CN119060956B

CN119060956B - Culture medium for in-vitro 3D micro-tumor model construction of thymus tumor, and method and application thereof

Info

Publication number: CN119060956B
Application number: CN202411561719.2A
Authority: CN
Inventors: 张毅; 卢高俊; 尹申意
Original assignee: Xuanwu Hospital; Genex Health Co Ltd
Current assignee: Xuanwu Hospital; Genex Health Co Ltd
Priority date: 2024-11-05
Filing date: 2024-11-05
Publication date: 2025-03-04
Anticipated expiration: 2044-11-05
Also published as: CN119060956A

Abstract

本发明公开了一种用于胸腺肿瘤体外3D微肿瘤模型构建的培养基及其方法与应用。本发明涉及生物技术领域，提供了一种用于胸腺肿瘤体外3D微肿瘤模型构建的培养基，由双抗P/S、HEPES、GlutaMax、MEM非必需氨基酸溶液、人重组蛋白EGF、人重组蛋白bFGF、人重组蛋白MSP、人重组蛋白HGF、人重组蛋白IL‑2、人重组蛋白IL‑15、人重组蛋白Noggin、人重组蛋白IGF、毛喉素、A83‑01、SB202190、B27、ITS‑X、Y‑27632和基础培养基组成。利用本发明培养基构建得到的胸腺肿瘤体外3D微肿瘤模型可以准确反应患者原病灶的各种特征，是肿瘤精准诊疗领域良好的科研实验模型和临床前实验模型。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

Culture medium for in-vitro 3D micro-tumor model construction of thymus tumor, and method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a culture medium for constructing an in-vitro 3D micro-tumor model of thymus tumor, and a method and application thereof.

Background

Thymus is taken as a place where T cells develop and mature, plays a central role in adaptive immunity, and is also a key part for producing pathogenic antibodies so as to cause autoimmune diseases. Thymus tumor is a relatively rare cancer, accounting for 0.2% -1.5% of all malignant tumors, which occurs primarily in the mediastinum region. The methods for histological classification of WHO thymus tumors can be broadly classified into type A, type AB, type B1, type B2, type B3 thymus tumors and type C thymus cancers.

Surgery is the primary treatment modality for patients with early stage thymus tumors, but systemic treatment is generally employed for patients with advanced or recurrent thymus tumors. According to NCCN guidelines, the first line regimen for thymoma is cisplatin, doxorubicin and cyclophosphamide, and the first line regimen for thymus cancer is carboplatin or paclitaxel. Correspondingly, after treatment with the above drugs, the objective remission rate of thymoma was about 50%, whereas the objective remission rate of thymoma was only 21.7%. This indicates that the clinical efficacy of current empirical treatment regimens is low and further improvement and enhancement is needed.

In recent years, the advent of various patient "reagent replacement" models has made personalized accurate treatment of tumors increasingly possible. For example, cancer cells are obtained directly from a patient's tumor sample, and their sensitivity to drugs can predict the clinical objective remission rate of colorectal, prostate and breast cancers. This technique has been applied to thymus biology research, including the use of artificial thymus organs to study T cell development, therapeutic engineering T cells, and the like. However, the development of new therapies is limited due to the rarity of the occurrence of thymic neoplastic diseases and the complexity of the pathological mechanisms. And, in vitro anthropomorphic models of thymus tumors are slower to develop than other solid tumors, and there are few reports of related documents and patents.

Currently, the mainstream tumor drug sensitivity detection technology in the market is based on a Patient-derived tumor allograft model (Patient-Derived tumor Xenograft, PDX) and a Patient-derived tumor organoid model (Patient-Derived Organoids, PDO). However, PDX has longer modeling period (2-4 months), lower success rate (50% -80%), high cost (5-10 ten thousand yuan), interference on immune related research due to heterogeneous cell infiltration, lack of immune microenvironment of PDO model, culture depending on matrigel, poor operation repeatability, large sample demand, limited test scheme flux (10), poor corresponding relation with clinic (about 80%), and inconvenience on related research of tumor metabolism secretion due to tumor matrix components (the culture medium is not a full liquid environment).

The prior art has the defects that (1) the sample requirement is large, the thymus tumor organoid generally needs a large sample (> 1 g) after operation to be cultivated, (2) the detection time is long, the thymus tumor organoid generally needs 7-10 days to be formed, drug sensitivity detection is completed after about 4 weeks, (3) the cultivation success rate is low, the overall cultivation success rate of the thymus tumor organoid is about 76.2% (16/21) from the reported data of the literature, (4) the construction cost is high, the cost of the thymus tumor organoid cultivated by taking matrigel as a raw material is greater than 1 ten thousand yuan per instance, (5) the immune microenvironment is lacked, the thymus tumor organoid is formed based on an induction differentiation mechanism of tumor stem cells, the main component of the thymus tumor organoid is tumor epithelial cells, the thymus tumor organoid almost does not contain other cell types, and (6) the clinical consistency is poor, the drug sensitivity detection result specificity of the thymus tumor organoid is 60% (3/5) from the reported data of the literature, and a large proportion of false negatives are present. Reference to the literature "Wu YH, Chao HS, Chiang CL, et al. Personalized cancer 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".

In view of the clinical application limitations of the current PDX and PDO models, it is highly desirable to develop a personified model that can overcome the above-mentioned drawbacks and fully represent the biological characteristics of the original tumor tissue of the patient, so as to promote the development of basic research and transformation application related to the thymus tumor.

Disclosure of Invention

In order to solve the problem that the existing tumor model PDX/PDO and the like are limited in clinical research, the invention provides a culture medium for constructing an in-vitro 3D micro-tumor model of thymus tumor and a method thereof, and based on the culture medium, the application effect of the culture medium in accurate tumor treatment and diagnosis is tested.

In a first aspect, the invention claims a medium for the construction of an in vitro 3D micro-tumor model of a thymus tumor.

The culture medium for constructing the thymus tumor in-vitro 3D micro-tumor model is composed 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 basic culture medium.

In the culture medium, the final concentration of HEPES is 8-12mM (e.g., 10 mM), the final concentration of GlutaMax is 0.8-1.2% (e.g., 1%) by volume, the final concentration of MEM nonessential amino acid solution is 0.8-1.2% (e.g., 1%) by volume, the final concentration of human recombinant protein EGF is 10-100ng/mL (e.g., 50 ng/mL), the final concentration of human recombinant protein bFGF is 10-50ng/mL (e.g., 20 ng/mL), the final concentration of human recombinant protein MSP is 5-25ng/mL (e.g., 20 ng/mL), the final concentration of human recombinant protein HGF is 10-100ng/mL (e.g., 20 ng/mL), the final concentration of human recombinant protein IL-2 is 10-100ng/mL (e.g., 20 ng/mL), the final concentration of human recombinant protein EGF-15 is 10-100ng/mL (e.g., 20 ng/mL), the final concentration of human recombinant protein bFGF is 10-50ng (e.g., 20 mg/mL), the final concentration of human recombinant protein MSP is 5-25 mg/mL (e.g., 20 mg/mL), the final concentration of human recombinant protein HGF is 10-100 mg/mL (e.g., 20 mg/mL), the final concentration of human recombinant protein IL-2 is 10-1% by volume is 10-100 mg/mL), the final concentration of human recombinant protein (e.g., 20 mg-2% is 10% of human recombinant protein IL-2, the final concentration is 10% and the final concentration is 5% and the final concentration is 2% of human protein is 5-1% and the final concentration is 2% is 5% 6% and the final concentration ) The final concentration of Y-27632 is 5-20 mu M (such as 10 mu M).

Further, the MEM nonessential amino acid solution is water as a solvent, and the solute and the concentration are 10mM glycine, 10mM L-alanine, 10mM L-asparagine, 10mM L-aspartic acid, 10mM L-glutamic acid, 10mM L-proline and 10mM L-serine. The GlutaMax is an advanced cell culture additive and can directly replace L-glutamine in a cell culture medium. The GlutaMax is "GlutaMAXTM Supplement" (e.g., gibco #35050061, or other product of the same composition). The component of 'GlutaMAXTM Supplement' is L-alanyl-L-glutamine, which is a substitute of L-glutamine, the concentration is 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" (e.g., sigma #S7067, or other products of the same composition). B27 is "B-27 (TM) support (50X), minus vitamin A" (e.g., gibco #12587010, or other products of the same composition). The "B-27 (TM) Supplement (50X), minus vitamin A" contains Biotin (Biotin), DL-Alpha-tocopherol acetate (DL Alpha Tocopherol Acetate), DL-Alpha-tocopherol (DL Alpha-Tocopherol), BSA (FATTY ACID FREE Fraction V), catalase (Catalase), human recombinant insulin (Human Recombinant Insulin), human transferrin (Human Transferrin), superoxide dismutase (Superoxide Dismutase), corticosterone (Corticosterone), D-galactose (D-Galactose), ethanolamine hydrochloride (Ethanolamine HCl), reduced glutathione (Glutathione (reduced)), L-carnitine hydrochloride (L-CARNITINE HCL), linoleic Acid (Linolenic Acid), linolenic Acid (Linolenic Acid), and pharmaceutical compositions, Progesterone (Progesterone), putrescine (Putrescine HCl), sodium selenite (Sodium Selenite), triiodothyronine (T3 (triodo-I-thyronine)). The solvent of ITS-X is EBSS solution (Earle's balanced salt solution), and the solute and concentration are as follows, insulin 1g/L, transferrin 0.55g/L, sodium selenite 0.00067g/L, and 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)" (e.g. MCE#129830-38-2, or other products of the same composition).

Further, the basal medium may be ADVANCED DMEM/F12 medium.

Further, the final concentration of penicillin in the dual antibody P/S may be 100-200U/mL (e.g., 100U/mL) and the final concentration of streptomycin in the dual antibody P/S may be 100-200 μg/mL (e.g., 100 μg/mL) in the medium.

Further, the culture medium may exist in two forms:

The culture medium is a solution formed by mixing the double antibody P/S, the HEPES, the GlutaMax, the MEM nonessential 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, the Forskolin (Forskolin), the A83-01, the SB202190, the B27, the ITS-X, the Y-27632 and the ADVANCED DMEM/F12 culture medium.

The medium was prepared and sterilized by filtration using a 0.22. Mu.M needle filter (Millipore SLGP033 RS) and stored at 4℃for two weeks.

And secondly, each component in the culture medium exists independently, and the culture medium is prepared according to a formula when in use.

Furthermore, the human recombinant proteins EGF, bFGF, MSP, HGF, IL-2, IL-15, noggin, IGF, forskolin, SB202190 and Y-27632 may be stored in the form of stock solution (mother solution) (80 ℃ for a long period), and may be 1000 times of stock solution (mother solution). Wherein A83-01 can exist in the form of stock solution (mother liquor), which can be stored at (-20 ℃ for a long period of time), specifically 100000 times stock solution (mother liquor).

1000 Xstock solution of human recombinant protein EGF consists of human recombinant protein EGF, BSA and PBS, wherein the final concentration of the human recombinant protein EGF is 20 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

1000 Xstock solution of human recombinant protein bFGF consists of human recombinant protein bFGF, BSA and PBS, wherein the final concentration of the human recombinant protein bFGF is 20 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

1000 Xstock solution of human recombinant protein MSP consists of human recombinant protein MSP, BSA and PBS, wherein the final concentration of human recombinant protein MSP is 20 mug/mL, the final concentration of BSA is 0.01g/mL, and the balance is PBS.

1000 Xstock solution of human recombinant protein HGF consists of human recombinant protein HGF, BSA and PBS, wherein the final concentration of the human recombinant protein HGF is 20 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

1000X stock solution of human recombinant protein IL-2 consists of human recombinant protein IL-2, BSA and PBS, wherein the final concentration of the human recombinant protein IL-2 is 20 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

1000X stock solution of human recombinant protein IL-15 comprises human recombinant protein IL-15, BSA and PBS, wherein the final concentration of the human recombinant protein IL-15 is 20 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

The 1000 Xhuman 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 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

1000 Xstock solution of human recombinant protein IGF consists of human recombinant protein IGF, BSA and PBS, wherein the final concentration of the human recombinant protein IGF is 40 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

Of the eight 1000-fold stock solutions, BSA was present in 100-fold stock (stock solution) form (as-prepared), and consisted of BSA and PBS, with final concentration of BSA (Sigma#A1933) of 0.1g/mL, and the balance PBS.

In addition, 1000 XForskolin consists of Forskolin and DMSO, where the final concentration of Forskolin is 10mM and the balance is DMSO.

1000 XSB 202190 stock solution consisted of SB202190 and DMSO, where the final concentration of SB202190 was 10mM, with the remainder being DMSO.

1000 XY-27632 consists of Y-27632 and ultrapure water, wherein the final concentration of Y-27632 is 10mM, and the balance is ultrapure water.

In addition, 100000 XA 83-01 stock solution is composed of A83-01 and DMSO, wherein the concentration of A83-01 is 25mM, and the balance is DMSO.

In a second aspect, the invention claims a kit for culturing an in vitro 3D microtumor model of a thymus tumor.

The kit for culturing the thymus tumor in-vitro 3D micro-tumor model, which is claimed by the invention, consists of the culture medium in the first aspect and all or part of sample dissociation liquid, sample preservation liquid, sample cleaning liquid, cell digestion liquid, digestion stop liquid and cell freezing liquid.

The sample preservation solution can be used for temporary preservation after the sample is isolated, and can maintain the activity of cells in the sample in a short time after the sample is isolated. The sample cleaning solution can be used for cleaning and disinfecting the sample. The sample dissociation liquid can be used for dissociation of a sample.

Further, the sample dissociation solution consists of collagenase I, collagenase II, collagenase IV and PBS, wherein the final concentration of the collagenase I is 150-250U/mL (such as 200U/mL), the final concentration of the collagenase II is 150-250U/mL (such as 200U/mL), the final concentration of the collagenase IV is 150-250U/mL (such as 200U/mL), and the balance is PBS.

Wherein the unit U of collagenase (said collagenase I or said collagenase II or said collagenase IV) is defined by the enzymatic activity of a protease, and L-leucine can be released by treating collagenase (said collagenase I or said collagenase II or said collagenase IV) with 1U of protease at 37℃and pH7.5 for 5 hours.

Further, 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% (such as 2%) 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 100-200 mug/mL (such as 100 mug/mL), the final concentration of HEPES is 8-12mM (such as 10 mM), and the balance is HBSS.

Further, the sample cleaning solution consists of double antibody P/S and PBS, wherein 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 100-200 mug/mL (such as 100 mug/mL), and the balance is PBS.

Further, the cell digestive juice comprises 4-6mL (e.g. 5 mL) of Ackutase, EDTA with a final concentration of 5mM, 1.5-2.5mL (e.g. 2 mL) of TrypLE Express, and the balance PBS in every 10mL of the cell digestive juice.

Wherein the Ackutase is "StemProTM AccutaseTM Cell Dissociation Reagent" (e.g., gibco #A11105-01, or other products of the same composition). The Ackutase is a single component enzyme dissolved in D-PBS,0.5mM EDTA solution. The TrypLE Express is "TrypLETM Express Enzyme (1X), no phenol red" (such as Gibco #12604013, or other products of the same composition). The "TrypLETM Express Enzyme (1X), no phenol red" contains 200mg/L KCl, 200mg/L KH ₂PO₄, 8000mg/L NaCl, 2160mg/L Na ₂HPO₄·7H₂ O and 457.6mg/L EDTA, and also contains recombinant protease.

Further, the digestion stopping solution consists of fetal bovine serum, double-antibody P/S and a 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 100-200 mug/mL (such as 100 mug/mL), and the balance is the DMEM culture medium.

Further, the cell freezing solution consists of ADVANCED DMEM/F12 culture medium, DMSO and 1% methyl cellulose solution, wherein the volume ratio of the ADVANCED DMEM/F12 culture medium to the DMSO to the 1% methyl cellulose solution is 20:2 (0.8-1.2), such as 20:2:1, and the 1% methyl cellulose solution is methyl cellulose water solution with the concentration of 1g/100 ml.

In a third aspect, the invention claims the use of a medium as described in the first aspect hereinbefore or a kit as described in the second aspect hereinbefore for the construction of an in vitro 3D micro-tumor model of a thymus tumor.

The thymus tumor can be A type thymus tumor, AB type thymus tumor, B1 type thymus tumor, B2 type thymus tumor, B3 type thymus tumor or C type thymus cancer.

In a fourth aspect, the invention claims a method of constructing an in vitro 3D microtumor model of a thymus tumor.

The method for constructing the thymus tumor in-vitro 3D micro-tumor model, which is claimed by the invention, can comprise the following steps:

(a1) Dissociating solid tumor tissue of the thymus tumor with the sample dissociation fluid described in the second aspect;

(a2) And (3) performing suspension culture on the single cells dissociated in the step (a 1) by using the culture medium in the first aspect to form cell clusters, thus obtaining the thymus tumor in-vitro 3D micro-tumor model.

In step (a 1), the thymus tumor solid tumor tissue can be dissociated with the sample dissociation liquid in a method comprising the step of dissociating the sheared thymus tumor solid tumor tissue with the sample dissociation liquid at 37 ℃ for 15 minutes to 2 hours (e.g., 1 hour) in an amount of not more than 0.5mg of tissue per 1mL of the sample dissociation liquid.

In step (a 2), the dissociated cells of (a 1) may be suspension-cultured with the medium according to a method comprising the step of culturing the dissociated cells of (a 1) in suspension with the medium using a cell culture vessel having a low adsorption surface at 37℃under 5% CO ₂.

Wherein the initial seeding density may be 10 ⁵ cells/cm ² container bottom area, for example, a six well plate, plated at a density of 10 ⁶ cells per well.

Further, the time of the cultivation in the step (a 2) is 3 to 5 days.

Further, before step (a 1), the method may further comprise a step of subjecting the solid tumor tissue of thymus to dissociation pretreatment, wherein the step of washing the surface of the solid tumor tissue of thymus with 70-75% ethanol by volume for 10-30 seconds, the step of washing the solid tumor tissue of thymus with the sample washing liquid according to the second aspect of the present invention for 5-10 times (e.g., 5 times), the step of washing the solid tumor tissue of thymus with a sterile PBS solution for 5-10 times (e.g., 5 times), and the step of removing impurities, connective tissue, adipose tissue, necrotic tissue, etc. affecting the micro-tumor culture in the solid tumor tissue of thymus.

The step of subjecting the thymus tumor solid tumor tissue to dissociation pretreatment needs to be performed on ice, and the whole operation step needs to be completed within 10 minutes.

Further, in step (a 1), the dissociation treatment of the solid tumor tissue of the thymus tumor with the sample dissociation solution further comprises the steps of stopping the dissociation reaction with 8 to 15 (e.g., 10) volumes of the digestion stop solution described in the second aspect, collecting the cell suspension, filtering the cell suspension with a 100 μm or 40 μm sterile cell filter to remove tissue debris and adherent cells, centrifuging 800 to 1000g (e.g., 800 g) at room temperature for 10 to 15 minutes (e.g., 10 minutes), discarding the supernatant, resuspending the cells with 3 to 5mL (e.g., 5 mL) sterile PBS, centrifuging 800 to 1000g (e.g., 800 g) at room temperature for 10 to 15 minutes (e.g., 10 minutes), discarding the supernatant, and resuspending the cell pellet with the medium described in the first aspect.

The sample of thymic tumor solid tumor tissue subjected to the dissociation pretreatment has an in vitro time of 24 hours or less and is stored in the sample storage solution described in the second aspect before the dissociation pretreatment is performed.

In a fifth aspect, the invention claims a method of obtaining primary cells of a solid tumor of the thymus.

The method for obtaining the primary cells of the solid tumor of the thymus, which is claimed by the invention, is to isolate the primary cells of the solid tumor of the thymus from the in-vitro 3D micro-tumor model of the thymus obtained by the method in the fourth aspect.

The method specifically comprises the following steps:

(b1) Digesting the thymus tumor micro-tumor model by using a cell digestive juice (such as the cell digestive juice) to obtain single cells;

Further, the method comprises the step of terminating the digestion. Digestion was terminated as described above with the digestion stop solution.

(B2) And (3) selecting CD326 positive cells from the single cells obtained in the step (b 1) to obtain the primary cells of the thymus tumor solid tumor.

Further, CD326 positive cells can be sorted by CD326 magnetic beads.

In a sixth aspect, the invention claims the use of an in vitro 3D microtumor model of a thymus tumor obtained by the method described in the fourth aspect above for screening for diagnostic and/or therapeutic drugs of a thymus tumor.

In a seventh aspect, the invention claims the use of a thymus tumor in vitro 3D microtumor model obtained using the method described in the fourth aspect above in an in vitro companion diagnosis.

The invention has the beneficial effects that:

(1) The sample demand is small, and the culture of micro samples such as puncture, biopsy (< 100 mg) and the like can be completed;

(2) The detection period is short, namely a micro tumor model of the thymus tumor can be formed only by 2-3 days, and drug sensitivity detection is completed within 10 days;

(3) The culture success rate of the culture medium for thymus tumor samples is 90.7 percent (88/97), wherein 9 failed cases are caused by objective reasons such as poor sampling quality (1 case of pollution and 8 cases of no tumor cells);

(4) The construction cost is low, the micro tumor model of the thymus tumor does not depend on matrigel, the culture medium contains specific cytokines and has no serum, and the total cost is only 0.2 ten thousand yuan per case;

(5) The micro tumor model of thymus tumor is characterized in that tumor tissue of a patient is firstly digested or separated into a single cell-based state, and then the single cell is subjected to suspension culture in a serum-free culture medium containing specific cytokines, so that the single cell is mutually aggregated and proliferated through self-assembly to obtain a cell cluster with a 3D structure, various cell types in a tumor sample of the patient can be reserved, including tumor epithelial cells, various immune cells (such as B cells and T cells) and the like, and the tumor micro environment can be simulated to a higher degree. In general, the tumor epithelial cells in a micro tumor model occupy a relatively large amount, and whether other cell types exist or not and how much the tumor epithelial cells occupy the tumor epithelial cells depend on the specific conditions (particularly the types and the proportions of immune cells) of a sample from a patient, so that the individual variability of a tumor patient is fully reduced.

(6) The invention detects the secretion level of AChR-Ab antibody in the culture supernatant of a micro tumor model of a patient type sample of the thymic tumor myoweakness (Thymoma-associated MYASTHENIA GRAVIS, TAMG), and the result shows that the relative content of the antibody shows obvious correlation with disease characteristics.

In a word, compared with the prior art, the external 3D micro-tumor model of the thymus tumor constructed by the invention can accurately reflect various characteristics of the original focus of a patient, is a scientific research experiment model and a preclinical experiment model which are good in the field of tumor accurate diagnosis and treatment, has the advantages of faster construction method, shorter detection period, lower cost, higher standardization and standardization degree and better operability and repeatability, can replace patient reagents, can search unconventional two-wire, three-wire or cross-indication medicines for partial drug-resistant patients, is provided by domestic reagent manufacturers, is simple to operate and suitable for large-area popularization, and can further accelerate the development of domestic related basic research and conversion application.

Drawings

FIG. 1 is a culture identification of an in vitro 3D microtumor model of thymus tumor. The bright field image depicts the phenotype of a breast tumor after tissue culture into a microtumor model in which solid clusters, solid dense clusters, and grape-like clusters are present. The cell diameter is 50-300 μm, and the number of cells of a single clone is several tens to hundreds of cells. The scale marks in the figures are all 100 μm,10 x bright field.

Fig. 2 is a comparison of morphological structural consistency of original tumor tissue with corresponding PTC microtumor model. AB, B2 and B3 thymoma is selected, and the morphological structure is verified to have obvious consistency through HE staining.

FIG. 3 is a comparison of the pathological feature consistency of the original tumor tissue with the corresponding PTC microtumor model (CK staining). AB, B2 and B3 type thymoma is selected, and through IHC staining of CK, obvious consistency in pathological characteristics is verified.

FIG. 4 is a comparison of pathological feature consistency (TdT staining) of original tumor tissue and corresponding PTC micro-tumor model. AB, B2 and B3 thymoma is selected, and through IHC staining of TdT, obvious consistency in pathological characteristics is verified.

FIG. 5 is an identification of immune cells in a PTC microtumor model. The PTC micro-tumor model of 1 TAMG which is successfully cultured is digested to prepare single-cell suspension, and various immune cells are marked through flow cytometry experiments, so that the existence and diversity of the immune cells in the model are determined.

Fig. 6 is a comparison of distribution of somatic mutation SNV in tumor tissue and PTC models in different cases. In the figure, "# number" indicates sample number corresponding to different thymus tumor patients, T indicates tumor tissue, and PTC is PTC micro-tumor model.

FIG. 7 shows comparison of chromosomal variation-CNV distribution of tumor tissue and corresponding PTC microtumors. Chr chromosome number. A is the CNV distribution map of tumor tissue, and B is the CNV distribution map of corresponding PTC. In the figure, each point represents a capture region of a gene, the horizontal axis represents the chromosomal location of the gene, and the vertical axis represents copy number.

FIG. 8 shows the isolation of primary thymus tumor cell lines from an in vitro 3D microtumor model of thymus tumors. The scale marks in the figures are all 100 μm,10 x bright field.

FIG. 9 shows AChR-Ab results in peripheral blood serum. ELISA was used to detect AChR-Ab levels in serum from TAMG patients. Red lines are drawn bounded by y=0.536, above which the line is positive for antibodies. The abscissas 1-29 represent the numbers of 29 TAMG patients.

FIG. 10 shows AChR-Ab results in PTC microtumor model culture supernatants. ELISA was used to detect AChR-Ab in the supernatant of the TAMG patient PTC model culture. A green line is drawn bounded by y=0.322, above which the concentration of antibody in PTC culture supernatant is indicated as meaning. * Indicating that the patient is using a glucocorticoid or immunosuppressant. The abscissas 1-29 represent the numbers of 29 TAMG patients.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1 preparation of reagents for use in the construction of an in vitro 3D micro-tumor model of thymus tumor

1. Sample preservation solution

The specific formulation of the sample storage solution (100 mL) is shown in Table 1.

After the sample preservation solution is prepared, split charging is carried out by using a 15mL centrifuge tube, and each tube is 5mL. Can be stored at 4deg.C for 1 month after sub-packaging.

2. Sample cleaning solution

The specific formulation of the sample rinse (100 mL) is shown in Table 2.

The sample cleaning liquid needs to be prepared at present.

3. Sample dissociation liquid

The specific formulation of the sample dissociation solution (10 mL) is shown in Table 3.

Note that the sample dissociation solution is ready for use.

In Table 3, the collagenase stock solutions were prepared as shown in tables 4 to 6.

After preparation of the 10 Xcollagenase I stock solution, 1.5mL sterile centrifuge tubes were used for split charging, 1mL per tube. The stock solution can be stored at-20deg.C for a long period.

After preparation of the 10 Xcollagenase II stock solution, 1.5mL sterile centrifuge tubes were used for split charging, 1mL per tube. The stock solution can be stored at-20deg.C for a long period.

After preparation of the 10 Xcollagenase IV stock solution, 1.5mL sterile centrifuge tubes were used for split charging, 1mL per tube. The stock solution can be stored at-20deg.C for a long period.

In tables 4 to 6, the unit U of collagenase (said collagenase I or said collagenase II or said collagenase IV) was defined by the enzyme activity of a protease, and L-leucine was released by treating collagenase (said collagenase I or said collagenase II or said collagenase IV) with 1U of protease at 37℃and pH 7.5 for 5 hours.

4. Cell digestive juice

The specific formulation of the cell digest (10 mL) is shown in Table 7.

The cell digestive juice is prepared at present.

5. Digestion stop solution

The specific formulation of the digestion terminator (100 mL) is shown in Table 8.

After the digestion stop solution is formulated, it can be stored for one month at 4 ℃.

6. Culture medium for constructing thymus tumor in-vitro 3D micro-tumor model

The specific formulation of the medium (100 mL) for constructing the thymus tumor in vitro 3D micro-tumor model is shown in Table 9.

After the preparation of the culture medium (100 mL) for constructing the thymus tumor in-vitro 3D micro-tumor model is completed, the culture medium is filtered and sterilized by a 0.22 mu M needle filter (Millipore SLGP033 RS), and can be stored for two weeks at 4 ℃.

In Table 9, the formulations of the human recombinant protein stock solutions are shown in tables 11 to 18 (the formulation of the BSA stock solution is shown in Table 10), the formulation of the Forskolin stock solution is shown in Table 19, the formulation of the A83-01 stock solution is shown in Table 20, the formulation of the SB202190 stock solution is shown in Table 21, and the formulation of the Y-27632 stock solution is shown in Table 22.

The 100 XBSA solution was ready for use.

After 1000 Xstock solution of human recombinant protein EGF was prepared, it was dispensed using a 1.5mL sterile centrifuge tube and the stock solution could be stored at-80℃for a long period of time.

After preparation of 1000 Xstock solution of human recombinant protein bEGF, the stock solution can be stored at-80℃for a long period of time by sub-packaging with a 1.5mL sterile centrifuge tube.

After 1000 Xhuman recombinant protein MSP stock solution was prepared, split-packed with 1.5mL sterile centrifuge tubes, the stock solution could be stored for a long period at-80 ℃.

After 1000 Xhuman recombinant protein HGF stock solution was prepared, split-packed with 1.5mL sterile centrifuge tubes, the stock solution could be stored for a long period at-80 ℃.

After 1000 Xhuman recombinant protein IL-2 stock solution was prepared, split-packed with 1.5mL sterile centrifuge tubes, the stock solution could be stored for a long period at-80 ℃.

After 1000 Xhuman recombinant protein IL-15 stock solution was prepared, split-packed with 1.5mL sterile centrifuge tubes, the stock solution could be stored for a long period at-80 ℃.

After the 1000 Xhuman recombinant protein Noggin stock solution is prepared, the stock solution is subpackaged by a 1.5mL sterile centrifuge tube, and the stock solution can be stored at-80 ℃ for a long time.

After 1000 Xhuman recombinant protein IGF stock solution was prepared, it was sub-packaged with 1.5mL sterile centrifuge tubes and the stock solution could be stored at-80℃for a long period of time.

After the 1000 XForskolin stock solution is prepared, it is dispensed with a 0.5mL sterile centrifuge tube and the stock solution can be stored for a long period of time at-20 ℃.

After 100000 XA 83-01 stock solution is prepared, the stock solution can be stored for a long period of time at-20 ℃ by sub-packaging with a 0.5mL sterile centrifuge tube.

After the 1000 XSB 202190 stock solution is prepared, the stock solution is split-packed by a 0.5mL sterile centrifuge tube, and the stock solution can be stored at-20 ℃ for a long time.

After the 1000 XY-27632 stock solution is prepared, the stock solution is split-packed by a 0.5mL sterile centrifuge tube, and the stock solution can be stored at-80 ℃ for a long time.

7. Cell freezing solution

The specific formulation of the cell cryopreservation solution is shown in table 23.

The cell freezing solution is prepared for use at present.

In Table 23, the formulation of the 1% methylcellulose solution is shown in Table 24.

The 1% methylcellulose solution can be stored for a long period of time at 4 ℃.

Example 2 culture identification of thymus tumor in vitro 3D micro tumor model

The operation flow of collecting samples of thymus tumor is as follows, the solid tumor is cut, punctured, biopsied and the like are collected, sampling is completed in an operating room, so that the samples are prevented from being polluted by microorganisms, the sampling is as follows, fresh cancer tissues rich in blood vessels (the fresh tissues should be cut in 30 minutes from body) are required to be taken during cutting of solid tumor tissues, necrosis areas, fibrosis areas, adipose tissues and tissues burnt by an electrotome are prevented, the cell viability in the tissues is maintained to the greatest extent, and the tumor tissues are required to be not less than 50mg (about 5mm multiplied by 5 mm). For puncture samples, more than 2 samples are needed, and each length is more than or equal to 1cm.

The excised solid tumor tissue of the thymus tumor should be subjected to oscillation cleaning in physiological saline to remove the dirty. Immediately after sample collection, the samples were stored in 15mL sterile centrifuge tubes containing 5mL sample preservation solution (see example 1) or stored in sterile cryopreservation tubes, transported at 4 ℃, and sent for inspection as soon as possible (completed within 24 hours).

The culture process of the thymus tumor in-vitro 3D micro-tumor model is specifically as follows:

first step, dissociation pretreatment of thymus tumor solid tumor tissue

The following operations are performed on ice, and the whole operation steps are completed within 10 minutes.

1. After weighing the sample, the surface of the sample was rinsed with medical alcohol (75% by volume) for 10 to 30 seconds.

2. The samples were washed 5 times with sample wash (see example 1) and 5 times with sterile PBS solution.

3. The adipose tissue, connective tissue and necrotic tissue in the sample are carefully peeled off by using an ophthalmic scissors, an ophthalmic forceps, a surgical knife and other devices.

Second step, dissociation of the thymus tumor solid tumor tissue sample

1. The tissue was cut into small pieces of about 0.5mm ³ with an ophthalmic scissors.

2. Tissue is treated with a sample dissociation solution (see example 1), 1mL of sample dissociation solution is used for tissue with a sample size of no more than 0.5mg, and 0.1mL of sample dissociation solution is required to increase each 0.1mg increase in tissue weight for tissue with a sample size of more than 0.5 mg. Sample dissociation solution treatment conditions were 37 ℃ and dissociation time was 1 hour. Dissociation of the sample was observed under a microscope every 15 minutes during dissociation until most cells were observed to be shed from the tissue.

3. The dissociation reaction was stopped with 10 volumes of digestion stop solution (see example 1), and after the cell suspension was filtered through a 100 μm sterile cell screen to remove tissue debris and adherent cells, the supernatant was discarded by centrifugation at 800g for 10 minutes at room temperature.

4. Cells were resuspended in 5mL of sterile PBS, centrifuged at 800g for 10 min at room temperature, and the supernatant discarded.

5. Cell pellet was resuspended in culture medium of thymus tumor in vitro 3D microtumor model (see table 9 in example 1), cell counts were performed, cell viability was determined by trypan blue staining, and cell inoculation culture was performed with isolated cell viability greater than 70%.

Third step, thymus tumor in vitro 3D micro tumor model culture

1. And (3) performing in-vitro 3D micro tumor model suspension culture by using a low adsorption surface (low-adsorption-surface), wherein the culture medium is the culture medium for constructing the in-vitro 3D micro tumor model of the thymus tumor in table 9 of example 1 (wherein the final concentration of penicillin in the dual antibody P/S is 100U/mL, the final concentration of streptomycin in the dual antibody P/S is 100 mug/mL, the final concentration of HEPES is 10mM, the final concentration of Glutamax is 1% by volume, the final concentration of MEM non-essential amino acid solution is 1% by volume, the final concentration of human recombinant protein EGF is 50ng/mL, the final concentration of human recombinant protein bFGF is 20ng/mL, the final concentration of human recombinant protein MSP is 20ng/mL, the final concentration of human recombinant protein HGF is 20ng/mL, the final concentration of human recombinant protein IL-2 is 20 g/mL, the final concentration of human recombinant protein IL-2 is 1% by volume, the final concentration of human recombinant protein is 20 mg/mL, the final concentration of human recombinant protein is 27 mg-27 mg/mL, the final concentration of human recombinant protein is 10 mg-25% by volume, the final concentration of human recombinant protein is 10 mg-25 mg/mL, and the final concentration of human recombinant protein is 10 mg-25 mg/mL, the final concentration of human recombinant protein is 10 mg/mL, the final concentration of human recombinant protein is 10 mg. The inoculated cells were cultured in a cell incubator at 37℃under 5% CO ₂.

2. The state of the cells was observed daily until the cells formed a mass with a diameter of about 100 μm, after which the medium was changed every 2-3 days to maintain the growth state of the thymic tumor micro-tumor.

The invention carries out in vitro culture on 97 fresh operation tissue samples, wherein 9 cases of A-type thymoma, 18 cases of AB-type thymoma, 12 cases of B1-type thymoma, 36 cases of B2-type thymoma, 17 cases of B3-type thymoma and 5 cases of C-type thymoma are related, the overall culture success rate is 90.7 percent (88/97), and 9 failed cases are caused by objective reasons such as poor sampling quality (1 case of pollution and 8 cases of tumor cell-free). The external 3D micro-tumor model bright field view of the thymus tumor is shown in figure 1, the diameter is 50-300 mu m, the cell number of a single microsphere is tens to hundreds of cells, at least 3 different types of cell clusters are observed in morphology, wherein the cell clusters comprise compact solid spheres, solid spheres with tube cavities and grape-shaped spheres with different sizes, and the individual difference characteristics of the tumor are fully reflected. Therefore, the invention has successfully constructed an in vitro 3D micro-tumor model of thymus tumor under a stable full-liquid culture environment.

Example 3 scientific characterization of thymus tumor in vitro 3D micro tumor model

In order to further determine the consistency of the external 3D micro tumor model of the thymus tumor (PTC micro tumor for short) constructed in the embodiment 2 of the present invention with respect to morphological structure, pathological characteristics, genetic information, etc. of the original tumor tissue, and to identify the diversity of cellular components in the PTC micro tumor model, a series of evaluation experiments were performed in the embodiment to ensure that the model has consistency characteristics with respect to the original tumor tissue in the in vivo environment, so that the model can be used for performing subsequent mechanism study, drug screening, etc.

1. Assessing morphological structure consistency

The morphological structure consistency between the original tumor tissue and the corresponding PTC micro tumor was assessed by HE staining. The invention selects AB type, B2 type and B3 type thymoma as the representative to evaluate the consistency of morphological structure. The results showed that the morphological structure of the PTC microtumor model had more pronounced features of consistency with the original tumor tissue (fig. 2). Wherein, the A type area of the AB type thymoma is not obvious, only the B type thymoma component is seen, and the neoplastic epithelial cells and scattered immature lymphocytes are seen. The B2 type thymoma can be seen under the microscope, a large amount of immature lymphocytes are scattered in the distribution of neoplastic epithelial cells, part of the epithelial cells are arranged in a net shape or a small nest shape, and a part of the epithelial cells can be seen as obvious nucleolus. The B3 type thymoma can be seen with a large amount of tumor epithelial cells under the microscope, the cells are arranged in a piece, a part of the cells can be seen with nucleolus, and the number of immature lymphocytes is obviously less than that of the B2 type thymoma.

2. Assessing consistency of pathological features

On the basis of evaluating the morphological structure consistency, the invention further explores the consistency of the PTC micro-tumor and the original tumor tissue in pathological characteristics. Thus, IHC staining was performed on selected 3 samples described above to assess the consistency of pathological features. The presence of tumor epithelial cells was assessed using CK commonly used clinically, and the presence of immature lymphocytes was assessed using TdT.

CK staining (FIG. 3) shows that the neoplastic epithelial cells of type AB, type B2 thymomas are scattered or arranged in a nest, and the neoplastic epithelial cells of type B3 thymomas are distributed in sheets.

TdT staining results (FIG. 4) showed a higher density of the distribution of immature lymphocyte sheets of AB-type thymoma. Immature lymphocytes of type B2 thymomas are scattered and have a high focal cell density. Immature lymphocytes of type B3 thymomas are scattered.

The result shows that the pathological features of the PTC micro-tumor model have obvious consistency with the original tumor tissue.

3. Characterization of the diversity of cellular components

It is well known that immune cells survive in vitro for a short period. In view of the high abundance of immune cells in thymoma tissue, helper T lymphocytes, memory T lymphocytes, regulatory T cells, B cells and memory B cells have been found to be involved in disease regulation many times. The present invention contemplates further determination of the presence or absence of the above immune cells in a self-assembled PTC microtumor model. The micro tumor model of 1 thymus tumor which is successfully cultured is digested, single cell suspension is prepared, and various immune cells are marked through flow cytometry experiments, so that the existence and diversity of the immune cells in the model are determined.

As shown in fig. 5, helper T cells (CD 3 ⁺CD4⁺), double positive T cells (CD 3 ⁺CD4⁺CD8⁺), double negative T cells (CD 3 ⁺CD4^-CD8^-), memory T cells (CD 3 ⁺CD4⁺CD45RA^-), total B cells (CD 19 ⁺), memory B cells (CD 19 ⁺CD27⁺), regulatory T cells (CD 3 ⁺CD4⁺CD25⁺CD127^-) and Th17 cells (CD 3 ⁺CD4⁺CD45RA^-CCR6⁺CXCR3^-CCR4⁺) were all present in the model in a certain proportion.

4. Assessing consistency of genetic information

As described above, the present invention has evaluated the consistency of tissue morphology and pathological features, and the present invention will continue to evaluate the consistency of genetic information from a molecular level on the original tumor tissue and the corresponding PTC microtumor model.

1. Somatic mutation- -SNV distribution results

SNV is a single base change site on the genome, widely distributed across the genome. And carrying out somatic cell SNV detection on the paired samples by using software VarScan, and filtering detection results by using software default parameters to finally obtain somatic cell SNV results with high credibility. The results of this study showed that the SNV results were approximately the same for each pair of samples (fig. 6). In particular, the frame shift mutation and the non-frame shift mutation between the original tumor tissue and the corresponding PTC micro tumor model, the synonymous SNV and the non-synonymous SNV, and the obtained relative content of termination and termination loss shows better consistency characteristics.

2. Chromosomal variation-CNV distribution results

After detecting single nucleotide polymorphisms, the present invention continues to detect copy number variation on chromosomes. CNV, also known as copy number polymorphism, refers to variations in DNA fragments of 1KB to several MB in the genome compared to a reference sequence, including complex chromosomal structural variations derived from insertions, deletions, amplifications, and combinations thereof. CNVs typically contain several times the amount of information as SNVs, greatly enriching the diversity of genomic genetic variations. The invention adopts the BIC-seq2 method to carry out CNV detection of somatic cells on whole genome data, and the CNV distribution of the whole genome data is shown in figure 7. The normal population CNV distribution is located between the two blue lines. The results of this example show that the distribution of red dots on the chromosomes of tumor tissue and PTC micro-tumors shows a more obvious consistency relationship.

3. MRNA expression correlation analysis results

The invention continues transcriptome sequencing of the original tumor tissue and the corresponding PTC microtumor model, and evaluates the similarity of mRNA expression between samples. The correlation coefficient is calculated by using the gene expression value after analyzing the mRNA sequencing data by using the Pearson and Spearman method, and the closer the correlation coefficient is to 1, the higher the similarity of the expression modes between samples is. Sequencing and analysis results show that the correlation coefficient of the mRNA expression values among samples is between 0.75 and 0.88, and the gene expression has certain similarity. As shown in table 25.

Example 4 Primary cell lines of thymus tumor microtumor model

The cell bead sorting procedure mentioned in the following examples uses the Methaemarrhena CD326 bead cationic selection kit (Methaemarrhena- # 130-061-101).

1. The thymus tumor microtumor model suspension-cultured in example 2 was collected, centrifuged at 800g at room temperature for 10 minutes, and the supernatant was discarded.

2. The cell pellet was washed once with sterile PBS solution, centrifuged at 800g for 10 min at room temperature and the supernatant discarded.

3. Cell pellet was re-selected with cell digests (see example 1), digested at 37 ℃ for 5-30 minutes, and cell pellet digestions were observed under a microscope every 5 minutes during digestion until most of the cell pellet was digested as single cells.

4. Digestion was stopped with 10 volumes of digestion stop solution (see example 1), and the cell suspension was centrifuged at 800g for 10 minutes at room temperature and the supernatant discarded.

5. And (3) separating the single cell suspension obtained by digestion by using a beauty and gentle CD326 magnetic bead positive selection kit to obtain CD326 positive tumor cells, namely re-suspending cell sediment by using a separation buffer (the formula is shown in table 26), adding 20 mu L of CD326 magnetic beads into a system when the cell quantity is less than 10 ⁷, and incubating on ice for 30 minutes. The cell pellet was washed with 2mL of sorting buffer. The separation column was washed with 5mL of separation buffer and placed on a magnetic rack. The cell suspension was passed through a cell sorting column, which was washed three times with 3mL of sorting buffer, and CD326 negative cells were eluted. The separation column was removed from the magnetic rack and washed once with 5mL of separation buffer, eluting CD326 positive cells.

It is noted that it is ready for use.

6. The sorted CD326 positive cells were centrifuged at 800g for 10 minutes at room temperature, and the supernatant was discarded. The primary thymus tumor cell line was obtained by inoculation and adherent culture in DMEM medium containing 10% serum at a density of 10 ⁵/3.5 cm cell culture dish (fig. 8).

Example 5 evaluation of correlation of antibody secretion levels with disease characteristics in PTC micro-tumor model

After the consistency characteristics are evaluated from the tissue structure, the pathological characteristics and the genetic information, the invention continuously explores the correlation between the AChR-Ab level in the culture supernatant of the PTC micro-tumor model and the disease characteristics. Previous studies suggest that the tumor tissue of patients with thymoma and myasthenia gravis (Thymoma-associated MYASTHENIA GRAVIS, TAMG) can continuously produce AChR-Ab, so that the invention adopts ELISA method to detect AChR-Ab respectively on the peripheral blood serum and PTC culture supernatant of the patients of the type. In order to facilitate the display of experimental results, the invention carries out logarithmic transformation on the AChR-Ab results, wherein y=log ₂ ^(x+1), x is the original result of the AChR-Ab in serum and PTC culture supernatant, and y is the analysis value after transformation.

As shown in the instruction book of the detection kit, AChR-Ab was considered positive for the antibody when it was not less than 0.45 nmol/L. Thus, y=0.536 is the converted antibody positive threshold after conversion according to the above formula, and a red line is drawn with 0.536 as a boundary.

As shown in FIG. 9, the results of the log-transformed analysis showed that AChR-Ab was positive in all TAMG patient sera (above the limit indicated by the red line), which remained completely consistent with the clinical profile of the disease.

As shown in FIG. 10, a total of 29 TAMG patients tested, excluding some TAMG patients (# 1, #2, #5, #7, #8, #17, #18, #24, # 26) that had been pre-operatively treated with glucocorticoid or immunosuppressant, were found to be successful in detection of AChR-Ab (near the limit indicated by the green line) in the PTC microtumor model culture supernatants of over 70% of TAMG patients (14/20).

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims

1. A culture medium for constructing an in vitro 3D micro-tumor model of thymic tumors, 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 basal 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 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.

4. A set of reagents for culturing an in vitro 3D micro-tumor model of thymic tumors, 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 the collagenase I is 150-250 U/mL; the final concentration of the collagenase II is 150-250 U/mL; the final concentration of the collagenase IV is 150-250 U/mL; and the remainder is PBS.

6. The reagent set according to claim 4 is 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 100-200 U/mL, and the final concentration of streptomycin in the double antibody P/S is 100-200 μg/mL; the final concentration of HEPES is 8-12 mM; and the remainder is HBSS.

7. The reagent set according to claim 4, characterized in that: the sample cleaning solution consists of double-antibody P/S and PBS; wherein 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 PBS.

8. The reagent set according to claim 4, characterized in that: the cell digestion solution is composed of: 4-6 mL Accutase, EDTA with a final concentration of 5 mM, 1.5-2.5 mL TrypLE Express, and the balance is PBS per 10 mL of the cell digestion solution.

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

10. The reagent set 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 1 g/100 ml.

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.