Construction method of tumor organoid and fibroblast high-flux drug sieve model

Abstract

The invention discloses a construction method of a tumor organoid and fibroblast high-flux drug sieve model. The construction method is simple in operation process, needs a small amount of cells, can shorten the experimental period, and is suitable for a co-culture system. In the co-culture of the tumor organoid and the fibroblast, the organoid is uniform in growth and consistent in density, and is flatly laid in the matrigel, so that the statistics of the growth condition of the subsequent organoid is facilitated; the fibroblasts serve as a main component of a tumor microenvironment, the fibroblasts are added into tumor organs for co-culture, the drug curative effect of a patient can be reflected more objectively and accurately, and the fibroblasts with fluorescence are used, so that the classification organs and the fibroblasts are distinguished, and the accuracy of drug sensitivity detection is improved; according to the invention, a high-throughput drug adding system can be used, a plurality of drugs with different concentrations can be directly added, and high-throughput drug screening under the condition of organoid co-culture can be realized in combination with a high-throughput photographing program.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for constructing a tumor organoid and fibroblast high-throughput drug screening model. Background Art

[0002] Currently, the research and development of drugs for tumors is developing rapidly, but the mechanism of action of tumor drugs is very complex, there are great individual differences among patients, and the diversity of the tumor microenvironment is also an important factor that leads to different responsiveness of patients to tumor drugs.

[0003] Tumor organoids are in vitro models derived from patient tumor tissues and highly consistent with the patient's tumor structure, physiology, and genetic characteristics. They have a wide range of applications in disease research, drug development, and precision medicine. High-throughput drug sensitivity testing of tumor organoids requires a large number of drugs to be tested in vitro to screen out drugs with strong specificity for patients.

[0004] The existing methods for detecting drug sensitivity of tumor organoids are mainly: (1) using the ATP luminescence method to detect the ATP value in cells after drug treatment, but the detection results of this method will be interfered by non-tumor cells and are not suitable for co-culture systems; (2) digesting tumor organoids into single cells, labeling them with Epcam and live and dead dyes, and using flow cytometry to detect the live and dead status of tumor cells after drug action, but this method is complicated to operate, requires a large number of cells, and cannot be used for high-throughput drug screening. Summary of the invention

[0005] The present invention provides a method for constructing a tumor organoid and fibroblast high-throughput drug screening model to solve the problems that the prior art is not suitable for a co-culture system, is complex to operate, requires a large amount of cells, and cannot perform high-throughput drug screening.

[0006] A method for constructing a tumor organoid and fibroblast high-throughput drug screening model, comprising: Step S1, extracting tumor cells from tumor tissue and culturing them into tumor organoids; Step S2, digesting the tumor tissue to obtain fibroblasts, and culturing them in a fibroblast culture medium. When the growth density of the fibroblasts reaches a preset value, adding a lentivirus carrying a pLV2-CMV-MCS-tdTomato-Puro plasmid to infect for a first preset time, then replacing the fibroblast culture medium, and after a second preset time, selecting with puromycin until all fibroblasts have red fluorescence, and then amplifying; Step S3, digesting the tumor organoids and the red fluorescently labeled fibroblasts into single cells respectively, and counting the cells, mixing the tumor organoids and fibroblasts digested into single cells according to a first preset ratio, and resuspending them in a basal culture medium to obtain a mixed cell resuspension; Step S4, mixing the matrix gel and the mixed cell resuspension at a second preset ratio, inoculating into a well plate after pipetting on ice, and placing in a 37° C. incubator, adding basal culture medium after the gel solidifies, and obtaining a co-culture of tumor organoids and fibroblasts after culturing for one day; Step S5, based on the co-culture of tumor organoids and fibroblasts, a blank group and drug-treated groups with different concentrations are set up, and the tumor drug is added to the drug-treated group at a high throughput, and then culture is continued for 5 days; Step S6, all groups are stained with live cell fluorescent dye. After the staining is completed, the dye is replaced with phosphate buffer, photos are taken, and the organoid activity-drug concentration gradient curve is statistically obtained to complete the construction of a high-throughput drug screening model for tumor organoids and fibroblasts, and then evaluate drug sensitivity.

[0007] Furthermore, step S2 specifically includes: Step S201, placing the tumor tissue in a cleaning solution for cleaning; Step S202, placing the cleaned tumor tissue on an inverted culture dish cover, chopping the tissue into 1 mm pieces, adding 1.5 mL of digestion solution, and placing in a metal bath for digestion for 30 min. The conditions of the metal bath are: 37° C., 1000 rpm; Step S203, after digestion is completed, a cleaning solution of the same volume as the digestion solution is added to terminate the digestion, and the suspension after the digestion is terminated is sucked by blowing evenly with a 1 mL pipette tip that has been cut off and rinsed, and then filtered with a 100 μm cell strainer; Step S204, taking the filtrate, centrifuging at 300 rcf, 4°C for 3 min, and discarding the supernatant; Step S205, add 500 μL of cleaning solution to resuspend the precipitate, centrifuge at 300 rcf, 4°C for 3 min, and discard the supernatant; Step S206, add 500 μL of red blood cell lysis solution, mix well, leave at room temperature for 5 minutes, centrifuge, and discard the supernatant; Step S207, add 500 μL of cleaning solution, mix well, centrifuge at 300 rcf, 4°C for 3 min, and discard the supernatant; Step S208, adding fibroblast culture medium to resuspend, inoculating in a cell culture dish, shaking in an 8-shape, and placing in an incubator; Step S209, replacing the fibroblast culture medium on the 3rd to 4th day; Step S210, when the growth density of fibroblasts reaches 60%, add lentivirus carrying pLV2-CMV-MCS-tdTomato-Puro plasmid for infection for 6 hours, and then replace the fibroblast culture medium; Step S211, after 48 hours, screen with 1 μg / mL of puromycin until all fibroblasts have red fluorescence, and then expand.

[0008] Furthermore, the components and contents of the fibroblast culture medium are as follows: α-MEM culture medium, 1X; penicillin-streptomycin mixture, 1X; and fetal bovine serum, 10%.

[0009] Furthermore, the composition and content of the basic culture medium are: Advanced DMEM / F12, 1X; B27, 1X; GluMAX, 1X; HEPES, 1X; N-acetylcysteine, 1.25mM; penicillin-streptomycin mixture, 100μg / mL.

[0010] Furthermore, the composition and content of the cleaning solution are: phosphate buffer, 1X; penicillin-streptomycin mixture, 1X; primocin antibiotic, 1X; The components and contents of the digestion solution are as follows: 1640 culture medium, 1X; collagenase IV, 2 mg / mL; and DNA enzyme, 200 U / mL.

[0011] Furthermore, the first preset ratio is: Tumor cells: fibroblasts = 3:1.

[0012] Furthermore, the second preset ratio is: Matrigel: mixed cell resuspension = 1:1.

[0013] Furthermore, in step S5, an Echo 650 acoustic pipetting system is used to achieve high-throughput drug addition.

[0014] Furthermore, in step S6, the living cell fluorescent dye is Calcein Am, which shows green fluorescence at a concentration of 1 μM, marking the living cells and distinguishing them from fibroblasts with red fluorescence at the same time, and the fluorescent dye staining time is 15-20 minutes.

[0015] Furthermore, in step S6, a PE high-content imaging system is used to take fluorescent photos, and the green fluorescence intensity of tumor organoids cultured at different drug concentrations is counted, and compared with the blank group without drug addition to obtain the organoid activity-drug concentration gradient curve.

[0016] Beneficial effects: 1. The present invention has a simple operation process, requires less cells, can shorten the experimental cycle, can meet the requirements of high-throughput drug screening, saves the cost of high-throughput drug screening, and is suitable for a co-culture system; 2. In the co-culture of tumor organoids and fibroblasts of the present invention, the organoids grow uniformly and have a consistent density and are flattened in the matrix gel, which is conducive to the subsequent statistics of the growth of the organoids; 3. Fibroblasts are the main component of the tumor microenvironment. Adding fibroblasts to tumor organoids for co-culture can more objectively and accurately reflect the efficacy of patients' drugs. The present invention uses fibroblasts with self-fluorescence, which is conducive to distinguishing between organoids and fibroblasts, and helps to improve the accuracy of drug sensitivity detection; 4. The present invention can use a high-throughput drug addition system to directly add multiple drugs of different concentrations, and combine with a high-throughput photography program to achieve high-throughput drug screening in the case of organoid co-culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Bright field images of lung cancer organoids co-cultured with fibroblasts and lung cancer organoids alone; Figure 2 Fluorescence images of the co-culture group and the single culture group; Figure 3 This is the organoid activity-drug concentration gradient curve of the co-culture group and the single culture group; Figure 4 The results are the statistical results of the sensitivity changes of 20 samples in the co-culture group and the single culture group to 6 drugs. DETAILED DESCRIPTION

[0018] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0019] The embodiment of the present invention provides a method for constructing a tumor organoid and fibroblast high-throughput drug screening model, wherein the tumor in this embodiment is lung cancer. Specifically, the method comprises the following steps S1 to S6: Step S1, extracting lung cancer cells from lung cancer tissue and culturing them into lung cancer organoids.

[0020] Step S2, digest the lung cancer tissue to obtain fibroblasts, and culture them in fibroblast culture medium. When the growth density of the fibroblasts reaches 60%, add lentivirus carrying pLV2-CMV-MCS-tdTomato-Puro plasmid to infect for 6 hours, then replace the fibroblast culture medium, and after 48 hours, select with puromycin until all fibroblasts have red fluorescence, and then amplify.

[0021] Wherein, step S2 specifically includes: Step S201, placing the lung cancer tissue in a cleaning solution for cleaning, wherein the cleaning solution comprises: phosphate buffer, 1X; penicillin-streptomycin mixture, 1X; primocin antibiotic, 1X.

[0022] Step S202, place the cleaned lung cancer tissue on the inverted culture dish cover, chop the tissue into 1mm size, add 1.5mL digestion solution, place in a metal bath for digestion for 30min, the metal bath conditions are: 37℃, 1000rmp (need to be preheated in advance). The composition and content of the digestion solution are: 1640 culture medium, 1X; collagenase IV, 2mg / mL; DNase, 200U / mL.

[0023] Step S203, after digestion is completed, a cleaning solution of the same volume as the digestion solution is added to terminate the digestion, and the suspension after the digestion is terminated is blown evenly with a 1 mL pipette tip that has been cut off and rinsed, and then the suspension is aspirated and filtered with a 100 μm cell strainer.

[0024] Step S204, take the filtrate, centrifuge at 300 rcf, 4°C for 3 min, and discard the supernatant.

[0025] Step S205, add 500 μL of cleaning solution to resuspend the precipitate, centrifuge at 300 rcf, 4°C for 3 min, and discard the supernatant.

[0026] Step S206, add 500 μL of red blood cell lysis solution, mix well, place at room temperature for 5 minutes, centrifuge, and discard the supernatant.

[0027] Step S207, add 500 μL of cleaning solution, mix well (rinse the tip), centrifuge at 300 rcf, 4°C for 3 min, and discard the supernatant.

[0028] Step S208, add fibroblast culture medium to resuspend, inoculate in a cell culture dish, shake in an 8-shape, and place in an incubator. The components and contents of the fibroblast culture medium are: α-MEM culture medium, 1X; penicillin-streptomycin mixture, 1X; fetal bovine serum, 10%.

[0029] Step S209, replace the fibroblast culture medium on the 3rd-4th day. In the specific implementation, it is necessary to observe whether adherent cells are formed under a microscope. The operation of replacing the fibroblast culture medium is as follows: remove the fibroblast culture medium, add an appropriate amount of 1X phosphate buffer to the culture dish, gently rinse it with a pipette to remove the miscellaneous cells that cannot grow on the wall, remove the 1X phosphate buffer, and add fibroblast culture medium preheated at 37°C.

[0030] Step S210, when the growth density of fibroblasts reaches 60%, add lentivirus carrying pLV2-CMV-MCS-tdTomato-Puro plasmid for infection for 6 hours, and then replace the fibroblast culture medium.

[0031] Step S211, after 48 hours, screen with 1 μg / mL of puromycin until all fibroblasts have red fluorescence, and then expand.

[0032] Through steps S201 to S211, it is convenient to distinguish fibroblasts from tumor organoids during the later co-culture. The growth rate of fibroblasts is faster than that of tumor organoids, and the infection efficiency is higher. The method of fluorescently labeling fibroblasts is more convenient and quick, which can better simplify the experimental steps, shorten the experimental cycle, and is more conducive to subsequent rapid high-throughput drug screening.

[0033] Step S3, digesting the lung cancer organoids and red fluorescently labeled fibroblasts into single cells respectively, and counting the cells, mixing the lung cancer organoids and fibroblasts digested into single cells according to a first preset ratio, and resuspending them with a basal culture medium to obtain a mixed cell resuspension.

[0034] Wherein, the first preset ratio is: Lung cancer cells: fibroblasts = 3:1.

[0035] Step S4, the matrix gel and the mixed cell resuspension were mixed at a second preset ratio, and then inoculated into a 96-well plate after pipetting on ice, 2 μL per well, 4500 lung cancer cells and 1500 fibroblasts per well, and placed in a 37°C incubator. After the gel solidified, basal culture medium was added. After one day of culture, a co-culture of lung cancer organoids and fibroblasts was obtained, and its bright field image can be referred to Figure 1 .

[0036] Specifically in this embodiment, 21 tumor organoids and fibroblasts were co-cultured in each 96-well plate to meet the requirements of setting different drug concentration gradients. In order to achieve the subsequent simultaneous determination of multiple drugs, they were inoculated simultaneously in multiple 96-well plates. In this embodiment, 6 drugs (paclitaxel, docetaxel, gemcitabine, vinorelbine, carboplatin, cisplatin) were screened, and 6 96-well plates were inoculated. After inoculation, they were placed in a 37°C incubator.

[0037] The composition and content of the basic culture medium are: Advanced DMEM / F12, 1X; B27, 1X; GluMAX, 1X; HEPES, 1X; N-acetylcysteine, 1.25mM; penicillin-streptomycin mixture, 100μg / mL. The basic culture medium has simple composition and does not contain additional growth factors compared with the reported lung cancer organoid culture medium.

[0038] Wherein, the second preset ratio is: Matrigel: mixed cell resuspension = 1:1.

[0039] Step S5, based on the co-culture of lung cancer organoids and fibroblasts, a blank group and drug-dosing groups with different concentrations are set up, lung cancer drugs are added to the drug-dosing groups at a high throughput, and then culture is continued for 5 days.

[0040] The blank group did not receive lung cancer drugs. In the drug-treated group, the Echo 650 acoustic liquid transfer system was used to achieve high-throughput drug addition.

[0041] Step S6, all groups are stained with live cell fluorescent dye. After the staining is completed, the dye is replaced with phosphate buffer, and photos are taken. The organoid activity-drug concentration gradient curve is statistically obtained to complete the construction of the high-throughput drug screening model of lung cancer organoids and fibroblasts, and then the drug sensitivity is evaluated. Among them, the live cell fluorescent dye is CalceinAm, which shows green fluorescence at a concentration of 1 μM, marking live cells and distinguishing them from fibroblasts with red fluorescence at the same time. The staining time of the fluorescent dye is 20 minutes.

[0042] In this example, a PE high-content imaging system was used for fluorescence photography. Taking paclitaxel as an example, the fluorescence image of the co-culture group obtained by photography can be found in Figure 2 At the same time, the green fluorescence intensity of lung cancer organoids under different drug concentrations was statistically analyzed, and compared with the blank group without drug addition, the organoid activity-drug concentration gradient curve was obtained. The results are as follows Figure 3 As shown, Figure 3 , activity ratio = (green fluorescence intensity of organoids in the drug-treated group / green fluorescence intensity of organoids in the non-drug-treated group) × 100%.

[0043] In addition, in order to compare the effect of fibroblasts on the drug sensitivity of tumor organoids, a separate culture group of lung cancer organoids was set up at the same time. The digested lung cancer organoid single cells were resuspended in 50% matrix gel and inoculated into a 96-well plate at 2μL / well (4500 tumor cells per well). The number of inoculated wells was the same as that of the co-culture, and the plate was placed in a 37°C incubator. After the gel solidified, lung cancer organoid culture medium was added. The composition and content of the lung cancer organoid culture medium were as follows: Advanced DMEM / F12, 1X; N-Acetylcysteine, 1.25mM; GlutaMax, 1X; HEPES, 10mM; penicillin-streptomycin mixture, 100μg / mL; FGF2, 20 ng / mL, B27, 1X. After one day of culture, a single lung cancer organoid was obtained, and its bright field image can be referred to. Figure 1 After obtaining the individual lung cancer organoids, the same operation process as steps S5 and S6 is followed, and paclitaxel is used as an example to obtain the fluorescence image of the individual culture group, as shown in FIG. Figure 2 As shown in the figure, the green fluorescence intensity of lung cancer organoids under different drug concentrations was statistically analyzed, and the organoid activity-drug concentration gradient curve was obtained by comparing it with the blank group without drug addition. The results are shown in Figure 3 shown.

[0044] from Figure 1 It can be seen that the co-culture can also generate uniform organoids after one day of co-culture, indicating that the fibroblast secretions can provide the cytokines required for organoid growth. Although the basal culture medium in this embodiment does not contain additional growth factors, it can also ensure the normal growth of fibroblasts and tumor organoids. The use of this basal culture medium can save the cost of high-throughput drug screening.

[0045] from Figure 2 It can be seen that fibroblasts have red fluorescence and lung cancer organoids have green fluorescence, which can well distinguish lung cancer organoids from fibroblasts, which is conducive to the subsequent statistics of organoid activity.

[0046] from Figure 3 It can be seen that based on the fluorescence area and fluorescence intensity of organoids under different paclitaxel concentration gradients, the sensitivity of lung cancer organoids to paclitaxel drugs was reduced after co-culture with fibroblasts.

[0047] In addition, this example tested the sensitivity changes of 20 co-culture groups and single culture groups to 6 drugs (paclitaxel, docetaxel, gemcitabine, vinorelbine, carboplatin, cisplatin), and calculated the area under the organoid activity-drug concentration curve to represent the drug sensitivity of the organoid. The results are as follows: Figure 4 As shown, from Figure 4It can be seen that fibroblasts generally reduce the sensitivity of lung cancer organoids to chemotherapeutic drugs, which also shows that the present invention can be used for high-throughput drug screening and evaluation of drug sensitivity.

[0048] In summary, the method for constructing a high-throughput drug screening model of tumor organoids and fibroblasts provided by the present invention has the following beneficial effects: 1. The present invention has a simple operation process, requires less cells, can shorten the experimental cycle, can meet the requirements of high-throughput drug screening, saves the cost of high-throughput drug screening, and is suitable for a co-culture system; 2. In the co-culture of tumor organoids and fibroblasts of the present invention, the organoids grow uniformly and have a consistent density and are flattened in the matrix gel, which is conducive to the subsequent statistics of the growth of the organoids; 3. Fibroblasts are the main component of the tumor microenvironment. Adding fibroblasts to tumor organoids for co-culture can more objectively and accurately reflect the efficacy of patients' drugs. The present invention uses fibroblasts with self-fluorescence, which is conducive to distinguishing between organoids and fibroblasts, and helps to improve the accuracy of drug sensitivity detection; 4. The present invention can use a high-throughput drug addition system to directly add multiple drugs of different concentrations, and combine with a high-throughput photography program to achieve high-throughput drug screening in the case of organoid co-culture.

[0049] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for constructing a high-throughput drug screening model of tumor organoids and fibroblasts, characterized in that: include: Step S1, extracting tumor cells from tumor tissue and culturing them into tumor organoids; Step S2, digesting the tumor tissue to obtain fibroblasts, and culturing them in a fibroblast culture medium. When the growth density of the fibroblasts reaches a preset value, adding a lentivirus carrying a pLV2-CMV-MCS-tdTomato-Puro plasmid to infect for a first preset time, then replacing the fibroblast culture medium, and after a second preset time, selecting with puromycin until all fibroblasts have red fluorescence, and then amplifying; Step S3, digesting the tumor organoids and the red fluorescently labeled fibroblasts into single cells respectively, and counting the cells, mixing the tumor organoids and fibroblasts digested into single cells according to a first preset ratio, and resuspending them in a basal culture medium to obtain a mixed cell resuspension; Step S4, mixing the matrix gel and the mixed cell resuspension at a second preset ratio, inoculating into a well plate after pipetting on ice, and placing in a 37° C. incubator, adding basal culture medium after the gel solidifies, and obtaining a co-culture of tumor organoids and fibroblasts after culturing for one day; Step S5, based on the co-culture of tumor organoids and fibroblasts, a blank group and drug-treated groups with different concentrations are set up, and the tumor drug is added to the drug-treated group at a high throughput, and then culture is continued for 5 days; Step S6, all groups are stained with live cell fluorescent dye. After the staining is completed, the dye is replaced with phosphate buffer, photos are taken, and the organoid activity-drug concentration gradient curve is statistically obtained to complete the construction of a high-throughput drug screening model for tumor organoids and fibroblasts, and then evaluate drug sensitivity.

2. The method for constructing a tumor organoid and fibroblast high-throughput drug screening model according to claim 1, characterized in that: Step S2 specifically includes: Step S201, placing the tumor tissue in a cleaning solution for cleaning; Step S202, placing the cleaned tumor tissue on an inverted culture dish cover, chopping the tissue into 1 mm pieces, adding 1.5 mL of digestion solution, and placing in a metal bath for digestion for 30 min. The conditions of the metal bath are: 37° C., 1000 rpm; Step S203, after digestion is completed, a cleaning solution of the same volume as the digestion solution is added to terminate the digestion, and the suspension after the digestion is terminated is sucked by blowing evenly with a 1 mL pipette tip that has been cut off and rinsed, and then filtered with a 100 μm cell strainer; Step S204, taking the filtrate, centrifuging at 300 rcf, 4°C for 3 min, and discarding the supernatant; Step S205, add 500 μL of cleaning solution to resuspend the precipitate, centrifuge at 300 rcf, 4°C for 3 min, and discard the supernatant; Step S206, add 500 μL of red blood cell lysis solution, mix well, leave at room temperature for 5 minutes, centrifuge, and discard the supernatant; Step S207, add 500 μL of cleaning solution, mix well, centrifuge at 300 rcf, 4°C for 3 min, and discard the supernatant; Step S208, adding fibroblast culture medium to resuspend, inoculating in a cell culture dish, shaking in an 8-shape, and placing in an incubator; Step S209, replacing the fibroblast culture medium on the 3rd to 4th day; Step S210, when the growth density of fibroblasts reaches 60%, add lentivirus carrying pLV2-CMV-MCS-tdTomato-Puro plasmid for infection for 6 hours, and then replace the fibroblast culture medium; Step S211, after 48 hours, screen with 1 μg / mL of puromycin until all fibroblasts have red fluorescence, and then expand.

3. The method for constructing a tumor organoid and fibroblast high-throughput drug screening model according to claim 1 or 2, characterized in that: The components and contents of the fibroblast culture medium are as follows: α-MEM culture medium, 1X; penicillin-streptomycin mixture, 1X; and fetal bovine serum, 10%.

4. The method for constructing a tumor organoid and fibroblast high-throughput drug screening model according to claim 1, characterized in that: The components and contents of the basic culture medium are: Advanced DMEM / F12, 1X; B27, 1X; GluMAX, 1X; HEPES, 1X; N-acetylcysteine, 1.25mM; and penicillin-streptomycin mixed solution, 100μg / mL.

5. The method for constructing a tumor organoid and fibroblast high-throughput drug screening model according to claim 2, characterized in that: The composition and content of the cleaning solution are as follows: phosphate buffer, 1X; penicillin-streptomycin mixture, 1X; primocin antibiotic, 1X; The components and contents of the digestion solution are as follows: 1640 culture medium, 1X; collagenase IV, 2 mg / mL; and DNA enzyme, 200 U / mL.

6. The method for constructing a tumor organoid and fibroblast high-throughput drug screening model according to claim 2, characterized in that: The first preset ratio is: Tumor cells: fibroblasts = 3:

1.

7. The method for constructing a tumor organoid and fibroblast high-throughput drug screening model according to claim 1, characterized in that: The second preset ratio is: Matrigel: mixed cell resuspension = 1:

1.

8. The method for constructing a tumor organoid and fibroblast high-throughput drug screening model according to claim 1, characterized in that: In step S5, high-throughput drug addition was achieved using the Echo 650 acoustic pipetting system.

9. The method for constructing a tumor organoid and fibroblast high-throughput drug screening model according to claim 1, characterized in that: In step S6, the living cell fluorescent dye is Calcein Am, which shows green fluorescence at a concentration of 1 μM, and is used to mark living cells and distinguish them from fibroblasts that also have red fluorescence. The staining time of the fluorescent dye is 15-20 minutes.

10. The method for constructing a tumor organoid and fibroblast high-throughput drug screening model according to claim 1, characterized in that: In step S6, a PE high-content imaging system is used to take fluorescent photos, and the green fluorescence intensity of tumor organoids cultured at different drug concentrations is counted, and compared with the blank group without drug addition to obtain the organoid activity-drug concentration gradient curve.

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