, and a red line is drawn with 0.536 as the boundary.

[0209] like Fig. 9 As shown, the logarithmic transformed analysis results showed that all TAMG patients had positive AChR-Ab in their serum (above the cut-off value shown by the red line), which was completely consistent with the clinical characteristics of the disease.

[0210] like Fig.10 As shown, a total of 29 TAMG patients were tested, excluding some TAMG patients who received glucocorticoid or immunosuppressant intervention before surgery (#1, #2, #5, #7, #8, #17, #18, #24, #26). AChR-Ab could be successfully detected in the culture supernatant of the PTC microtumor model of more than 70% of TAMG patients (14 / 20) (near the boundary value shown by the green line).

[0211] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A culture medium for constructing an in vitro 3D micro-tumor model of thymic tumor, characterized in that: The culture medium consists of double-antibody P / S, HEPES, GlutaMax, MEM non-essential amino acid solution, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein MSP, human recombinant protein HGF, human recombinant protein IL-2, human recombinant protein IL-15, human recombinant protein Noggin, human recombinant protein IGF, forskolin, A83-01, SB202190, B27, ITS-X, Y-27632 and basal culture medium; In the culture medium, the final concentration of HEPES is 8-12 mM; the final concentration of GlutaMax is 0.8-1.2% by volume; the final concentration of the MEM non-essential amino acid solution is 0.8-1.2% by volume; the final concentration of the human recombinant protein EGF is 10-100 ng / mL; the final concentration of the human recombinant protein bFGF is 10-50 ng / mL; the final concentration of the human recombinant protein MSP is 5-25 ng / mL; the final concentration of the human recombinant protein HGF is 10-100 ng / mL; the final concentration of the human recombinant protein IL-2 is 10-100 ng / mL. L; the final concentration of the human recombinant protein IL-15 is 10-100 ng / mL; the concentration of the human recombinant protein Noggin is 100-200 ng / mL; the final concentration of the human recombinant protein IGF is 10-40 ng / mL; the final concentration of forskolin is 2-10 μM; the concentration of A83-01 is 0.25-1.25 μM; the concentration of SB202190 is 5-10 μM; the final concentration of B27 is 1.5-2.5% by volume; the final concentration of ITS-X is 0.8-1.2% by volume; the final concentration of Y-27632 is 5-20 μM.

2. The culture medium according to claim 1, characterized in that: The basic culture medium is Advanced DMEM / F12 culture medium.

3. The culture medium according to claim 1 or 2, characterized in that: In the culture medium, the final concentration of penicillin in the double-antibody P / S is 100-200 U / mL; the final concentration of streptomycin in the double-antibody P / S is 100-200 μg / mL.

4. A set of reagents for culturing an in vitro 3D micro-tumor model of thymic tumor, comprising the culture medium of claim 1 and all or part of the following: Sample dissociation solution, sample preservation solution, sample cleaning solution, cell digestion solution, digestion termination solution and cell freezing solution.

5. The reagent set according to claim 4, characterized in that: The sample dissociation solution consists of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of collagenase I is 150-250 U / mL; the final concentration of collagenase II is 150-250 U / mL; the final concentration of collagenase IV is 150-250 U / mL; and the remainder is PBS.

6. The reagent kit according to claim 4, characterized in that: The sample preservation solution consists of fetal bovine serum, double-antibody P / S, HEPES and HBSS; wherein the final concentration of the fetal bovine serum is 1-5% by volume; the final concentration of penicillin in the double-antibody P / S is 100-200U / mL, and the final concentration of streptomycin in the double-antibody P / S is 100-200μg / mL; the final concentration of HEPES is 8-12mM; and the remainder is HBSS.

7. The reagent kit according to claim 4, characterized in that: The sample cleaning solution is composed of dual-antibody P / S and PBS; wherein the final concentration of penicillin in the dual-antibody P / S is 100-200 U / mL; the final concentration of streptomycin in the dual-antibody P / S is 100-200 μg / mL; and the remainder is PBS.

8. The reagent kit according to claim 4, characterized in that: The cell digestion solution has the following composition: each 10 mL of the cell digestion solution contains 4-6 mL Accutase, EDTA with a final concentration of 5 mM, 1.5-2.5 mL TrypLE Express, and the remainder is PBS.

9. The reagent set according to claim 4, characterized in that: The digestion stop solution is composed of fetal bovine serum, double-antibody P / S and DMEM culture medium; wherein the final concentration of the fetal bovine serum is 8-12% by volume; the final concentration of penicillin in the double-antibody P / S is 100-200 U / mL; the final concentration of streptomycin in the double-antibody P / S is 100-200 μg / mL; and the remainder is DMEM culture medium.

10. The reagent kit according to claim 4, characterized in that: The cell freezing solution consists of Advanced DMEM / F12 culture medium, DMSO and 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM / F12 culture medium, the DMSO and the 1% methylcellulose solution is 20:2:(0.8-1.2); and the 1% methylcellulose solution is a methylcellulose aqueous solution with a concentration of 1g / 100ml.

11. Use of the culture medium according to any one of claims 1 to 3 or the reagent set according to any one of claims 4 to 10 in constructing an in vitro 3D micro-tumor model of thymic tumor.

12. The use according to claim 11, characterized in that: The thymic tumor is type A thymoma, type AB thymoma, type B1 thymoma, type B2 thymoma, type B3 thymoma or type C thymic carcinoma.

13. A method for constructing an in vitro 3D micro-tumor model of thymic tumor, comprising the following steps: (a1) dissociating thymic tumor solid tumor tissue using the sample dissociation solution described in claim 4 or 5; (a2) Using the culture medium described in any one of claims 1 to 3 to suspend and culture the single cells dissociated in step (a1) to form cell clusters, thereby obtaining an in vitro 3D micro-tumor model of thymic tumor.

14. The method according to claim 13, characterized in that: The thymic tumor is type A thymoma, type AB thymoma, type B1 thymoma, type B2 thymoma, type B3 thymoma or type C thymic carcinoma.

15. A method for obtaining thymic tumor solid tumor primary cells, comprising isolating thymic tumor solid tumor primary cells from an in vitro 3D micro-tumor model of thymic tumor obtained by the method of claim 13 or 14.

16. Use of an in vitro 3D micro-tumor model of thymic tumor obtained by the method of claim 13 or 14 in screening diagnostic and / or therapeutic drugs for thymic tumor.

Images