Pancreatic cancer tissue organoid drug sensitivity detection method
The method addresses the issue of non-uniform pancreatic cancer organoid cultures by using pancreatic cancer tissue digestion and controlled culture conditions to achieve uniform organoids, enhancing drug sensitivity testing accuracy.
Patent Information
- Application Number
- CN202411123360.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-15
- Publication Date
- 2024-12-27
AI Technical Summary
Existing methods for drug sensitivity testing of pancreatic cancer organoids lack specificity and uniformity, leading to low culture success rates and inconsistent results due to poor organoid culture and uneven density.
A method involving pancreatic cancer tissue organoid digestion, followed by controlled inoculation and culture in basement gel, with regular medium changes and ATP detection after drug treatment, to achieve uniform organoid cultures and accurate sensitivity assessment.
The method ensures uniform pancreatic cancer organoids with improved culture success and precise drug sensitivity evaluation by adjusting culture conditions and observation times, reducing errors in drug efficacy judgment.
Smart Images
Figure CN119193489A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of drug sensitivity detection, and in particular to a method for detecting drug sensitivity of pancreatic cancer tissue organoids. Background Art
[0002] Pancreatic cancer is the fifth most common cancer in the world and the second most deadly malignant tumor in China, which seriously endangers human health. Patients with early pancreatic cancer have no obvious symptoms, and most pancreatic cancer patients have already developed to the middle and late stages when diagnosed. Pancreatic cancer cells are less sensitive to chemotherapy drugs, so their chemotherapy effects are poor, resulting in a low five-year survival rate. Organoid culture is a variety of cell populations contained in specific tissues and organs cultured in a three-dimensional environment. The composition is similar to that of organs in the body, and its culture system is more closely connected to the in vivo microenvironment. In recent years, with the rapid development of organoid technology, organoid models have been widely used in basic research, precision medicine, drug screening and development, gene therapy and other fields. However, the long operation time of organoids and the low culture success rate have greatly reduced their application in the field of precision medicine.
[0003] The patent with application number CN202311023031.4 discloses a method for culturing body fluid tumor organoids and a method for drug sensitivity detection. The patent with application number CN202111189130.0 discloses a method for drug sensitivity detection of tumor organoids based on staining. The patent mixes tumor organoids with anti-tumor drugs and cultures them in a drug-sensitive culture medium for a period of time, then uses a cell fluorescent dye to stain the tumor organoids, and then processes the stained organoids into single cells for activity detection and drug sensitivity analysis evaluation. The tumor organoid drug sensitivity detection method of the present invention is based on cell staining, using organoids derived from tumor patients, directly calculating the number of live and dead cells, and performing vitality analysis to determine the sensitivity of the sample to the drug.
[0004] However, they all lack specificity for pancreatic organoids, and the uneven density can easily reduce the success rate of organoid activity detection. The root cause is that the organoids have poor uniformity and are difficult to culture. During the drug sensitivity test, the number of organoids is insufficient, resulting in large differences in results. Therefore, the number and size of organoids are crucial in the process of organoid drug sensitivity testing. Summary of the invention
[0005] The purpose of the present invention is to provide a method for detecting the drug sensitivity of pancreatic cancer tissue organoids in response to the deficiencies in the prior art.
[0006] To achieve the above object, the technical solution adopted by the present invention is:
[0007] A method for detecting drug sensitivity of pancreatic cancer tissue organoids is provided, wherein pancreatic cancer organoids are cultured using pancreatic cancer tissue organoid digestion fluid, and the method comprises the following steps:
[0008] S1. Obtain pancreatic cancer organoid tissue samples, wash the pancreatic cancer tissue organoid samples repeatedly with pre-cooled PBS buffer, cut them into pieces, and add pancreatic cancer tissue organoid digestion solution;
[0009] S2. Add pre-cooled PBS buffer to terminate the primary digestion, repeatedly blow and beat until the tissue fragments disappear, take out the partially digested solution, and when it is observed as a uniform cell cluster under a microscope, resuspend the cell cluster by low-temperature low-speed centrifugation, count the number of cell clusters under a microscope, and transfer the corresponding cell clusters to a centrifuge tube for centrifugation according to the number of wells to be inoculated;
[0010] S3, add the enzymatically hydrolyzed cell mass to the basement gel to resuspend it, and inoculate it into the TC cell culture plate. When the basement gel solidifies, add Advanced DMEM / F-12 culture medium to culture it, and observe and change the medium regularly during the period;
[0011] S4. After culturing the pancreatic cancer tissue organoids for 5-7 days, add pre-cooled PBS buffer to the TC cell culture plate, transfer the base gel to a centrifuge tube, pipette repeatedly, transfer to a refrigerator and let stand, centrifuge and remove the supernatant to obtain pancreatic cancer tissue organoids;
[0012] S5, enzymatically digesting the pancreatic cancer tissue organoids obtained in step S4 to obtain pancreatic cancer tissue organoid fragments;
[0013] S6, resuspending the pancreatic cancer tissue organoid fragments obtained in step S5 with basement gel, adjusting the cell concentration to 200-300 cells / 25 μl, inoculating into a 24-well plate, and adding the culture medium of the pancreatic cancer tissue organoid after the basement gel solidifies;
[0014] S7, resuspending the pancreatic cancer tissue organoid fragments obtained in step S6 with basement gel, adjusting the cell concentration to 30-50 cells / 5 μl, inoculating into a 96-well plate, and adding the culture medium of the pancreatic cancer tissue organoid after the basement gel solidifies;
[0015] S8, completely removing the culture medium of the pancreatic cancer tissue organoids in step S7, adding culture medium containing pancreatic cancer tissue organoids with different drug concentrations, selecting the whole-well field of view to continuously take pictures after adding the drug, and recording the morphology and size of the organoids;
[0016] S9. At the 72nd hour of drug action, add the culture medium containing pancreatic cancer tissue organoids with different drug concentrations again; after 120 hours of drug action on organoids, discard the culture medium, add the ATP detection kit, and detect the activity of the organoids.
[0017] Furthermore, in S1, the digestion conditions are: 37° C., shaking digestion for 10-20 min.
[0018] Furthermore, in S3, the TC cell culture plate is selected from one of 6-well, 12-well, 24-well, and 48-well.
[0019] Furthermore, in S4, the temperature of the refrigerator is set to 4°C.
[0020] Furthermore, in S5, the obtained organoid fragments are enzymatically digested using TrypLE Express.
[0021] Furthermore, the enzymatic digestion operation is as follows: add 1-2 ml of TrypLE Express, digest for 2-3 min, digest while blowing, add 6-8 ml of pre-cooled PBS buffer, centrifuge at 300 g for 5 min, and terminate the enzymatic digestion.
[0022] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:
[0023] The detection method of the present invention targets pancreatic organoids. By combining pancreatic cancer tissue organoid digestion fluid with culture medium to culture organoids, pancreatic tissue organoids with better uniformity are obtained for drug sensitivity detection. The drug treatment time and observation time are adjusted according to the drug action mechanism and organoid characteristics to obtain the best drug efficacy evaluation, thus overcoming the problem of large errors in organoid drug sensitivity judgment in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 The organoids are cultured in the pancreatic cancer tissue organoid digestion fluid combined with culture medium in Example 1; wherein, P1 is the first generation of pancreatic cancer organoids, and P3 is the third generation of pancreatic cancer organoids.
[0025] Figure 2 These are HE images of organoids and pancreatic cancer tissues in Example 1.
[0026] Figure 3 These are pictures of the organoids before and after drug administration in Example 1.
[0027] Figure 4 This is a graph showing the ATP detection activity analysis of organoids after drug administration in Example 1. DETAILED DESCRIPTION
[0028] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.
[0029] The raw materials and reagents involved in the embodiments of the present invention are all commercially available products unless otherwise specified, and the operations involved are all routine operations in the art unless otherwise specified.
[0030] The names, specifications and sources of the raw materials used in the embodiments of the present invention are as follows:
[0031] 1. Advanced DMEM (Dulbecco's modified Eagle medium) / F12 culture medium, purchased from Gibco, catalog number 12634010.
[0032] 2. Matrigel, purchased from Corning, was packaged and stored at -20 degrees Celsius before use. It was thawed in a refrigerator at 4 degrees Celsius before use.
[0033] 3. Pancreatic cancer organoid culture medium was purchased from Shanghai Jiefang Kairui Biotechnology Co., Ltd. with the catalog number JFKR-PADC-100.
[0034] 4. Pancreatic cancer organoid tissue digestion fluid was purchased from Shanghai Jiefang Kairui Biotechnology Co., Ltd. with the catalog number JFKR-ZXHY-100.
[0035] 5. Organoid activity detection kit, purchased from Promega (Beijing) Biotechnology Co., Ltd., catalog number G9681
[0036] Example 1
[0037] Pancreatic cancer organoid drug sensitivity test: In this example, two pancreatic cancer tissues (Patient 1 and Patient 2) were used to test the drug sensitivity of 8 compounds. The specific operation is as follows:
[0038] S1. Obtain pancreatic cancer organoid tissue samples, wash them repeatedly with pre-cooled PBS buffer, cut them into pieces and cut them into 1mm 2 Add 3-5 ml of pancreatic cancer tissue organoid digestion solution to the fragments to obtain a tissue mixture;
[0039] S2, add 3 times the volume of the above tissue mixture to the pre-cooled PBS buffer to terminate the primary digestion, repeatedly blow with a pipette until the tissue fragments disappear, take out the partially digested solution, and when it is observed as a uniform cell cluster under a microscope, resuspend the cell cluster by low-temperature low-speed centrifugation, count the number of cell clusters under a microscope, and transfer the corresponding cell clusters to a 1.5ml or 2ml centrifuge tube according to the number of wells to be inoculated, and centrifuge at 300g for 5 minutes;
[0040] S3, add the enzymatically hydrolyzed cell mass to the basement gel to resuspend it, and inoculate it into the TC cell culture plate. When the basement gel solidifies, add Advanced DMEM / F-12 culture medium to culture it, and observe and change the medium regularly during the period;
[0041] S4. After culturing pancreatic cancer tissue organoids for 5-7 days, add 0.8-1 ml of pre-cooled PBS buffer to the TC cell culture plate, transfer the base gel to a 15 ml or 50 ml centrifuge tube, pipette repeatedly, transfer to a refrigerator and let stand for 10 min, centrifuge at 300 g for 5 min, remove the supernatant, and obtain pancreatic cancer tissue organoids;
[0042] S5, enzymatically digesting the pancreatic cancer tissue organoids obtained in step S4 to obtain pancreatic cancer tissue organoid fragments;
[0043] S6. Resuspend the pancreatic cancer tissue organoid fragments obtained in step S5 with Matrigel, adjust the cell concentration to 200-300 cells / 25 μl, inoculate into a 24-well plate, and add the culture medium of the pancreatic cancer tissue organoids after the Matrigel solidifies;
[0044] S7, resuspending the pancreatic cancer tissue organoid fragments obtained in step S6 with Matrigel, adjusting the cell concentration to 30-50 cells / 5 μl, inoculating into a 96-well plate, and adding the culture medium of the pancreatic cancer tissue organoid after the Matrigel solidifies;
[0045] S8. Completely remove the culture medium of pancreatic cancer tissue organoids in step S7, add culture medium containing pancreatic cancer tissue organoids with different drug concentrations, select the whole-well field of view and take pictures continuously from 0h to 120h after drug addition to record the morphology and size of the organoids;
[0046] S9. At the 72nd hour of drug action, add the culture medium containing pancreatic cancer tissue organoids with different drug concentrations again; after 120 hours of drug action on organoids, discard the culture medium, add the ATP detection kit, and detect the activity of the organoids.
[0047] The results are as follows Figure 1-4 As shown: Figure 1 The images show that the pancreatic cancer organoids cultured from primary pancreatic cancer tissue are of uniform size and large in number. After passaging, the number of organoids increased significantly and their size became larger. Figure 2 The HE of pancreatic cancer organoids and pancreatic cancer tissues is shown. By comparing the pancreatic cancer organoids with the cancer tissues, the pancreatic organoids and tissues are highly consistent. Figure 3 The pictures show two pancreatic cancer organoids from different tissue sources before and after drug treatment. The blank control group and the drug group were photographed continuously before and 120 hours after the treatment. The organoids showed different growth states. The blank group had an increased volume and a high refractive index, while the drug-sensitive group had shrunk, a weak refractive index, and a significantly decreased activity of the organoids. Figure 4 This is the ATP detection activity analysis diagram of organoids after drug administration, combined with Figure 3Bright field images were taken using the ATP detection method to detect organoid activity. Compared with the blank control group, the activity of organoids in the sensitive drug treatment group was significantly decreased. Continuous photography and tracking were used, and bright field image analysis combined with chemiluminescence was used to perform qualitative and quantitative analysis of organoid activity. The organoid drug sensitivity results were more accurate and uniform.
[0048] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for detecting drug sensitivity of pancreatic cancer tissue organoids, using pancreatic cancer tissue organoid digestion fluid to culture pancreatic cancer organoids, characterized in that: The steps include: S1. Obtain pancreatic cancer organoid tissue samples, wash the pancreatic cancer tissue organoid samples repeatedly with pre-cooled PBS buffer, cut them into pieces, and add pancreatic cancer tissue organoid digestion solution; S2. Add pre-cooled PBS buffer to terminate the primary digestion, repeatedly blow and beat until the tissue fragments disappear, take out the partially digested solution, and when it is observed as a uniform cell cluster under a microscope, resuspend the cell cluster by low-temperature low-speed centrifugation, count the number of cell clusters under a microscope, and transfer the corresponding cell clusters to a centrifuge tube for centrifugation according to the number of wells to be inoculated; S3, add the enzymatically hydrolyzed cell mass to the basement gel to resuspend it, and inoculate it into the TC cell culture plate. When the basement gel solidifies, add Advanced DMEM / F-12 culture medium to culture it, and observe and change the medium regularly during the period; S4. After culturing the pancreatic cancer tissue organoids for 5-7 days, add pre-cooled PBS buffer to the TC cell culture plate, transfer the base gel to a centrifuge tube, pipette repeatedly, transfer to a refrigerator and let stand, centrifuge and remove the supernatant to obtain pancreatic cancer tissue organoids; S5, enzymatically digesting the pancreatic cancer tissue organoids obtained in step S4 to obtain pancreatic cancer tissue organoid fragments; S6, resuspending the pancreatic cancer tissue organoid fragments obtained in step S5 with basement gel, adjusting the cell concentration to 200-300 cells / 25 μl, inoculating into a 24-well plate, and adding the culture medium of the pancreatic cancer tissue organoid after the basement gel solidifies; S7, resuspending the pancreatic cancer tissue organoid fragments obtained in step S6 with basement gel, adjusting the cell concentration to 30-50 cells / 5 μl, inoculating into a 96-well plate, and adding the culture medium of the pancreatic cancer tissue organoid after the basement gel solidifies; S8, completely removing the culture medium of the pancreatic cancer tissue organoids in step S7, adding culture medium containing pancreatic cancer tissue organoids with different drug concentrations, selecting the whole-well field of view to continuously take pictures after adding the drug, and recording the morphology and size of the organoids; S9. At the 72nd hour of drug action, add the culture medium containing pancreatic cancer tissue organoids with different drug concentrations again; after 120 hours of drug action on organoids, discard the culture medium, add the ATP detection kit, and detect the activity of the organoids.
2. The method for detecting drug sensitivity of pancreatic cancer tissue organoids according to claim 1, characterized in that: In S1, the digestion conditions are: 37° C., shaking digestion for 10-20 min.
3. The method for detecting drug sensitivity of pancreatic cancer tissue organoids according to claim 1, characterized in that: In said S3, the TC cell culture plate is selected from one of 6-well, 12-well, 24-well and 48-well plates.
4. The method for detecting drug sensitivity of pancreatic cancer tissue organoids according to claim 1, characterized in that: In S4, the temperature of the refrigerator is set to 4°C.
5. The method for detecting drug sensitivity of pancreatic cancer tissue organoids according to claim 1, characterized in that: In S5, the obtained organoid fragments were enzymatically digested using TrypLE Express.
6. The method for detecting drug sensitivity of pancreatic cancer tissue organoids according to claim 5, characterized in that: The enzymatic digestion operation is as follows: add 1-2 ml of TrypLE Express, digest for 2-3 min, digest while blowing, add 6-8 ml of pre-cooled PBS buffer, centrifuge at 300 g for 5 min, and terminate the enzymatic digestion.