Bladder cancer lymph node metastasis derived tumor organoid culture medium, culture method and rapid drug detection method

Abstract

The invention discloses a bladder cancer lymph node metastasis derived tumor organoid culture medium, a culture method and a rapid drug detection method, and belongs to the technical field of organoid and cell culture. The method comprises the following steps: culturing a bladder cancer lymph node metastasis-derived tumor organoid by using a bladder cancer lymph node metastasis-derived tumor organoid culture medium, immersing matrigel drops in the culture medium, and obtaining the bladder cancer lymph node metastasis-derived tumor organoid after the cultured organoid grows to 100-150 microns, to-be-detected drugs with different concentrations are directly added into the cell pore plate inoculated with the tumor organoids, the influence of the to-be-detected drugs on cell growth or metabolism is analyzed through a full-view bright-field imaging method, and the detection result of the drug for treating bladder cancer lymph node metastasis is obtained. Compared with a traditional ENR organ-like culture medium, the method has the advantages that the effect is better, and the cost is lower; the kit is used for screening medicines for bladder cancer lymph node metastasis, is simple and efficient, and can be used for accurately detecting medicines for efficiently killing bladder cancer lymph node metastasis cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoids and cell culture, and particularly relates to a tumor organoid culture medium, a culture method and a drug rapid detection method derived from lymph node metastases of bladder cancer. Background Art

[0002] Bladder cancer is a common malignant tumor in the urinary system, characterized by high heterogeneity and high recurrence rate, seriously threatening human health. Patient-derived organoids of bladder cancer (PDO; Patient Derived Organoid), as an emerging three-dimensional cell culture model, can highly retain the genetic characteristics, cell composition and biological behavior of tumor tissues, providing an ideal model for the basic research of bladder cancer. Particularly crucial is that due to its high retention of tumor tissue heterogeneity and biological characteristics, it can more truly reflect the response of tumor cells to different drugs, giving it a unique advantage in drug sensitivity testing.

[0003] Lymph nodes are an important part of the human immune system, a place where lymphocytes and macrophages gather, and at the same time an important route and target organ for distant metastasis of tumors. Lymphatic metastasis is one of the common and key factors affecting the prognosis of bladder cancer during the metastasis process, and is closely related to tumor invasiveness, growth rate, drug resistance and treatment response, etc. By culturing lymph node metastasis organoids, the biological mechanism of tumor lymphatic metastasis can be deeply explored, the microenvironment of the metastasis can be analyzed, and valuable information can be provided for the development of new treatment strategies. However, the reported bladder cancer organoid culture systems usually use surgically resected tumor tissues as starting materials, including muscle-invasive bladder cancer and non-muscle-invasive bladder cancer. Although there have been reports on the culture of bladder cancer organoids, and the samples cover various stages from T1 to T3, none of them involve lymph node metastatic samples. Therefore, a method for culturing tumor organoids derived from lymphatic metastases is needed.

[0004] Currently, the classic ENR culture system is widely used in the process of culturing tumor organoids. It is composed of three key factors, namely epidermal growth factor (EGF), noggin, and R-spondin 1 (Roofplate-specific Spondin 1), combined with a variety of other growth factors. By adding EGF, Noggin, and Rspondin-1 to the basal medium, problems such as the inability to form structures, low cell viability, and failed passage during the culturing of organoids can be solved. However, when using this medium to culture organoids from lymph node metastases, due to: 1) lymphatic metastatic lesions are usually small, the biopsy sample size is small, and normal lymphoid tissue may be mixed, resulting in low purity of tumor cells; 2) there are a large number of immune cells in lymphoid tissue, making the tumor cells metastasized into it highly specialized, resulting in difficulty in culturing tumor organoids derived from lymph node metastases.

[0005] After achieving the organoids of bladder cancer lymph node metastases, if the general method of digestion - plating - cell viability detection is still used for drug testing, the following problems will also exist: 1) The operation of digestion and re-plating will disrupt the local microenvironment for the growth of organoids, affecting the cell viability and re-aggregation of organoids, thus affecting the accuracy of drug testing on organoids; 2) There are problems of complicated operation and long process in the method of digestion and re-plating, which is not conducive to quickly obtaining drug test results; 3) This method reflects the average response of the overall cell population, ignoring the cell heterogeneity within tumor organoids and the morphological and structural changes of organoids caused by drugs, and cannot intuitively and accurately reflect the response of organoids to drugs. Summary of the Invention

[0006] In order to overcome the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a culture medium, a culture method, and a rapid drug detection method for tumor organoids derived from bladder cancer lymph node metastases, so as to solve the technical problems of difficult culturing and low purity of tumor organoids derived from bladder cancer lymph node metastases, as well as the technical problems of complicated operation, long time, and inaccurate detection existing in the existing drug testing method based on tumor organoids.

[0007] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, a culture medium for tumor organoids derived from bladder cancer lymph node metastases is disclosed, which is composed of a basal medium, amino acids, cytokines, small molecule inhibitors, and a buffer solution, and the cytokines do not contain EGF, Noggin, and Rspondin-1.

[0009] Preferably, the basal medium is Advanced DMEM / F-12 or RMPI-1640.

[0010] Preferably, the amino acid is any one or more of L-Glutamax, N-acetylcysteine and nicotinamide.

[0011] Preferably, the cytokines include Penicillin-Streptomycin, B27, Nicotinamide, N-Acetylcysteine, FGF2 and FGF10.

[0012] Preferably, the small molecule inhibitor is any one or more of A83-01, Y-27632 and SB431542.

[0013] Preferably, the buffer is HEPES buffer or NaHCO3 buffer.

[0014] Preferably, the tumor organoid culture medium derived from bladder cancer lymph node metastasis foci is composed of Advanced DMEM / F-12, HEPES, Glutamax, Penicillin-Streptomycin, B27, Nicotinamide, N-Acetylcysteine, FGF2, FGF10 and A83-01.

[0015] In the second aspect of the present invention, the application of the above-mentioned tumor organoid culture medium in the culture of tumor organoids derived from bladder cancer lymph node metastasis foci is disclosed.

[0016] In the third aspect of the present invention, a method for culturing tumor organoids derived from bladder cancer lymph node metastasis foci based on the above-mentioned tumor organoid culture medium is disclosed, including the following steps:

[0017] Step 1: Crush, digest, resuspend, filter and lyse red blood cells of the tumor tissue of bladder cancer lymph node metastasis foci, centrifuge to discard the supernatant, and take the cells at the bottom;

[0018] Step 2: Take the tumor organoid culture medium to resuspend the cells at the bottom in Step 1 to obtain a cell suspension; add Matrigel to the cell suspension, mix well and inoculate it into a cell well plate, and incubate until the Matrigel solidifies;

[0019] Step 3: Continuously add the tumor organoid culture medium to the cell well plate until the Matrigel is submerged. When the cultured organoids grow to 100-150 μm, the tumor organoids derived from bladder cancer lymph node metastasis foci are obtained.

[0020] Preferably, in Step 2, the inoculation speed is 10 μL / drop, and the inoculation density is 1×10 4 viable cells / μL.

[0021] Preferably, in step two, incubate for 10 - 15 min in an incubator at 37°C with 5% CO2.

[0022] Preferably, in step three, culture in an incubator at 37°C with 5% CO2.

[0023] Preferably, change the culture medium every two days in step three.

[0024] In the fourth aspect of the present invention, a rapid detection method for drugs for treating lymph node metastasis of bladder cancer based on the above tumor organoid culture medium is disclosed. After culturing tumor organoids derived from lymph node metastases of bladder cancer using the tumor organoid culture medium, directly add test drugs with different concentrations to the cell well plates inoculated with the tumor organoids, and analyze the effects of the test drugs on cell growth or metabolism by the whole - field bright - field imaging method to obtain the detection results of drugs for treating lymph node metastasis of bladder cancer.

[0025] Preferably, use an integrated live - cell imaging system for whole - field bright - field imaging.

[0026] Preferably, use the tumor organoid culture medium derived from lymph node metastases of bladder cancer as a diluent to perform gradient concentration dilution of the test drugs to obtain test drugs with different concentrations.

[0027] Preferably, add 100 μL / well of the test drug to the wells to be tested.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] A tumor organoid culture medium derived from lymph node metastases of bladder cancer provided by the present invention is formed by removing the ENR factors (EGF, Noggin, and Rspondin - 1) in the widely used ENR organoid culture medium. Using this tumor organoid culture medium, tumor organoids derived from lymph node metastases of bladder cancer can be successfully cultured. Through comparative experiments, it is found that compared with the traditional ENR organoid culture medium, this tumor organoid culture medium shows better culture effects both in terms of the number and size of organoid growth, and the purity of the cultured tumor cells is high. Therefore, this tumor organoid culture medium has a lower cost, can be used for culturing tumor organoids derived from lymph node metastases of bladder cancer, and has a better culture effect.

[0030] The method for culturing tumor organoids derived from lymph node metastases of bladder cancer based on the tumor organoid culture medium provided by the present invention uses the tumor organoid culture medium derived from lymph node metastases of bladder cancer to culture tumor organoids derived from lymph node metastases of bladder cancer, which is simple and efficient.

[0031] The rapid detection method of drugs for treating lymph node metastasis of bladder cancer based on tumor organoid culture medium provided by the present invention does not require additional digestion, plating, and cell viability detection. After inoculating tumor organoids derived from lymph node metastases of bladder cancer by the dome embedding method, different concentrations of the drug to be tested are directly added into the original wells, and the killing effects of different drugs on tumor organoids are analyzed by the whole-field bright-field imaging method. Compared with the traditional drug testing method, this method has the following advantages: first, it does not damage the co-culture microenvironment of the cultured tumor organoids and immune cells; second, it is simple to operate and has a short process; third, it can pay attention to the cell heterogeneity within the tumor organoids and the morphological structure changes of the organoids caused by the drugs. On the basis of retaining the in-situ culture of the organoids, a more intuitive and accurate organoid drug evaluation method is established based on the real-time imaging method, realizing the requirements of rapid, intuitive, and accurate drug detection, and providing a reference for the culture and application of lymph node metastasis organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 FIG. is the bright-field result diagram of tumor organoids derived from lymph node metastases of bladder cancer cultured in different culture media; among them, A is the culture result of tumor organoid culture medium derived from lymph node metastases of bladder cancer, and B is the culture result of traditional tumor organoid culture medium (containing ENR factor), and the scale bars are both 200 μm.

[0033] Figure 2 FIG. is the statistical result diagram of the number of tumor organoids derived from lymph node metastases of bladder cancer cultured in different culture media.

[0034] Figure 3 FIG. is the flow chart of whole-field bright-field imaging of the rapid detection method of drugs for treating lymph node metastasis of bladder cancer.

[0035] Figure 4 FIG. is the image stitching result diagram after whole-field imaging; among them, A is before stitching, and B is after stitching.

[0036] Figure 5 FIG. is the result diagram of morphological recognition and comparison of tumor organoids derived from lymph node metastases of bladder cancer before and after drug treatment; among them, A is the stitched image before adding the drug, and B is the stitched image after adding the drug, and the organoid morphology is circled in pink.

[0037] Figure 6 FIG. is the drug effect curve diagram obtained by the rapid detection method of drugs for treating lymph node metastasis of bladder cancer. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0038] To enable those skilled in the art to understand the features and effects of the present invention, the following provides a general description and definition of the terms and phrases mentioned only in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein shall have the ordinary meanings understood by those skilled in the art with respect to the present invention. In case of conflicts, the definitions in this specification shall prevail.

[0039] The theories or mechanisms described and disclosed herein, whether right or wrong, shall not in any way limit the scope of the present invention, that is, the content of the present invention can be implemented without being limited by any specific theory or mechanism.

[0040] In this article, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are only for the sake of brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be regarded as having covered and specifically disclosed all possible sub-ranges and individual numerical values (including integers and fractions) within the ranges.

[0041] In this article, unless otherwise specified, the terms "comprising", "including", "containing", "having", or similar terms cover the meanings of "consisting of" and "consisting essentially of". For example, "A comprises a" covers the meanings of "A comprises a and others" and "A consists only of a".

[0042] In this article, for the sake of brevity of description, all possible combinations of all technical features in each embodiment or example are not described. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each embodiment or example can be combined arbitrarily, and all possible combinations should be considered as within the scope described in this specification.

[0043] A tumor organoid culture medium derived from a lymph node metastasis focus of bladder cancer, which is composed of a basal medium, amino acids, cytokines, small molecule inhibitors, and a buffer, and the cytokines do not contain EGF, Noggin, and Rspondin-1;

[0044] Among them, the basal medium includes, but is not limited to, one of Advanced DMEM / F-12 and RMPI-1640; the amino acids can provide essential and non-essential amino acids required for cell protein synthesis, including, but not limited to, any one or more of L-Glutamax, N-acetylcysteine, and nicotinamide; the cytokines are Penicillin-Streptomycin, B27, Nicotinamide, N-Acetylcysteine, FGF2, and FGF10; the small molecule inhibitors include, but are not limited to, any one or more of A83-01, Y-27632, and SB431542; the buffer includes, but is not limited to, one of HEPES buffer (Hepes Buffer) and NaHCO3 buffer.

[0045] The present invention provides a method for culturing tumor organoids derived from bladder cancer lymph node metastases, comprising the following steps:

[0046] Step 1: Cut the fresh tissue into pieces of 1-3 mm 3 and digest it at a constant temperature for 30-60 min, and invert and mix it up and down every 5-10 min;

[0047] Step 2: On the basis of Step 1, add basal medium for culture, centrifuge, and discard the supernatant; resuspend the cells, filter, centrifuge, and discard the supernatant; lyse red blood cells, centrifuge, and discard the supernatant; resuspend the cells again, centrifuge, and discard the supernatant, and take the cells at the bottom;

[0048] Step 3: Resuspend the bottom cells in Step 2 with the tumor organoid medium derived from bladder cancer lymph node metastases, pre-cool on ice to obtain a cell suspension; add Matrigel to the cell suspension and mix well, and inoculate it into a pre-warmed cell plate at 10 μL / drop, and the cell seeding density is 1×10 4 viable cells / μL;

[0049] Step 4: After inoculation, quickly turn over the cell plate inoculated in Step 3, add the tumor organoid medium derived from bladder cancer lymph node metastases, and place the plate in an incubator at 37 °C and 5% CO2 for 10-15 min to solidify the Matrigel.

[0050] Step 5: After the Matrigel solidifies, continue to add the tumor organoid medium derived from bladder cancer lymph node metastases until the Matrigel drops are submerged, transfer the inoculated plate to an incubator at 37 °C and 5% CO2 for culture, observe the culture condition of the organoids under the microscope, change the medium every 2 days, and wait until the organoids grow to about 100 μm to complete the culture of tumor organoids derived from bladder cancer lymph node metastases.

[0051] The present invention provides a method for detecting a drug for treating lymph node metastases of bladder cancer, comprising the following steps:

[0052] Step 1: Wash and cut the lymph node metastatic tissue of bladder cancer into minced meat (1 - 3 mm 3 ), and then digest it at a constant temperature for 30 - 60 min. Invert and mix it up and down every 5 - 10 min;

[0053] Step 2: On the basis of Step 1, add basal medium for culture, centrifuge, and discard the supernatant; resuspend the cells, filter, centrifuge, and discard the supernatant; lyse, centrifuge, and discard the supernatant; resuspend the cells again, centrifuge, and discard the supernatant, and take the cells at the bottom;

[0054] Step 3: Take 30 - 50 μL of tumor organoid medium from the lymph node metastases of bladder cancer to resuspend the cells at the bottom in Step 2, pre-cool on ice to obtain a cell suspension; add Matrigel to the cell suspension and mix well, and inoculate it into a pre-warmed cell well plate at 10 μL / drop, and the cell seeding density is 1×10 4 viable cells / μL;

[0055] Step 4: After inoculation, quickly invert the cell well plate inoculated in Step 3, add the tumor organoid medium from the lymph node metastases of bladder cancer, and place the cell well plate in an incubator at 37°C and 5% CO2 for 10 - 15 min to solidify the Matrigel.

[0056] Step 5: After the Matrigel solidifies, continue to add the tumor organoid medium from the lymph node metastases of bladder cancer until the Matrigel drops are submerged, transfer the inoculated well plate to an incubator at 37°C and 5% CO2 for culture, observe the culture condition of the organoids under bright field microscopy, change the medium every 2 days, and wait until the organoids grow to about 100 - 150 μm to obtain tumor organoids from the lymph node metastases of bladder cancer.

[0057] Step 6: In an integrated live cell imaging system, according to the growth and distribution of tumor organoids and gel drops from the lymph node metastases of bladder cancer, set 7 automatic focusing layers along the Z-axis direction for a single well to capture organoid morphology images of different layers, set 15 fields of view imaging for a single well to cover the entire gel drop imaging, then automatically generate pictures of tumor organoids from the lymph node metastases of bladder cancer for the whole plate, and use the built-in stitching program of the machine to generate full-field images of each culture well.

[0058] Step 7: Using the tumor organoid medium from the lymph node metastases of bladder cancer as a diluent, prepare different test drugs according to a 5-fold concentration gradient, and add 100 μL / well to the wells to be tested.

[0059] Step 8. After the drug has acted for 4 to 6 days, transfer the well plate to the integrated live cell imaging system again and repeat step 6. Then, the system's built-in software automatically identifies and outlines spherical cell clusters larger than 50 μm, and then manually calibrates the edges of the outlined cell clusters.

[0060] Step 9: Analyze the area of ​​organoids derived from bladder cancer lymph node metastases before and after drug addition, and normalize the values ​​compared with the control wells. Use GraphPad software to draw the drug action curve and IC 50 .

[0061] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0062] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, are conventional commercial products, and their specifications are conventional specifications in the art.

[0063] Example 1

[0064] A method for culturing tumor organoids derived from bladder cancer lymph node metastases, comprising the following steps:

[0065] 1. Place fresh tissue in a centrifuge tube and rinse several times in PBS (containing 1% double-antibody and amphotericin B); then transfer the tissue to a 10 cm sterile culture dish and chop it into minced meat (1-3 mm 3 ) and transferred into a 15 mL centrifuge tube.

[0066] 2. First, add 5 mL of tumor tissue digestive solution (R6002, purchased from Mingao Biology), incubate at 37 °C for 30 - 60 min, and invert the mixture up and down every 5 - 10 min; Second, add 5 mL of Advanced DMEM / F12 (containing 10% FBS), mix well, centrifuge at 300 g for 4 min, and carefully aspirate and discard the supernatant; Third, add 5 mL of PBS to resuspend the cells, filter through a 100 μm cell strainer, aspirate 5 mL of PBS to wash the strainer, centrifuge the filtrate at 300 g for 4 min, and carefully aspirate and discard the supernatant; Third, add 1 mL of red blood cell lysate (R8002, purchased from Mingao Biology), gently vortex or invert to mix well, let stand at room temperature for 5 min, centrifuge at 300 g for 4 min, and carefully aspirate and discard the supernatant: Finally, resuspend the cells with 5 mL of PBS, aspirate a small amount of the suspension, and calculate the total number of cells and the ratio of viable cells using an automatic cell counter or a manual counting plate. After counting, centrifuge at 300 g for 4 min, and carefully aspirate and discard the supernatant.

[0067] 3. Take 30 - 50 μL of the tumor organoid medium from bladder cancer lymph node metastasis foci to resuspend the bottom cells according to Table 1, and place it on ice for pre-cooling to obtain a pre-cooled cell suspension. Pre-cool the pipette tips and aspirate an appropriate amount of MACreGel, carefully mix the pre-cooled cell suspension, and use an electric continuous pipette to inoculate 10 μL per drop into a pre-warmed cell culture plate. The cell seeding density is 1×10 4 viable cells / μL, and the whole process is carried out on ice.

[0068] Table 1 Tumor organoid medium from bladder cancer lymph node metastasis foci

[0069] Component Function Advanced DMEM / F-12(1×) Basal medium 1% 1M HEPES Buffer 1% Glutamax Amino acid 1% Penicillin-Streptomycin Cytokine 2％B27(50×) Cytokine 10 mM Nicotinamide Cytokine 1.25 mM N-Acetylcysteine Cytokine 5 ng / mL FGF2 Cytokine 10 ng / mL FGF10 Cytokine 500 nM A83-01 Small molecule inhibitor

[0070] 4. After inoculation, quickly invert the inoculated cell culture plate, add 100 μL of the tumor organoid medium from bladder cancer lymph node metastasis foci (prepared according to Table 1), and place the plate in an incubator at 37 °C and 5% CO2 for 10 - 15 min to solidify the Matrigel.

[0071] 5. After the Matrigel solidifies, add the tumor organoid medium from bladder cancer lymph node metastasis foci (prepared according to Table 1) to submerge the Matrigel droplets, transfer the inoculated plate to an incubator at 37 °C and 5% CO2 for culture; observe the culture status of the organoids under the microscope and change the medium every 2 days; after about one week, most of the organoids grow to about 100 μm, and tumor organoids from bladder cancer lymph node metastasis foci are obtained.

[0072] Comparative Example 1

[0073] The difference from Example 1 is that a traditional tumor organoid medium (containing ENR factor) is prepared according to Table 2.

[0074] Table 2 Traditional Organoid Culture Medium (Containing ENR Factor)

[0075] Component Function Advanced DMEM / F-12(1×) Basal medium 1% 1M HEPES Buffer 1% Glutamax Amino acid 1% Penicillin-Streptomycin Cytokine 2％B27(50×) Cytokine 10 mM Nicotinamide Cytokine 1.25 mM N-Acetylcysteine Cytokine 5 ng / mL FGF2 Cytokine 10 ng / mL FGF10 Cytokine 50 ng / mL EGF Cytokine 100 ng / mL Noggin Cytokine 1 μg / mL R-Spondin1 Cytokine 500 nM A83-01 Small molecule inhibitor

[0076] The bright-field shooting results of tumor organoids derived from lymph node metastases of bladder cancer in Example 1 and Comparative Example 1 are as Figure 1 shown. It can be seen that the tumor organoid culture medium (lacking ENR culture system) derived from lymph node metastases of bladder cancer can better support the growth state of metastatic bladder cancer organoids.

[0077] The quantitative statistical results of tumor organoids derived from lymph node metastases of bladder cancer in Example 1 and Comparative Example 1 are as Figure 2 shown. It can be seen that the tumor organoid culture medium (lacking ENR culture system) derived from lymph node metastases of bladder cancer can better support the growth of metastatic bladder cancer organoids, and the number of organoids grown is significantly more than that of the traditional organoid culture medium (containing ENR culture system).

[0078] In summary, through culture comparison, it is found that whether in terms of the number or size of organoids, the tumor organoid culture medium without ENR shows better culture performance.

[0079] Example 2

[0080] A detection method for a drug treating lymph node metastases of bladder cancer, comprising the following steps:

[0081] 1. Place fresh tissue of lymph node metastases of bladder cancer in a centrifuge tube, and rinse it several times with PBS (containing 1% double antibiotics and amphotericin B); then transfer the tissue to a 10-cm sterile culture dish, cut it into minced meat (1 - 3 mm 3 ) and transfer it into a 15-mL centrifuge tube.

[0082] 2. First, add 5 mL of tumor tissue digestive solution (R6002, purchased from Mingao Biotech), incubate at 37 °C for 30 - 60 min, and invert the mixture up and down every 5 - 10 min to mix well. Secondly, add 5 mL of Advanced DMEM / F12 (containing 10% FBS), mix well, centrifuge at 300 g for 4 min, and carefully aspirate and discard the supernatant. Thirdly, add 5 mL of PBS to resuspend the cells, filter through a 100 μm cell strainer, aspirate 5 mL of PBS to wash the strainer, centrifuge the filtrate at 300 g for 4 min, and carefully aspirate and discard the supernatant. Thirdly, add 1 mL of red blood cell lysate (R8002, purchased from Mingao Biotech), gently vortex or invert to mix well, let stand at room temperature for 5 min, centrifuge at 300 g for 4 min, and carefully aspirate and discard the supernatant. Finally, resuspend the cells with 5 mL of PBS, aspirate a small amount of the suspension, and calculate the total cell count and viable cell ratio using an automatic cell counter or a manual counting plate. After counting, centrifuge at 300 g for 4 min, and carefully aspirate and discard the supernatant.

[0083] 3. Take 30 - 50 μL of tumor organoid medium (prepared according to Table 1) from bladder cancer lymph node metastasis foci to resuspend the bottom cells, and place it on ice for pre-cooling to obtain a pre-cooled cell suspension. Pre-cool the pipette tips and aspirate an appropriate amount of MACreGel (purchased from Mingao Biotech), carefully mix the pre-cooled cell suspension, and use an electric continuous pipette to inoculate 10 μL per drop into a pre-warmed cell culture plate. The cell seeding density is 1×10 4 viable cells / μL, and the whole process is carried out on ice.

[0084] 4. After inoculation, quickly invert the inoculated cell culture plate, add 100 μL of tumor organoid medium (prepared according to Table 1) from bladder cancer lymph node metastasis foci, and place the plate in an incubator at 37 °C and 5% CO2 for 10 - 15 min to allow the matrix gel to solidify.

[0085] 5. After the matrix gel has solidified, add the tumor organoid medium (prepared according to Table 1) from bladder cancer lymph node metastasis foci until the matrix gel drops are submerged. Transfer the inoculated plate to an incubator at 37 °C and 5% CO2 for culture; observe the culture status of the organoids under bright field microscopy and change the medium every 2 days; after about one week, most of the organoids grow to about 100 μm, obtaining tumor organoids from bladder cancer lymph node metastasis foci.

[0086] 6. As Figure 3As shown in the figure, the tumor organoids derived from the bladder cancer lymph node metastasis obtained in step 5 were transferred to the integrated live cell imaging system (KEYENCE). According to the growth distribution of the organoids and the gel droplets, 7 autofocus layers were set in the Z-axis direction of the single hole to capture the morphology of the organoids in different layers; 15 field of view imaging of the single hole was set to cover the entire gel droplet imaging, and then the whole plate of tumor organoids derived from the bladder cancer lymph node metastasis was automatically generated, and the full field of view image of each culture well was generated using the machine's own stitching program. The results are shown in the figure below. Figure 4 shown.

[0087] 7. Using the tumor organoid culture medium derived from bladder cancer lymph node metastases as the diluent, different concentrations (10 μM, 2 μM, 0.4 μM, 0.08 μM, 0.016 μM, 0.0032 μM) of the test drug Erdafitinib (HY-18708, purchased from MCE) were prepared according to a 5-fold concentration gradient and added to the test wells at 100 μL / well.

[0088] At the same time, the test wells without drug addition were used as control wells.

[0089] 8. After 5 days of drug action, transfer the well plate to the integrated live cell imaging system again and repeat step 6; then use the system's built-in software to automatically identify and outline spherical cell clusters larger than 50 μm, and then manually calibrate the edges of the outlined cell clusters.

[0090] 9. Analyze the size of tumor organoids before and after drug addition, and normalize the values ​​compared with the control wells. Use GraphPad software to draw drug action curves and IC 50 .

[0091] The size of the tumor organoids derived from bladder cancer lymph node metastases before and after drug addition Figure 5 As shown in the figure, it can be seen that before the addition of the drug, the number of tumor organoids was large, and the spheroids were more regular in shape and had clear edges. However, after the addition of the drug, most of the in situ tumor organoids had poor morphology, reduced area, and some tumor organoids even disappeared. This result shows that under the action of the drug Erdafitinib, tumor organoids derived from bladder cancer lymph node metastases underwent universal cell death.

[0092] Drug action curves Figure 6 As shown in the figure, it can be seen that the activity values ​​of tumor organoids in different wells at the same concentration are slightly different, and the effects of different concentrations of drugs on the activity of tumor organoid cells are concentration-dependent, indicating that this detection method has good accuracy. The drug action curve was fitted by nonlinear regression analysis, showing that the drug action curve was well fitted, and the IC 50It is 0.1049 μM, and it has an obvious killing effect on tumor organoids.

[0093] The above content is only to illustrate the technical idea of the present invention, and the protection scope of the present invention cannot be limited thereby. Any modification made on the basis of the technical solution according to the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

Claims

1. A tumor organoid culture medium derived from lymph node metastases of bladder cancer, characterized in that, It is composed of a basal medium, amino acids, cytokines, small molecule inhibitors and a buffer, and the cytokines do not contain EGF, Noggin and Rspondin-1.

2. The tumor organoid culture medium derived from lymph node metastasis sites of bladder cancer according to claim 1, wherein The basal medium is Advanced DMEM / F-12 or RMPI-1640.

3. A tumor organoid culture medium derived from lymph node metastases of bladder cancer according to claim 1, characterized in that, The amino acids are any one or more of L-Glutamax, N-acetylcysteine and nicotinamide.

4. A tumor organoid culture medium derived from bladder cancer lymph node metastases according to claim 1, characterized in that, The cytokines include Penicillin-Streptomycin, B27, Nicotinamide, N-Acetylcysteine, FGF2 and FGF10.

5. The tumor organoid culture medium derived from bladder cancer lymph node metastases according to claim 1, wherein The small molecule inhibitors are any one or more of A83-01, Y-27632 and SB431542.

6. The tumor organoid culture medium derived from bladder cancer lymph node metastases according to claim 1, wherein, The buffer is HEPES buffer or NaHCO3 buffer.

7. The tumor organoid culture medium derived from bladder cancer lymph node metastases according to claim 1, characterized in that, The tumor organoid culture medium derived from bladder cancer lymph node metastasis foci is composed of Advanced DMEM / F-12, HEPES, Glutamax, Penicillin-Streptomycin, B27, Nicotinamide, N-Acetylcysteine, FGF2, FGF10 and A83-01.

8. Use of the tumor organoid culture medium according to any one of claims 1 to 7 in the culture of tumor organoids derived from bladder cancer lymph node metastasis foci.

9. The method for culturing tumor organoids derived from lymph node metastases of bladder cancer using the tumor organoid culture medium according to any one of claims 1 to 7, characterized in that, It includes the following steps: Step 1: Cut, digest, resuspend, filter and lyse red blood cells of the tumor tissue of bladder cancer lymph node metastasis foci, centrifuge and discard the supernatant, and take the cells at the bottom; Step 2: Take the tumor organoid culture medium to resuspend the cells at the bottom in Step 1 to obtain a cell suspension; add Matrigel to the cell suspension, mix well and inoculate it into a cell well plate, and incubate until the Matrigel solidifies; Step 3: Add the tumor organoid culture medium to the cell well plate to submerge the Matrigel. When the cultured organoids grow to 100-150 μm, the tumor organoids derived from bladder cancer lymph node metastasis foci are obtained.

10. A rapid detection method for a drug for treating lymph node metastasis of bladder cancer with the tumor organoid culture medium according to any one of claims 1 to 7, characterized in that, After culturing the tumor organoids derived from bladder cancer lymph node metastasis foci using the tumor organoid culture medium, directly add different concentrations of the test drug to the cell well plate inoculated with the tumor organoids, and analyze the effect of the test drug on cell growth or metabolism by the whole-field bright-field imaging method to obtain the detection result of the drug for treating bladder cancer lymph node metastasis.

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