Special culture medium containing Mucin family recombinant protein for tumor organoids and culture method of special culture medium

Abstract

The invention belongs to the technical field of biological culture, and particularly relates to a special culture medium containing Mucin family recombinant protein for tumor organoids and a culture method of the special culture medium. The invention relates to a Mucin family recombinant protein-containing special culture medium for tumor organs, which is prepared from a basic culture medium Advanced DMEM / F12 and R-helix protein 1, a fibroblast growth factor 7, a fibroblast growth factor 10, a head protein, a small-molecule inhibitor A83-01, a small-molecule inhibitor Y-27632, a small-molecule inhibitor SB202190, glutathione precursor N-acetylcysteine, nicotinamide and a cell culture additive 50 *. The invention relates to an anti-fungal composition which is composed of an L-glutamine substitute additive 100 *, a pH buffer solution, an antibiotic and an antifungal agent-plerimosin, penicillin / streptomycin and Mucin family recombinant protein. The Mucin family recombinant protein is specifically added into the culture medium, especially under the conditions of small sample size and few tumor cells, the success rate of tumor organoid culture is greatly increased, and the proliferation rate of tumor organoids is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of biological culture, and particularly relates to a special culture medium for tumor organoids containing Mucin family recombinant proteins and a culture method thereof. Background Art

[0002] According to statistics, my country ranks first in the world in new cancer cases and deaths from cancer each year. Although there are many treatments for tumors, treatment still faces huge challenges. In recent years, clinical treatment of tumors has made significant progress due to clinical models of basic research and next-generation sequencing technology for precise individualized treatment. However, there are still differences among tumor patients, so seeking personalized medicine or precision medicine methods will be more conducive to improving patient survival rates.

[0003] Patient-derived organoids (PDOs) are 3D tumor models cultured in vitro using patient tumor tissues or malignant pleural effusions and ascites. Compared with other preclinical models, PDOs can highly simulate the pathological and genetic characteristics of the source tumor tissues. More importantly, they can reveal the heterogeneity of tumors, and the model can be established in a shorter time.

[0004] The mucin family is a class of highly glycosylated macromolecular proteins that are widely distributed in various tissues and body fluids of the human body and play an important role in physiological and pathological processes. Studies have shown that multiple proteins in the Mucin family can be used as markers for tumor diagnosis and prognosis, and play an important role in tumor progression. At present, various tumor organoid culture systems are mainly based on the addition of various cytokines, which cannot better simulate the complex conditions of the tumor microenvironment, and the success rate of tumor organoid culture varies greatly. How to better improve the tumor organoid culture system, improve the success rate of tumor organoid culture, and realize the clinical application transformation of tumor organoids is a problem that needs to be solved urgently.

[0005] Therefore, there is an urgent need to provide a tumor organoid-specific culture medium containing Mucin family recombinant proteins and a culture method thereof. Summary of the invention

[0006] In order to make up for the deficiencies of the prior art and improve the culture success rate and proliferation efficiency of tumor organoids, the present invention aims to provide a tumor organoid-specific culture medium containing Mucin family recombinant proteins and a culture method thereof.

[0007] The tumor organoid-specific culture medium containing the Mucin family recombinant protein of the present invention adopts the following technical scheme:

[0008] A tumor organoid-specific culture medium containing Mucin family recombinant proteins, consisting of basal culture medium Advanced DMEM / F12 and R-Spondin 1, fibroblast growth factor 7 (FGF 7), fibroblast growth factor 10 (FGF 10), Noggin, small molecule inhibitor A83-01, small molecule inhibitor Y-27632, small molecule inhibitor SB202190, glutathione precursor N-acetylcysteine, nicotinamide, cell culture supplement B27 supplement 50×, L-glutamine replacement supplement GlutaMax 100×, pH buffer Hepes, antibiotics and antifungal agents-Primocin, Penicillin / Streptomycin and Mucin family recombinant proteins.

[0009] Preferably, the content of each component is as follows:

[0010] R-helix protein 1, 250±5ng / mL; fibroblast growth factor 7, 25ng / mL; fibroblast growth factor 10, 20±5ng / mL; noggin, 100±5ng / mL; small molecule inhibitor A83-01, 500±5nM; small molecule inhibitor Y-27632, 5±1μM; small molecule inhibitor SB202190, 500±5nM; glutathione precursor N-acetylcysteine, 1.25±0.25mM; nicotinamide, 10±5mM; cell culture supplement B27 supplement, 1×; L-glutamine replacement supplement GlutaMax, 1×; pH buffer Hepes, 10mM; antibiotics and antifungal agents-primomycin, 50μg / mL; penicillin / streptomycin, 100mg / mL; Mucin family recombinant proteins are all 20ng / mL; basic culture medium AdvancedDMEM / F12, 1×.

[0011] The method for culturing tumor organoids containing Mucin family recombinant proteins of the present invention adopts the following technical scheme:

[0012] A method for culturing tumor organoids containing Mucin family recombinant proteins, comprising the following steps:

[0013] (1) The tumor tissue removed by surgery was washed twice with D-PBS buffer in a 6 cm diameter culture dish, and then placed in a 1.5 mL EP tube and cut into 1 mm 3 of fragments;

[0014] (2) Resuspend the tumor tissue fragments in 500 μL tissue preservation solution and transfer them to a 15 mL centrifuge tube; add 10-15 mL digestion solution and digest in a 37°C water bath for 1-2 h. Filter the digested tumor tissue with a 70 μm cell sieve and add an equal volume of tissue preservation solution to terminate digestion. Centrifuge at 1400 rpm for 5 min at room temperature to collect the cell pellet.

[0015] (3) Add 8-10 mL of red blood cell lysis buffer to the primary cell pellet, let it stand for 10 min, then add an equal volume of D-PBS buffer to terminate lysis, centrifuge at 1400 rpm for 5 min at room temperature, and collect the secondary cell pellet;

[0016] (4) Prepare a tumor organoid-specific culture medium according to the composition of the above-mentioned tumor organoid-specific culture medium, add 50 to 500 μL of the culture medium to the secondary cell pellet to resuspend the cells, count the cells, take 10,000 cells and mix them in a ratio of cell suspension to Matrigel matrix gel = 1:1, and spot the suspension into the well plate.

[0017] Preferably, in step (1), the tumor tissue is breast cancer tissue, lung cancer tissue, gastrointestinal cancer, head and neck cancer or gynecological tumor.

[0018] Preferably, in step (1), the tissue preservation solution is a basic culture medium Advanced DMEM / F12 to which 10 mM pH buffer Hepes, 1×L-glutamine replacement additive GlutaMax, 100 mg / mL penicillin / streptomycin, and 5 μM Y-27632 are added.

[0019] Preferably, in step (2), the digestion solution is prepared by adding 20 μL of 5 mg / mL type II collagenase and 10 μL of 100 μg / mL DNase I to every 1 mL of the above tissue preservation solution.

[0020] A method for culturing organoids from patient punctured tumor tissue comprises the following steps:

[0021] (1) Add 7 mL of digestion solution directly to the tumor tissue puncture strip and digest in a 37°C water bath for 1 h. Add an equal volume of tissue preservation solution to terminate digestion, centrifuge at 1400 rpm for 5 min at room temperature, and collect the cell pellet;

[0022] 2) Add 7 mL of red blood cell lysis buffer to the cell pellet in step (1), let stand for 10 min, then add an equal volume of D-PBS to terminate lysis, centrifuge at 1400 rpm for 5 min at room temperature, and collect the cell pellet;

[0023] 3) Prepare a culture medium according to the composition of the above-mentioned tumor organoid-specific culture medium, add 50 μL of the culture medium to the cell pellet in step (2) to resuspend the cells, count the cells, take 10,000 cells and mix them in a ratio of cell suspension: Matrigel = 1:1, and spot the suspension into a 24-well plate.

[0024] A method for culturing a patient's malignant pleural effusion and ascites organoids, comprising the following steps:

[0025] 1) The malignant pleural effusion of lung cancer patients was centrifuged at room temperature and 1400 rpm for 5 min to collect the cell pellet;

[0026] 2) Add 7 mL of red blood cell lysis buffer to the cell pellet in step (1), let stand for 10 min, then add an equal volume of D-PBS to terminate lysis, centrifuge at 1400 rpm for 5 min at room temperature, and collect the cell pellet.

[0027] 3) Prepare a culture medium according to the composition of the above-mentioned tumor organoid-specific culture medium, add 50 μL of the culture medium to the cell pellet in step (2) to resuspend the cells, count the cells, take 10,000 cells and mix them in a ratio of cell suspension: Matrigel = 1:1, and spot the suspension into a 24-well plate.

[0028] Beneficial effects:

[0029] The culture medium of the present invention specifically adds Mucin family recombinant proteins, especially when the tumor tissue volume is less than 0.1cm 3 The success rate of tumor organoid culture is greatly improved when the sample size is small (such as punctured tissue, etc.) and the tumor cell amount is less than 10,000 cells (such as malignant effusion, etc.), and the proliferation rate of tumor organoids is promoted. In addition, the genotyping of tumor organoids cultured by the culture medium of the present invention is highly consistent with the patient's tumor tissue, providing a good and beneficial choice for personalized medical guidance for tumor patients.

[0030] The present invention also provides methods for processing and culturing tumor organoids from three tissue sources, which cover a wide range and are simple and easy to operate. They can form tumor organoid models relatively quickly, providing a good model foundation for clinical and mechanism research based on this. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Bright field images of breast cancer organoids formed using the culture steps and culture medium of Example 2, wherein the figure (left) shows breast cancer tissue cultured for 1 day, and the figure (right) shows breast cancer tissue cultured for 14 days;

[0032] Figure 2Bright field images of lung cancer organoids formed using the culture steps and culture medium of Example 3, wherein the image (left) shows lung cancer puncture tissue cultured for 1 day, and the image (right) shows lung cancer puncture tissue cultured for 14 days;

[0033] Figure 3 Bright field images of lung cancer organoids formed using the culture steps and culture medium of Example 3, wherein the image (left) shows lung cancer malignant pleural effusion tissue cultured for 1 day, and the image (right) shows lung cancer malignant pleural effusion tissue cultured for 14 days;

[0034] Figure 4 Bright field images of breast cancer organoids formed using the culture steps and culture medium of Comparative Example 1, wherein the figure (left) shows breast cancer tissue cultured for 1 day, and the figure (right) shows breast cancer tissue cultured for 14 days;

[0035] Figure 5 Bright field images of lung cancer puncture tissue organoids formed using the culture steps and culture medium of Comparative Example 2, wherein the figure (left) shows lung cancer puncture tissue cultured for 1 day, and the figure (right) shows lung cancer puncture tissue cultured for 14 days. DETAILED DESCRIPTION

[0036] Example 1

[0037] In this example, Mucin 1 in the Mucin family was used as an example to prepare a recombinant protein.

[0038] In this example, the preparation of the Mucin 1 recombinant protein was carried out with reference to a method for preparing a nucleic acid, a recombinant plasmid, and a recombinant human IL-5 protein disclosed in Chinese invention patent application CN 118853713 A.

[0039] 1) Construction of eukaryotic expression vector of Mucin 1 recombinant protein

[0040] The amino acid sequence of human Mucin 1 was obtained from the Uniprot database, and its gene sequence was obtained from the NCBI database. 6×His was connected to the C-terminus for Ni column purification. The expression vector pCDNA3.1 was selected, and the appropriate restriction sites were selected and connected to the expression vector according to the characteristics of each protein gene sequence. Gene synthesis and sequencing were completed by Sangon Biotechnology Co., Ltd.

[0041] 2) Transfection, expression and harvest of Mucin 1 recombinant protein plasmid

[0042] Take cells in the logarithmic growth phase, prepare transfection reagent according to the instructions of the transfection reagent used, transfect the plasmid into the cells, and change the medium after 12 hours. Collect the cell culture supernatant 36 hours, 48 ​​hours, and 72 hours after transfection and filter it through a 0.22μm filter membrane.

[0043] 3) Purification of Mucin 1 recombinant protein

[0044] Prepare the required nickel column and pre-equilibrate the nickel column. Purify the Mucin 1 recombinant protein using the sample purification method disclosed in Chinese invention patent application CN 118853713 A, and detect the protein concentration using an ultraviolet spectrophotometer.

[0045] The amino acid sequence of the Mucin 1 recombinant protein is as follows:

[0046] MTPGTQSPFFLLLLLLTVLTVVTGSGHASSTPGGEKETSATQRSSVPSSTEKNAVS

[0047] MTSSVLSSHSPSGSSTTQGQDVTLAPAATEPASGSAATWGQDVTSVPVTRPALGSTTP

[0048] PAHDVTSAPDNKPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGST

[0049] APPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPG

[0050] STAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAP

[0051] GSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRP

[0052] APGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDT

[0053] RPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAP

[0054] DTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTS

[0055] APDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGV

[0056] TSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHG

[0057] VTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAH

[0058] GVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPA

[0059] HGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPP

[0060] AHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTA

[0061] PPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGST

[0062] APPAHGVTSAPDNRPALGSTAPPVHNVTSASGSASGSASTLVHNGTSARATTTPASKS

[0063] TPFSIPSHHSDTPTTLASHSTKTDASSTHHSSVPPLTSSNHSTSPQLSTGVSFFFLSFHISN

[0064] LQFNSSLEDPSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAFREG

[0065] TINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVC

[0066] VLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDR

[0067] SPYEKVSAGNGGSSLSYTNPAVAATSANL.

[0068] Example 2

[0069] This embodiment provides a method for culturing tumor tissue organoids derived from surgically removed tissue of a breast cancer patient, comprising the following steps:

[0070] 1) The surgically removed breast cancer tissue was washed twice with D-PBS buffer in a 6 cm diameter culture dish, and then the tumor tissue was placed in a 1.5 mL EP tube and cut into 1 mm pieces in 500 μL tissue preservation solution. 3 The tissue preservation solution is the basic culture medium Advanced DMEM / F12 with the addition of 10mM pH buffer Hepes, 1×L-glutamine replacement additive GlutaMax, 100mg / mL penicillin / streptomycin (Penicillin / Streptomycin), and 5μM Y-27632.

[0071] 2) Transfer the breast cancer tissue fragment suspension to a 15 mL centrifuge tube and add 10 mL of digestion solution. The digestion solution is prepared by adding 20 μL of 5 mg / mL type II collagenase and 10 μL of 100 μg / mL DNase I to each 1 mL of the above tissue preservation solution. Digest in a 37°C water bath for 1.5 h, filter the digested breast cancer tissue with a 70 μm cell sieve, and add 10 mL of tissue preservation solution to terminate digestion. Centrifuge at 1400 rpm for 5 min at room temperature to collect the cell pellet.

[0072] 3) Add 8 mL of red blood cell lysis buffer to the primary cell pellet, let it stand for 10 min, then add 8 mL of D-PBS to terminate the lysis. Centrifuge at 1400 rpm for 5 min at room temperature to collect the secondary cell pellet.

[0073] 4) Add 100 μL of culture medium to the secondary cell pellet to resuspend the cells, count the cells, take 10,000 cells and mix them in a ratio of 1:1, then spot the suspension on a 24-well plate. Place the 24-well plate in a 37°C water bath for 2 minutes, wipe the bottom dry, and invert it in a CO 2 Incubate in the incubator for 10 min, add a certain amount of the above culture medium to each well, and incubate at 37°C and 5% CO 2 Tumor organoids were obtained by culturing at the same concentration for 14 days.

[0074] The components of the above culture medium are R-Spondin 1, 250 ng / mL; FGF 7, 25 ng / ml; FGF 10, 20 ng / ml; Noggin, 100 ng / ml; A83-01, 500 nM; Y-27632, 5 μM; SB202190, 500 nM; N-Acetylcysteine, 1.25 mM; Nicotinamide, 10 mM; B27 supplement, 1×; GlutaMax, 1×; Hepes, 10 mM; Primocin, 50 μg / mL; Penicillin / Streptomycin, 100 mg / mL; Mucin 1, 20 ng / ml; and the basic culture medium is Advanced DMEM / F12, 1×. Among them, Mucin 1 comes from Example 1.

[0075] Figure 1 This is a bright field image of breast cancer organoids formed using the culture steps and culture medium of Example 2. Figure 1 It can be seen that the formed breast cancer organoids are irregular and dense spherical. After 14 days of culture, breast cancer organoids with a diameter of more than 100 μm account for 70% of the total number of breast cancer organoids. This shows that the use of the culture method and special culture medium of this embodiment can achieve rapid growth of breast cancer organoids.

[0076] Example 3

[0077] This embodiment provides a method for culturing tumor organoids derived from puncture tissue of a lung cancer patient, comprising the following steps:

[0078] 1) A 1 cm long and 0.25 mm cross section 2 7 mL of digestion solution was directly added to the lung cancer patient's puncture strip and digested in a 37°C water bath for 1 hour, and 7 mL of tissue preservation solution was added to terminate the digestion. Centrifuge at 1400 rpm for 5 minutes at room temperature to collect the cell pellet. The digestion solution was prepared by adding 20 μL of type II collagenase at a concentration of 5 mg / mL and 10 μL of DNase I at a concentration of 100 μg / mL to each 1 mL of the above tissue preservation solution. The tissue preservation solution was a basic culture medium Advanced DMEM / F12 with 10 mM pH buffer Hepes, 1×L-glutamine replacement additive GlutaMax, 100 mg / mL penicillin / streptomycin, and 5 μM Y-27632.

[0079] 2) Add 7 mL of red blood cell lysis buffer to the cell pellet collected in step (1), let it stand for 10 min, then add 7 mL of D-PBS to terminate the lysis. Centrifuge at 1400 rpm for 5 min at room temperature to collect the cell pellet.

[0080] 3) Add 50 μL of culture medium to the cell pellet collected in step (2) to resuspend the cells, count the cells, take 10,000 cells and mix them in a ratio of cell suspension: Matrigel = 1:1, and spot the suspension on a 24-well plate. Place the 24-well plate in a 37°C water bath for 2 minutes, wipe the bottom dry, and invert it in a CO 2 Incubate in the incubator for 10 min, add a certain amount of the above culture medium to each well, and incubate at 37°C and 5% CO 2 Tumor organoids were obtained by culturing at the same concentration for 14 days.

[0081] The components of the above culture medium are R-Spondin 1, 250 ng / mL; FGF 7, 25 ng / ml; FGF 10, 20 ng / ml; Noggin, 100 ng / ml; A83-01, 500 nM; Y-27632, 5 μM; SB202190, 500 nM; N-Acetylcysteine, 1.25 mM; Nicotinamide, 10 mM; B27 supplement, 1×; GlutaMax, 1×; Hepes, 10 mM; Primocin, 50 μg / mL; Penicillin / Streptomycin, 100 mg / mL; Mucin 1, 20 ng / ml; and the basic culture medium is Advanced DMEM / F12, 1×. Among them, Mucin 1 comes from Example 1.

[0082] Figure 2 This is a bright field image of lung cancer organoids formed using the culture steps and culture medium of Example 3. Figure 2 It can be seen that after 14 days of culture, most of the lung cancer organoids derived from the patient's lung cancer puncture tissue are regular and dense spherical, a few are vacuolar, and the lung cancer organoids with a diameter of more than 60 μm account for 85% of the total lung cancer organoids. It can be seen that the use of the culture method of this embodiment and the culture medium of the present invention can achieve rapid and uniform growth of lung cancer organoids with a small sample size.

[0083] Example 4

[0084] This embodiment provides a method for culturing tumor organoids derived from malignant pleural effusion of lung cancer patients, comprising the following steps:

[0085] 1) The malignant pleural effusion of lung cancer patients was centrifuged at room temperature at 1400 rpm for 5 minutes to collect the cell precipitate.

[0086] 2) Add 7 mL of red blood cell lysis buffer to the cell pellet collected in step (1), let it stand for 10 min, then add 7 mL of D-PBS to terminate the lysis. Centrifuge at 1400 rpm for 5 min at room temperature to collect the cell pellet.

[0087] 3) Add 50 μL of culture medium to the cell pellet collected in step (2) to resuspend the cells, count the cells, take 10,000 cells and mix them in a ratio of cell suspension: Matrigel = 1:1, and spot the suspension on a 24-well plate. Place the 24-well plate in a 37°C water bath for 2 minutes, wipe the bottom dry, and invert it in a CO 2 Incubate in the incubator for 10 min, add a certain amount of the above culture medium to each well, and incubate at 37°C and 5% CO 2 Tumor organoids were obtained by culturing at the same concentration for 14 days.

[0088] The components of the above culture medium are R-Spondin 1, 250 ng / mL; FGF 7, 25 ng / ml; FGF 10, 20 ng / ml; Noggin, 100 ng / ml; A83-01, 500 nM; Y-27632, 5 μM; SB202190, 500 nM; N-Acetylcysteine, 1.25 mM; Nicotinamide, 10 mM; B27 supplement, 1×; GlutaMax, 1×; Hepes, 10 mM; Primocin, 50 μg / mL; Penicillin / Streptomycin, 100 mg / mL; Mucin 1, 20 ng / ml; and the basic culture medium is Advanced DMEM / F12, 1×. Among them, Mucin 1 comes from Example 1.

[0089] Figure 3 This is a bright field image of a tumor organoid formed using the culture steps and culture medium of Example 4. Figure 3 It can be seen that the tumor cells from the malignant pleural effusion of lung cancer patients formed dense spherical lung cancer organoids after 14 days of culture. Compared with the first day of culture, the formation rate of lung cancer organoids was as high as 95%. It can be seen that the use of the culture method of this embodiment and the culture medium of the present invention can achieve the rapid growth of tumor organoids from special sample sources (such as malignant effusion, etc.),

[0090] Comparative Example 1

[0091] This comparative example provides a method for culturing tumor organoids, which uses a DMEN / F12 culture medium supplemented with 10% serum. The culture process is the same as that of Example 2, and the tumor tissue is breast cancer tissue.

[0092] Figure 4To form a bright field image of breast cancer organoids using the culture steps and culture medium of Comparative Example 1, Figure 4 It can be seen that tumor cells cannot form breast cancer organoids during culture.

[0093] Comparative Example 2

[0094] The culture medium provided in this comparative example is minus the Mucin 1 recombinant protein, and the other culture processes and culture medium are the same as those in Example 3, and the tumor tissue is lung cancer tissue.

[0095] The state of the tumor organoids obtained after culturing for 14 days using the above culture medium according to the method of Example 3 is as follows Figure 5 As shown by Figure 5 It can be seen that lung cancer organoids grow slowly and have a small diameter, with an average diameter of 25μm-45μm.

[0096] In summary, the tumor-specific culture medium of the present invention can achieve rapid growth of tumor organoids, uniform size and long-term stable culture. Tumor organoids from different patients have different morphologies and can well preserve the heterogeneity of patient tumor tissues in vitro.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A special culture medium for tumor organoids containing Mucin family recombinant proteins, characterized in that: It consists of the basic medium Advanced DMEM / F12 and R-helix protein 1, fibroblast growth factor 7, fibroblast growth factor 10, noggin, small molecule inhibitor A83-01, small molecule inhibitor Y-27632, small molecule inhibitor SB202190, glutathione precursor N-acetylcysteine, nicotinamide, cell culture supplement B27 supplement 50×, L-glutamine replacement supplement GlutaMax 100×, pH buffer Hepes, antibiotics and antifungal agents - primomycin, penicillin / streptomycin and Mucin family recombinant proteins.

2. The tumor organoid-specific culture medium containing Mucin family recombinant proteins according to claim 1, characterized in that: The content of each ingredient is as follows: R-helix protein 1, 250±5ng / mL; fibroblast growth factor 7, 25ng / mL; fibroblast growth factor 10, 20±5ng / mL; noggin, 100±5ng / mL; small molecule inhibitor A83-01, 500±5nM; small molecule inhibitor Y-27632, 5±1μM; small molecule inhibitor SB202190, 500±5nM; glutathione precursor N-acetylcysteine, 1.25±0.25mM; nicotinamide, 10±5mM; cell culture supplement B27 supplement, 1×; L-glutamine replacement supplement GlutaMax, 1×; pH buffer Hepes, 10mM; antibiotics and antifungal agents-primomycin, 50μg / mL; penicillin / streptomycin, 100mg / mL; Mucin family recombinant proteins are all 20ng / mL; basic culture medium AdvancedDMEM / F12, 1×.

3. A method for culturing tumor organoids containing Mucin family recombinant proteins, characterized in that: The following steps are involved: (1) The tumor tissue removed by surgery was washed twice with D-PBS buffer in a 6 cm diameter culture dish, and then placed in a 1.5 mL EP tube and cut into 1 mm 3 of fragments; (2) Resuspend the tumor tissue fragments in 500 μL tissue preservation solution and transfer them to a 15 mL centrifuge tube; add 10-15 mL digestion solution and digest in a 37°C water bath for 1-2 h. Filter the digested tumor tissue with a 70 μm cell sieve and add an equal volume of tissue preservation solution to terminate digestion. Centrifuge at 1400 rpm for 5 min at room temperature to collect the cell pellet. (3) Add 8-10 mL of red blood cell lysis buffer to the primary cell pellet, let it stand for 10 min, then add an equal volume of D-PBS buffer to terminate lysis, centrifuge at 1400 rpm for 5 min at room temperature, and collect the secondary cell pellet; (4) According to claim 1 or 2, 50-500 μL of the culture medium for tumor organoids is added to the secondary cell pellet to resuspend the cells, and the cells are counted. 10,000 cells are taken and mixed in a ratio of cell suspension to Matrigel matrix gel = 1:1, and the suspension is spotted into a well plate.

4. The method for culturing tumor organoids containing Mucin family recombinant proteins according to claim 3, characterized in that: In step (1), the tumor tissue is breast cancer tissue, lung cancer tissue, gastrointestinal cancer, head and neck cancer or gynecological tumor.

5. The method for culturing tumor organoids containing Mucin family recombinant proteins according to claim 3, characterized in that: In step (1), the tissue preservation solution is a basic culture medium Advanced DMEM / F12 to which 10 mM pH buffer Hepes, 1×L-glutamine replacement additive GlutaMax, 100 mg / mL penicillin / streptomycin, and 5 μM Y-27632 are added.

6. The method for culturing tumor organoids containing Mucin family recombinant proteins according to claim 3, characterized in that: In step (2), the digestion solution is prepared by adding 20 μL of 5 mg / mL type II collagenase and 10 μL of 100 μg / mL DNase I to every 1 mL of the above tissue preservation solution.

7. A method for culturing organoids from patient punctured tumor tissue, characterized in that: The following steps are involved: (1) Add 7 mL of digestion solution directly to the tumor tissue puncture strip and digest in a 37°C water bath for 1 h. Add an equal volume of tissue preservation solution to terminate digestion, centrifuge at 1400 rpm for 5 min at room temperature, and collect the cell pellet; 2) Add 7 mL of red blood cell lysis buffer to the cell pellet in step (1), let stand for 10 min, then add an equal volume of D-PBS to terminate lysis, centrifuge at 1400 rpm for 5 min at room temperature, and collect the cell pellet; 3) Add 50 μL of the culture medium for tumor organoids prepared according to claim 1 or 2 to the cell pellet in step (2) to resuspend the cells, count the cells, take 10,000 cells and mix them in a ratio of cell suspension: Matrigel = 1:1, and spot the suspension into a 24-well plate.

8. A method for culturing organoids of malignant pleural effusion and ascites from a patient, characterized in that: The following steps are involved: 1) The malignant pleural effusion of lung cancer patients was centrifuged at room temperature and 1400 rpm for 5 min to collect the cell pellet; 2) Add 7 mL of red blood cell lysis buffer to the cell pellet in step (1), let stand for 10 min, then add an equal volume of D-PBS to terminate lysis, centrifuge at 1400 rpm for 5 min at room temperature, and collect the cell pellet. 3) Add 50 μL of the culture medium for tumor organoids prepared according to claim 1 or 2 to the cell pellet in step (2) to resuspend the cells, count the cells, take 10,000 cells and mix them in a ratio of cell suspension: Matrigel = 1:1, and spot the suspension into a 24-well plate.

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