Novel operation method for performing drug sensitivity test on breast cancer organoid

Abstract

The invention discloses a novel operation method for performing drug sensitivity test on breast cancer organoid, and particularly relates to the technical field of breast cancer organoid drug determination, which comprises the following steps: step 1, obtaining a fresh enough breast cancer tissue sample through needle biopsy; 2, immediately putting the tissue sample into a sterile container containing a culture medium, keeping the activity of cells, and gently washing the sample with sterile normal saline to remove bloodstains and pollutants on the surface; and step 3, cutting the tissue sample into small blocks to facilitate subsequent digestion and separation, digesting at 37 DEG C by using digestive enzymes until the tissue blocks are dispersed into cell clusters, and performing cell activity detection by using a trypan blue staining method to ensure that the activity and quantity of cells meet experimental requirements. According to the invention, the test precision of the drug sensitivity test on the breast cancer organoid is high enough, and the test is carried out in seven concentration gradients at an interval of 25 [mu] M, so that the test result is accurate enough, and the subsequent use of drugs is facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of breast cancer organoid drug determination, and more specifically, to a new operating method for performing drug sensitivity testing on breast cancer organoids. Background Art

[0002] The drug sensitivity test of breast cancer organoids is carried out by constructing patient-derived breast cancer organoids to simulate the tumor microenvironment in the patient's body, and then evaluating the inhibitory effects of different drugs on the growth or activity of organoids. This test method can reflect the patient's sensitivity to drugs and provide a basis for precision treatment. The existing methods for drug sensitivity testing of breast cancer organoids are not accurate enough, resulting in inaccurate test results, which affects the subsequent use of drugs.

[0003] Therefore, a new operating method for drug sensitivity testing of breast cancer organoids is urgently needed to solve the above problems. Summary of the invention

[0004] In order to overcome the above-mentioned defects of the prior art, an embodiment of the present invention provides a new operating method for drug sensitivity testing of breast cancer organoids. The test accuracy of the drug sensitivity testing of breast cancer organoids of the present invention is high enough, and the test is divided into seven concentration gradients separated by 25 μM, so that the test results are accurate enough, which is helpful for the subsequent use of drugs to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a new operation method for drug sensitivity testing of breast cancer organoids, comprising the following steps: Step 1: Obtain a sufficient amount of fresh breast cancer tissue sample through puncture biopsy; Step 2: Immediately place the tissue sample in a sterile container containing culture medium to maintain cell activity, and gently rinse the sample with sterile saline to remove blood and contaminants on the surface; Step 3: Cut the tissue sample into small pieces to facilitate subsequent digestion and separation. Use digestive enzymes to digest at 37°C until the tissue blocks are dispersed into cell clusters. Use trypan blue staining to detect cell activity to ensure that the viability and number of cells meet the experimental requirements. Step 4: After digestion is completed, the digestion solution is removed by centrifugation, and the cells are resuspended in culture medium to prepare a single-cell suspension for subsequent organoid culture. The treated cell suspension is tested for microbial contamination to ensure that there is no bacterial or fungal contamination; Step 5: Record in detail the sample collection time, location, patient information, and key steps and parameters in the sample processing process to ensure the traceability and repeatability of the experiment; Step 6: Select Matrigel matrix gel to simulate the in vivo microenvironment and provide support for the formation of organoids. Inoculate the treated breast cancer cell clusters on the matrix gel to ensure that the cells are evenly distributed and have a moderate density. Step 7: According to the growth requirements of breast cancer organoids, select a culture medium containing essential nutrients, growth factors and hormones, maintain the culture environment temperature at 37°C, CO2 concentration at 5%, and maintain appropriate humidity to simulate the physiological conditions in vivo; Step 8: Regularly replace the culture medium, remove metabolites and supplement nutrients. Regularly use a microscope to observe the morphological characteristics of the organoids to ensure that their structure is similar to that of native breast cancer tissue. When the organoids grow to a certain extent, perform a subculture operation, that is, disperse the organoids into individual small pieces, re-inoculate them on new matrix gel for continued culture, and regularly test the culture for contamination to ensure that there is no contamination by bacteria, fungi and other microorganisms. Record the growth of the organoids in detail, including morphological changes and growth rate, for subsequent analysis; Step 9: Select the drug to be tested and set seven concentration gradients of 50μM, 75μM, 100μM, 125μM, 150μM, 175μM and 200μM to comprehensively evaluate the effect of the drug on organoids at different concentrations. Set up a control group without drug treatment to accurately evaluate the drug effect. Step 10: Select a transparent, sterile, and non-toxic culture plate to ensure the normal growth and observation of organoids. When plating, control the density of organoids to avoid overcrowding or sparseness to ensure the normal growth of organoids and the accuracy of drug sensitivity testing. Step 11: The drug is diluted according to a predetermined concentration gradient and evenly added to the culture plate; Step 12: Assess cell viability by measuring ATP levels in organoids, non-destructively image organoids using a high-resolution microscope to observe morphological changes and growth of organoids, stain organoids with AOPI fluorescent dyes, and observe the distribution of live and dead cells using a fluorescent microscope; Step 13: Calculate the IC50 value using linear regression method based on drug concentration and corresponding cell viability data, collect inhibition rate data at different drug concentrations, calculate the average inhibition rate at each concentration, organize the concentrations and corresponding average inhibition rates into a table, and draw a scatter plot with the logarithm of drug concentration as the horizontal axis and the inhibition rate as the vertical axis. On the scatter plot, find the best fitting straight line so that it passes through and is as close to all data points as possible. Through linear regression analysis, find the slope (a) and intercept (b) of the straight line, and then use the IC50 calculation formula IC50=10^((0.5b) / a) to calculate the IC50 value, and analyze the inhibitory effects of different concentrations of drugs on organoid growth or activity.

[0006] In a preferred embodiment, the digestive enzyme in step three is trypsin.

[0007] Technical effects and advantages of the present invention: The present invention obtains a fresh and sufficient amount of breast cancer tissue samples by puncture biopsy, immediately puts the tissue samples into a sterile container containing a culture medium to maintain the activity of the cells, and gently rinses the samples with sterile physiological saline to remove blood stains and contaminants on the surface, cuts the tissue samples into small pieces for subsequent digestion and separation, uses digestive enzymes to digest at 37°C until the tissue blocks are dispersed into cell clusters, uses trypan blue staining to detect cell activity to ensure that the activity and number of cells meet the experimental requirements, removes the digestion solution by centrifugation after digestion, and adds culture medium to resuspend the cells to prepare a single cell suspension for subsequent organoid culture, performs microbial contamination detection on the treated cell suspension to ensure that there is no bacterial or fungal contamination, and records the sample in detail. The time, location, patient information, and key steps and parameters in the sample processing process were collected to ensure the traceability and repeatability of the experiment. Matrigel was selected to simulate the in vivo microenvironment and provide support for the formation of organoids. The treated breast cancer cell clusters were inoculated on the matrix gel to ensure uniform cell distribution and moderate density. According to the growth requirements of breast cancer organoids, a culture medium containing essential nutrients, growth factors, and hormones was selected. The culture environment temperature was maintained at 37°C, the CO2 concentration was 5%, and the humidity was maintained appropriately to simulate the in vivo physiological conditions. The culture medium was replaced regularly to remove metabolites and supplement nutrients. The morphological characteristics of the organoids were observed regularly under a microscope to ensure that their structure was similar to that of native breast cancer tissue. When the organoids grow to a certain size, they are subcultured, that is, the organoids are dispersed into small individual pieces and re-seeded on new matrix gel for continued culture. The cultures are regularly tested for contamination to ensure that there is no contamination from bacteria, fungi, and other microorganisms. The growth of the organoids, including morphological changes and growth rate, is recorded in detail for subsequent analysis. The drugs to be tested are selected, and seven concentration gradients of 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 175 μM, and 200 μM are set to comprehensively evaluate the effects of drugs on organoids at different concentrations. A control group without drug treatment is set up to accurately evaluate the drug effect. Transparent, sterile, and non-toxic culture plates are selected to ensure the normal growth and observation of the organoids. , control the density of organoids to avoid overcrowding and oversparseness, ensure the normal growth of organoids and the accuracy of drug sensitivity detection, dilute the drug according to a predetermined concentration gradient and evenly add it to the culture plate, evaluate cell viability by measuring the ATP level in the organoids, use a high-resolution microscope to non-destructively image the organoids, observe the morphological changes and growth of the organoids, use AOPI fluorescent dye to stain the organoids, observe the distribution of live and dead cells under a fluorescent microscope, calculate the IC50 value based on the drug concentration and the corresponding cell viability data using a linear regression method, and analyze the inhibitory effect of different concentrations of drugs on the growth or activity of the organoids. The present invention has a sufficiently high test accuracy for drug sensitivity testing of breast cancer organoids,The test was divided into seven concentration gradients separated by 25 μM, so that the test results were accurate enough to facilitate the subsequent use of drugs. DETAILED DESCRIPTION

[0008] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0009] Example 1 The present invention provides a new operation method for performing drug sensitivity testing on breast cancer organoids, comprising the following steps: Step 1: Obtain a sufficient amount of fresh breast cancer tissue sample through puncture biopsy; Step 2: Immediately place the tissue sample in a sterile container containing culture medium to maintain cell activity, and gently rinse the sample with sterile saline to remove blood and contaminants on the surface; Step 3: Cut the tissue sample into small pieces to facilitate subsequent digestion and separation. Use digestive enzymes to digest at 37°C until the tissue blocks are dispersed into cell clusters. Use trypan blue staining to detect cell activity to ensure that the viability and number of cells meet the experimental requirements. Step 4: After digestion is completed, the digestion solution is removed by centrifugation, and the cells are resuspended in culture medium to prepare a single-cell suspension for subsequent organoid culture. The treated cell suspension is tested for microbial contamination to ensure that there is no bacterial or fungal contamination; Step 5: Record in detail the sample collection time, location, patient information, and key steps and parameters in the sample processing process to ensure the traceability and repeatability of the experiment; Step 6: Select Matrigel matrix gel to simulate the in vivo microenvironment and provide support for the formation of organoids. Inoculate the treated breast cancer cell clusters on the matrix gel to ensure that the cells are evenly distributed and have a moderate density. Step 7: According to the growth requirements of breast cancer organoids, select a culture medium containing essential nutrients, growth factors and hormones, maintain the culture environment temperature at 37°C, CO2 concentration at 5%, and maintain appropriate humidity to simulate the physiological conditions in vivo; Step 8: Regularly replace the culture medium, remove metabolites and supplement nutrients. Regularly use a microscope to observe the morphological characteristics of the organoids to ensure that their structure is similar to that of native breast cancer tissue. When the organoids grow to a certain extent, perform a subculture operation, that is, disperse the organoids into individual small pieces, re-inoculate them on new matrix gel for continued culture, and regularly test the culture for contamination to ensure that there is no contamination by bacteria, fungi and other microorganisms. Record the growth of the organoids in detail, including morphological changes and growth rate, for subsequent analysis; Step 9: Select the drug to be tested and set seven concentration gradients of 50μM, 75μM, 100μM, 125μM, 150μM, 175μM and 200μM to comprehensively evaluate the effect of the drug on organoids at different concentrations. Set up a control group without drug treatment to accurately evaluate the drug effect. Step 10: Select a transparent, sterile, and non-toxic culture plate to ensure the normal growth and observation of organoids. When plating, control the density of organoids to avoid overcrowding or sparseness to ensure the normal growth of organoids and the accuracy of drug sensitivity testing. Step 11: The drug is diluted according to a predetermined concentration gradient and evenly added to the culture plate; Step 12: Assess cell viability by measuring ATP levels in organoids, non-destructively image organoids using a high-resolution microscope to observe morphological changes and growth of organoids, stain organoids with AOPI fluorescent dyes, and observe the distribution of live and dead cells using a fluorescent microscope; Step 13: Calculate the IC50 value using linear regression method based on drug concentration and corresponding cell viability data, collect inhibition rate data at different drug concentrations, calculate the average inhibition rate at each concentration, organize the concentrations and corresponding average inhibition rates into a table, and draw a scatter plot with the logarithm of drug concentration as the horizontal axis and the inhibition rate as the vertical axis. On the scatter plot, find the best fitting straight line so that it passes through and is as close to all data points as possible. Through linear regression analysis, find the slope (a) and intercept (b) of the straight line, and then use the IC50 calculation formula IC50=10^((0.5b) / a) to calculate the IC50 value, and analyze the inhibitory effects of different concentrations of drugs on organoid growth or activity.

[0010] The digestion enzyme in step three is set to be trypsin.

[0011] The present invention obtains a fresh and sufficient amount of breast cancer tissue samples by puncture biopsy, immediately puts the tissue samples into a sterile container containing a culture medium to maintain the activity of the cells, and gently rinses the samples with sterile physiological saline to remove blood stains and contaminants on the surface, cuts the tissue samples into small pieces for subsequent digestion and separation, uses digestive enzymes to digest at 37°C until the tissue blocks are dispersed into cell clusters, uses trypan blue staining to detect cell activity to ensure that the activity and number of cells meet the experimental requirements, removes the digestion solution by centrifugation after digestion, and adds culture medium to resuspend the cells to prepare a single cell suspension for subsequent organoid culture, performs microbial contamination detection on the treated cell suspension to ensure that there is no bacterial or fungal contamination, and records the sample in detail. The time, location, patient information, and key steps and parameters in the sample processing process were collected to ensure the traceability and repeatability of the experiment. Matrigel was selected to simulate the in vivo microenvironment and provide support for the formation of organoids. The treated breast cancer cell clusters were inoculated on the matrix gel to ensure uniform cell distribution and moderate density. According to the growth requirements of breast cancer organoids, a culture medium containing essential nutrients, growth factors, and hormones was selected. The culture environment temperature was maintained at 37°C, the CO2 concentration was 5%, and the humidity was maintained appropriately to simulate the in vivo physiological conditions. The culture medium was replaced regularly to remove metabolites and supplement nutrients. The morphological characteristics of the organoids were observed regularly under a microscope to ensure that their structure was similar to that of native breast cancer tissue. When the organoids grow to a certain size, they are subcultured, that is, the organoids are dispersed into small individual pieces and re-seeded on new matrix gel for continued culture. The cultures are regularly tested for contamination to ensure that there is no contamination from bacteria, fungi, and other microorganisms. The growth of the organoids, including morphological changes and growth rate, is recorded in detail for subsequent analysis. The drugs to be tested are selected, and seven concentration gradients of 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 175 μM, and 200 μM are set to comprehensively evaluate the effects of drugs on organoids at different concentrations. A control group without drug treatment is set up to accurately evaluate the drug effect. Transparent, sterile, and non-toxic culture plates are selected to ensure the normal growth and observation of the organoids. , control the density of organoids to avoid overcrowding and oversparseness, ensure the normal growth of organoids and the accuracy of drug sensitivity detection, dilute the drug according to a predetermined concentration gradient and evenly add it to the culture plate, evaluate cell viability by measuring the ATP level in the organoids, use a high-resolution microscope to non-destructively image the organoids, observe the morphological changes and growth of the organoids, use AOPI fluorescent dye to stain the organoids, observe the distribution of live and dead cells under a fluorescent microscope, calculate the IC50 value based on the drug concentration and the corresponding cell viability data using a linear regression method, and analyze the inhibitory effect of different concentrations of drugs on the growth or activity of the organoids. The present invention has a sufficiently high test accuracy for drug sensitivity testing of breast cancer organoids,The test was divided into seven concentration gradients separated by 25 μM, so that the test results were accurate enough to facilitate the subsequent use of drugs.

[0012] Finally: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A new method for drug sensitivity testing of breast cancer organoids, characterized in that: The following steps are involved: Step 1: Obtain a sufficient amount of fresh breast cancer tissue sample through puncture biopsy; Step 2: Immediately place the tissue sample in a sterile container containing culture medium to maintain cell activity, and gently rinse the sample with sterile saline to remove blood and contaminants on the surface; Step 3: Cut the tissue sample into small pieces to facilitate subsequent digestion and separation. Use digestive enzymes to digest at 37°C until the tissue blocks are dispersed into cell clusters. Use trypan blue staining to detect cell activity to ensure that the viability and number of cells meet the experimental requirements. Step 4: After digestion is completed, the digestion solution is removed by centrifugation, and the cells are resuspended in culture medium to prepare a single-cell suspension for subsequent organoid culture. The treated cell suspension is tested for microbial contamination to ensure that there is no bacterial or fungal contamination; Step 5: Record in detail the sample collection time, location, patient information, and key steps and parameters in the sample processing process to ensure the traceability and repeatability of the experiment; Step 6: Select Matrigel matrix gel to simulate the in vivo microenvironment and provide support for the formation of organoids. Inoculate the treated breast cancer cell clusters on the matrix gel to ensure that the cells are evenly distributed and have a moderate density. Step 7: According to the growth requirements of breast cancer organoids, select a culture medium containing essential nutrients, growth factors and hormones, maintain the culture environment temperature at 37°C, CO2 concentration at 5%, and maintain appropriate humidity to simulate the physiological conditions in vivo; Step 8: Regularly replace the culture medium, remove metabolites and supplement nutrients. Regularly use a microscope to observe the morphological characteristics of the organoids to ensure that their structure is similar to that of native breast cancer tissue. When the organoids grow to a certain extent, perform a subculture operation, that is, disperse the organoids into individual small pieces, re-inoculate them on new matrix gel for continued culture, and regularly test the culture for contamination to ensure that there is no contamination by bacteria, fungi and other microorganisms. Record the growth of the organoids in detail, including morphological changes and growth rate, for subsequent analysis; Step 9: Select the drug to be tested and set seven concentration gradients of 50μM, 75μM, 100μM, 125μM, 150μM, 175μM and 200μM to comprehensively evaluate the effect of the drug on organoids at different concentrations. Set up a control group without drug treatment to accurately evaluate the drug effect. Step 10: Select a transparent, sterile, and non-toxic culture plate to ensure the normal growth and observation of organoids. When plating, control the density of organoids to avoid overcrowding or sparseness to ensure the normal growth of organoids and the accuracy of drug sensitivity testing. Step 11: The drug is diluted according to a predetermined concentration gradient and evenly added to the culture plate; Step 12: Assess cell viability by measuring ATP levels in organoids, non-destructively image organoids using a high-resolution microscope to observe morphological changes and growth of organoids, stain organoids with AOPI fluorescent dyes, and observe the distribution of live and dead cells using a fluorescent microscope; Step 13: Calculate the IC50 value using linear regression method based on drug concentration and corresponding cell viability data, collect inhibition rate data at different drug concentrations, calculate the average inhibition rate at each concentration, organize the concentrations and corresponding average inhibition rates into a table, and draw a scatter plot with the logarithm of drug concentration as the horizontal axis and the inhibition rate as the vertical axis. On the scatter plot, find the best fitting straight line so that it passes through and is as close to all data points as possible. Through linear regression analysis, find the slope (a) and intercept (b) of the straight line, and then use the IC50 calculation formula IC50=10^((0.5b) / a) to calculate the IC50 value, and analyze the inhibitory effects of different concentrations of drugs on organoid growth or activity.

2. A novel method for drug sensitivity testing of breast cancer organoids according to claim 1, characterized in that: The digestion enzyme in step three is set to be trypsin.