Efficient drug sensitivity detection method based on tumor organoids

Abstract

The invention discloses an efficient drug sensitivity detection method based on tumor organoids, which comprises the following steps: laying a certain number of cultured cell clusters digested by the tumor organoids, culturing for 48-72 hours, adding a drug to be detected, continuously culturing for 48-96 hours, and detecting the cell activity, according to the method, the tumor microenvironment of the organoid is utilized, the tumor state can be simulated, high-throughput drug screening can be carried out in vitro, operation is easy and convenient, standard and easy to master, reagent consumable consumption is smaller, cost is reduced, the number of needed cells is smaller, and the organoid detection period is shortened.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a tumor organoid drug sensitivity detection method. BACKGROUND

[0002] An organoid refers to a tissue analog with a certain spatial structure formed by in vitro 3D culture of adult stem cells or pluripotent stem cells. The organoid has more advantages than traditional 2D culture and can show physiological cell composition and behavior.

[0003] As a new technology, the tumor organoid is proved to be a valuable diagnostic tool in precision medicine application, which can screen the in vitro drug response of patients through tumor organoid drug sensitivity detection, aiming to provide guidance for cancer patients and predict the treatment effect. It is of great significance to quickly and correctly select effective chemotherapy drugs for patients during chemotherapy and realize individualized differential treatment.

[0004] The specific drug sensitivity detection operation in the prior art is not clear, or several drug sensitivity detection methods proposed are not conducive to clinical use. For example, the patent with the patent number CN202110344521.9 and the patent name A novel MTT cell viability detection kit and its application provides an organoid drug sensitivity detection method. The patent adds factors twice to continue culturing for 4-6h to detect the organoid viability after culturing and dosing the organoid in a 96-well culture plate for 72-96h. Another example is the patent with the patent number CN201910445410.X and the patent name A peritoneal effusion organoid culture medium, a culture method and a drug sensitivity test method. The patent detects after culturing and dosing the organoid in a 96-well culture plate for 8 days. The cell consumption of the above two patents is too large, and a longer time is needed for expansion and culture, which consumes more reagents and consumables. In addition, the dosing time and detection period are too long, and the two detection methods are not conducive to clinical use. Clinicians need fast, reliable and effective low-cost services.

[0005] Currently, there is an ATP chemiluminescence drug sensitivity detection method for 2D cells, but there is no detailed introduction of the application of the ATP drug sensitivity detection method to tumor organoids, which makes it difficult for experimental operators to operate and not standardized. In addition, the methods are quite different from each other, so there is an urgent need for an efficient drug sensitivity detection method for tumor organoids. SUMMARY

[0006] In view of this, the present application aims to provide a drug sensitivity efficient detection method based on tumor organoids to solve the above problems, and the technical scheme is as follows: (1) Set up an experimental group (6 gradient concentrations of each drug, 3 replicates), a control group and a blank group.

[0007] (2) Digest the cultured tumor organoids into cell clusters of 3-5 cells and seed them into 384-well culture plates. Only the experimental group and the control group are seeded. The volume of matrix gel per well is 3-10 μL, and the number of cells resuspended in each well is 1000-3000. After gelation, add 10-20 μL of the corresponding cancer culture medium per well and place it in an incubator at 37°C, 5% CO2, and saturated humidity.

[0008] (3) After 48-72 hours of inoculation, the organoid culture plate was removed, the test drug was added, and the plate was cultured at 5% CO2 and 37°C for 48-96 hours.

[0009] (4) Remove the organoid culture plate, equilibrate the cell culture plate to room temperature for 10 min, and add 5-10 μL of ATP detection reagent to the blank well, control well and drug test well of the culture plate.

[0010] (5) Cells were shaken at room temperature to promote cell lysis. After incubation, chemiluminescence detection was performed using a multi-functional microplate reader with chemiluminescence detection function. The data were analyzed to calculate cell viability and drug inhibition rate, and a growth inhibition curve was plotted.

[0011] This invention provides a highly efficient drug sensitivity detection method based on tumor organoids, which has the following advantages compared with traditional drug sensitivity detection methods: 1. This method provides an ATP chemiluminescence method for 3D organoids, making the operation simple, standardized, and easy to learn.

[0012] 2. This method uses fewer reagents and consumables, reduces costs, requires fewer cells, and saves organoid culture time.

[0013] 3. The present invention has a shorter drug administration and detection time in organoid drug sensitivity testing, making it faster. Attached Figure Description

[0014] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 The curve of human colorectal cancer organoid cell viability as a function of irinotecan concentration; Figure 2 The curve of human breast cancer organoid cell viability as a function of gemcitabine concentration; Figure 3 The curve showing the change in cell viability of human lung cancer organoids with etoposide concentration; Figure 4 Curve showing the change in cell viability of human ovarian cancer organoids with 5-fluorouracil concentration; Figure 5 The curve of human colorectal cancer organoid cell viability as a function of irinotecan concentration; Figure 6 Human breast cancer organoid cell viability as a function of gemcitabine drug concentration; Figure 7 Human lung cancer organoid cell viability as a function of etoposide drug concentration; Figure 8 Human ovarian cancer organoid cell viability as a function of 5-fluorouracil drug concentration. DETAILED DESCRIPTION

[0015] In the description of the present application, it should be noted that, in the examples, no specific conditions are specified, and the conventional conditions or the conditions recommended by the manufacturer are used. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be purchased on the market. EXAMPLE

[0016] Set up experimental groups (6 concentrations of each drug, three replicates), control groups, and blank groups.

[0017] The cultured intestinal cancer organoids were digested into 3-5 cell cell clusters and inoculated in 384-well culture plates, only the experimental groups and the control groups were inoculated, the volume of Matrigel per well was 3 μL, the number of resuspended cells per well was 2000, after gelation, 10 μL / well of corresponding cancer medium was added, and the plates were placed in a 37°C, 5% CO2, saturated humidity incubator for culture.

[0018] After 48 hours of inoculation, the organoid culture plates were removed, and the test drugs were added, and then incubated at 37°C in 5% CO2 for 48 hours.

[0019] The organoid culture plates were removed, and the cell culture plates were equilibrated at room temperature for 10 min, and 5 μL of ATP detection reagent was added to the blank wells, control wells, and drug test wells of the culture plates.

[0020] Room temperature shaking was used to promote cell lysis, and after incubation, a multifunctional enzyme labeler with chemiluminescence detection function was used for chemiluminescence detection. Analyze the data, calculate the cell survival rate, the drug inhibition rate, and draw the growth inhibition curve.

[0021] Figure 1 The human intestinal cancer organoid cell viability as a function of irinotecan drug concentration is provided EXAMPLE

[0022] Set up experimental groups (6 concentrations of each drug, three replicates), control groups, and blank groups.

[0023] The cultured breast cancer organoids were digested into 3-5 cell cell clusters and inoculated in 384-well culture plates, only the experimental group and the control group, 3 μL of Matrigel per well, 1000 resuspended cells per well, after gel, 10 μL / well of corresponding cancer culture medium was added, and placed in a 37°C, 5% CO2, saturated humidity incubator for culture.

[0024] After 48 hours of inoculation, the organoid culture plates were removed, and after adding the drug to be tested, they were incubated at 5% CO2, 37°C for 48 hours.

[0025] The organoid culture plates were removed, and the cell culture plates were equilibrated at room temperature for 10 min, and 5 μL of ATP detection reagent was added to the blank wells, control wells, and drug test wells of the culture plates.

[0026] Room temperature shaking was used to promote cell lysis, and after incubation, a multifunctional enzyme labeler with chemiluminescence detection function was used for chemiluminescence detection. Analyze the data, calculate the cell survival rate, drug inhibition rate, and draw the growth inhibition curve.

[0027] Figure 2 The human breast cancer organoid cell viability curve with gemcitabine drug concentration is provided Embodiment

[0028] The experimental group (6 concentrations of each drug, three replicates), control group, and blank group were set.

[0029] The cultured lung cancer organoids were digested into 3-5 cell cell clusters and inoculated in 384-well culture plates, only the experimental group and the control group, 5 μL of Matrigel per well, 3000 resuspended cells per well, after gel, 20 μL / well of corresponding cancer culture medium was added, and placed in a 37°C, 5% CO2, saturated humidity incubator for culture.

[0030] After 48 hours of inoculation, the organoid culture plates were removed, and after adding the drug to be tested, they were incubated at 5% CO2, 37°C for 72 hours.

[0031] The organoid culture plates were removed, and the cell culture plates were equilibrated at room temperature for 10 min, and 10 μL of ATP detection reagent was added to the blank wells, control wells, and drug test wells of the culture plates.

[0032] Room temperature shaking was used to promote cell lysis, and after incubation, a multifunctional enzyme labeler with chemiluminescence detection function was used for chemiluminescence detection. Analyze the data, calculate the cell survival rate, drug inhibition rate, and draw the growth inhibition curve.

[0033] Figure 3 The human lung cancer organoid cell viability curve with etoposide drug concentration is provided Embodiment

[0034] Set up experimental groups (6 concentrations of each drug, three replicates), control group, blank group.

[0035] The cultured ovarian cancer organoids were digested into 3-5 cell cell clusters and inoculated in 384-well culture plates, only the experimental group and the control group were inoculated, the volume of matrigel in each well was 10 μL, the number of resuspended cells in each well was 3000, after the gel, 20 μL / well of corresponding cancer medium was added, and the plate was placed in a 37°C, 5% CO2, saturated humidity incubator for culture.

[0036] After 48 hours of inoculation, the organoid culture plate was taken out, the drug to be tested was added, and the culture was continued at 5% CO2, 37°C for 96 hours.

[0037] The organoid culture plate was taken out, the cell culture plate was equilibrated at room temperature for 10 min, and 10 μL of ATP detection reagent was added to the blank wells, control wells and drug test wells of the culture plate.

[0038] Room temperature shaking to promote cell lysis, after incubation, a multifunctional enzyme labeler with chemiluminescence detection function was used for chemiluminescence detection. Analyze the data, calculate the cell survival rate, drug inhibition rate, and draw the growth inhibition curve.

[0039] Figure 4 The curve of the cell viability of the human ovarian cancer organoids with the change of the concentration of 5-fluorouracil drug is provided Comparative example 1 Set up experimental groups (6 concentrations of each drug, three replicates), control group, blank group.

[0040] The cultured intestinal cancer organoids were digested into 3-5 cell cell clusters and inoculated in 96-well culture plates, only the experimental group and the control group were inoculated, the volume of matrigel in each well was 20 μL, the number of resuspended cells in each well was 3000, after the gel, 100 μL / well of corresponding cancer medium was added, and the plate was placed in a 37°C, 5% CO2, saturated humidity incubator for culture.

[0041] After 48 hours of inoculation, the organoid culture plate was taken out, the drug to be tested was added, and the culture was continued at 5% CO2, 37°C for 96 hours.

[0042] The organoid culture plate was taken out, the cell culture plate was equilibrated at room temperature for 10 min, and 50 μL of ATP detection reagent was added to the blank wells, control wells and drug test wells of the culture plate.

[0043] Room temperature shaking to promote cell lysis, after incubation, a multifunctional enzyme labeler with chemiluminescence detection function was used for chemiluminescence detection. Analyze the data, calculate the cell survival rate, drug inhibition rate, and draw the growth inhibition curve.

[0044] Figure 5The human breast cancer organoid cell viability as a function of gemcitabine drug concentration is provided Comparative Example 2 Set up experimental groups (6 concentrations of each drug, three replicates), control groups, and blank groups.

[0045] The cultured breast cancer organoids were digested into 3-5 cell cell clusters and inoculated into 96-well culture plates, only the experimental and control groups were inoculated, 10 μL of Matrigel was added to each well, 3000 resuspended cells were added to each well, and after the gel was added 50 μL / well of the corresponding cancer culture medium, and placed in a 37°C, 5% CO2, saturated humidity incubator for culture.

[0046] After 48 hours of inoculation, the organoid culture plates were removed, the test drugs were added, and the culture was continued at 5% CO2, 37°C for 96 hours.

[0047] The organoid culture plates were removed, the cell culture plates were equilibrated at room temperature for 10 min, and 25 μL of ATP detection reagent was added to the blank wells, control wells, and drug test wells of the culture plates.

[0048] Room temperature shaking was used to promote cell lysis, and after incubation, a multifunctional enzyme labeler with chemiluminescence detection function was used for chemiluminescence detection. Analyze the data, calculate the cell survival rate, drug inhibition rate, and draw the growth inhibition curve.

[0049] Figure 6 The human breast cancer organoid cell viability as a function of gemcitabine drug concentration is provided Comparative Example 3 Set up experimental groups (6 concentrations of each drug, three replicates), control groups, and blank groups.

[0050] The cultured lung cancer organoids were digested into 3-5 cell cell clusters and inoculated into 96-well culture plates, only the experimental and control groups were inoculated, 30 μL of Matrigel was added to each well, 5000 resuspended cells were added to each well, and after the gel was added 100 μL / well of the corresponding cancer culture medium, and placed in a 37°C, 5% CO2, saturated humidity incubator for culture.

[0051] After 48 hours of inoculation, the organoid culture plates were removed, the test drugs were added, and the culture was continued at 5% CO2, 37°C for 96 hours.

[0052] The organoid culture plates were removed, the cell culture plates were equilibrated at room temperature for 10 min, and 50 μL of ATP detection reagent was added to the blank wells, control wells, and drug test wells of the culture plates.

[0053] Incubate at room temperature for 10 min, then add 25 μL ATP detection reagent to the blank wells, control wells and drug test wells of the cell culture plate.

[0054] Figure 7 The human lung cancer organoid cell viability curve with etoposide drug concentration is provided Comparative Example 4 Set up experimental groups (6 concentrations of each drug, three replicate wells), control groups, and blank groups (6 wells).

[0055] The cultured ovarian cancer organoids were digested into 3-5 cell cell clusters and inoculated in a 96-well culture plate, only the experimental group and the control group were inoculated, 10 μL of Matrigel was added to each well, the number of resuspended cells in each well was 2000, after the gel was placed, 50 μL / well of corresponding cancer culture medium was added, and the plate was placed in a 37°C, 5% CO2, saturated humidity incubator for culture.

[0056] After 48 h of inoculation, the organoid culture plate was taken out, and after the addition of the drug to be tested, it was continued to be cultured at 5% CO2, 37°C for 96 h.

[0057] The organoid culture plate was taken out, and the cell culture plate was equilibrated at room temperature for 10 min, and 25 μL of ATP detection reagent was added to the blank wells, control wells, and drug test wells of the culture plate.

[0058] Incubate at room temperature for 10 min, then add 25 μL ATP detection reagent to the blank wells, control wells and drug test wells of the cell culture plate.

[0059] Figure 8 The human ovarian cancer organoid cell viability curve with 5-fluorouracil drug concentration is provided Table 1 Comparison of results of human intestinal cancer organoid drug sensitivity detection method Irinotecan (IC50 μM) R square Cellular consumption (W) Matrigel consumption (μL) Medium consumption (μL) ATP detection reagent consumption (μL) Detection period (h) Example 1 2.248 0.883 4.8 72 300 150 96 Comparative Example 1 2.216 0.875 7.2 480 3000 1500 144 Table 2 Comparison of results of human breast cancer organoid drug sensitivity detection method Gemcitabine (IC50 μM) R square Cellular consumption (W) Matrigel consumption (μL) Medium consumption (μL) ATP detection reagent consumption (μL) Detection period (h) Example 2 1.02 0.815 2.4 72 300 150 96 Comparative Example 2 1.539 0.471 7.2 240 1500 750 144 Table 3 Comparison of results of human lung cancer organoid drug sensitivity detection method Etoposide (IC50 μM) R square Cellular consumption (W) Matrigel consumption (μL) Medium consumption (μL) ATP detection reagent consumption (μL) Detection period (h) Example 3 3.887 0.955 7.2 120 600 300 120 Comparative Example 3 3.45 0.955 12 720 3000 1500 144 Table 4 Drug sensitivity detection results of human ovarian cancer organoids in Example 4 5-Fluorouracil (IC50 μM) R square Cellular consumption (W) Matrigel consumption (μL) Medium consumption (μL) ATP detection reagent consumption (μL) Detection period (h) Example 4 108.3 0.894 7.2 240 600 300 120 Comparative Example 4 117.6 0.762 4.8 240 1500 750 144 The above example and comparative example results show that the drug sensitivity culture method has little difference in results (IC50, curve inhibition rate) compared with other traditional methods, but the R square (evaluation model fitting degree) of the method is closer to 1 compared with the traditional method, and the detection period is shortened, the cell and reagent consumption is reduced, and the cost is reduced.

[0060] The above examples only express several embodiments of the present application, which are described in more detail and in more detail, but cannot be understood as limiting the scope of the patent of the present application. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A method for efficient tumor organoid drug sensitivity test, characterized by, Specifically comprising the following steps: Step one, the tumor organoids are digested into 3-5 cell cell clusters, and the digested cells are inoculated in 384-well culture plates, only the experimental group (6 gradient concentrations for each drug) and the control group are inoculated; Step two, after 48-72h of tumor organoid culture, add drugs, add 5μL of a certain concentration of the drug to be tested to the experimental group, and add 5μL of culture medium to the control group and the blank group, and continue to culture for 48-96h; Step three, when testing the activity of the organoids, add 5-10μL of ATP detection reagent to the blank wells, control wells, and drug test wells of the culture plate. Incubate at room temperature to promote cell lysis, and then use a multifunctional enzyme labeler with chemiluminescence detection function to perform chemiluminescence detection. Use GraphPad Prism software to analyze the data, calculate the cell survival rate and drug inhibition rate, and draw a growth inhibition curve. 2.The method for efficient drug sensitivity test of tumor organoids according to claim 1, characterized in that: The volume of Matrigel for inoculating the tumor organoids in each well is 3-10μL, and after the gel is formed, 10-20μL / well of the corresponding cancer culture medium is added, and the culture is placed in a 37℃, 5% CO2, saturated humidity incubator. 3.The method for efficient drug sensitivity test of tumor organoids according to claim 1, characterized in that: The tumor organoids are digested and inoculated with 1000-3000 cells per well.

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