Drug toxicity detection method based on kidney organoids

Abstract

The invention discloses a drug toxicity detection method based on kidney organoid. The method comprises the following steps: performing target drug treatment on kidney organoid cultured in advance; determining the estimated toxicity of the target drug based on the morphological difference before and after the drug administration of the kidney organoid; performing cell viability detection on the kidney organoid to obtain the cell viability inhibition ratio of the kidney organoid; and based on the estimated toxicity and the cell activity inhibition ratio, performing molecular pathology detection on the kidney organoid, and determining the real toxicity of the target drug. The toxicity of the target drug is preliminarily estimated according to the morphological difference before and after drug administration of the kidney organoid, then the cell activity inhibition rate of the kidney organoid is obtained, molecular pathology detection is performed on the kidney organoid based on the preliminarily estimated toxicity and the cell activity inhibition rate, and finally the toxicity of the target drug is determined. Through differential gradient classification comparison detection, the toxicity of a target drug is accurately detected.

Description

Technical Field

[0001] The present invention relates to a drug toxicity detection method based on kidney organoids, belonging to the technical field of biomedicine. Background Art

[0002] Drug-induced renal injury, also known as drug-induced renal damage disease, is an adverse drug reaction unrelated to the therapeutic effect caused by the interaction between a certain drug or several drugs. The kidney is the main organ for drug excretion. It has the most contact with drugs during the process of drug intake, transportation, accumulation and excretion, and the drug concentration in the kidney is also high, so drug-induced damage is very likely to occur.

[0003] Current drug nephrotoxicity testing results are often simply classified as toxic or non-toxic, lacking differential gradient classification comparisons and failing to accurately evaluate the drug's own molecular mechanisms. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies in the prior art and to provide a drug toxicity detection method based on kidney organoids. Through differential gradient classification and comparative detection, accurate detection of the toxicity of the target drug can be achieved, solving the problems of low reliability and low accuracy of current drug renal toxicity detection.

[0005] In order to achieve the above purpose / solve the above technical problems, the present invention is implemented by adopting the following technical solutions: A drug toxicity detection method based on kidney organoids, comprising: Treat kidney organoids that have been cultured from pluripotent stem cells with targeted drugs; Determine the estimated toxicity of the target drug based on the morphological differences of kidney organoids before and after administration; Conduct cell viability tests on kidney organoids to obtain the cell activity inhibition rate of kidney organoids; Based on the estimated toxicity and the cell activity inhibition rate, molecular pathology testing is performed on the kidney organoids to determine the actual toxicity of the target drug.

[0006] Furthermore, the method for culturing kidney organoids pre-cultured based on pluripotent stem cells comprises the following steps: (1) Preparing pluripotent stem cells into single-cell suspension; (2) Inoculate the single cell suspension onto a 24-well plate coated with a lactose dehydrogenase-free virus-enhanced low-growth factor basement membrane matrix and culture for 3 days; (3) Transfer the cell suspension in step (2) to a culture medium containing L-glutamine, CHIR99021, Dorsomorphin and Noggin protein factors, and culture the medium every 2-3 days until the cell morphology changes into loose dense cell clusters; (4) Resuspend the dense cell clusters in step (3) and inoculate them into a 96-well plate for static culture for 2-3 days; (5) converting the culture medium in step (4) into a culture medium containing L-glutamine and fibroblast growth factor-9, and culturing for 3-5 days; (6) Obtain cultured kidney organoids.

[0007] Furthermore, the step of preparing the pluripotent stem cells into a single cell suspension comprises the following steps: (1.1) Inoculate pluripotent stem cells into 6-well plates coated with lactose dehydrogenase-free virus-low growth factor basement membrane matrix and add feeder-free culture medium containing FGF factor; (1.2) Subculture the cells in step (1.1) every 5-7 days, and replace the feeder-free medium containing fresh FGF every 2-3 days; (1.3) When the cells in step (1.2) show a round colony morphology with clear boundaries and the cell density has expanded to 80-90%, add Accutase cell digestion solution and resuspend in medium containing Y27632; (1.4) to obtain a single cell suspension.

[0008] Furthermore, the estimated toxicity of the target drug is determined based on the morphological differences of the kidney organoids before and after administration, including: Performing bright-field three-dimensional imaging on the kidney organoid before drug administration to obtain first bright-field three-dimensional imaging data; After the preset drug administration cycle of the kidney organoid ends, performing bright field three-dimensional imaging of the drug-administered kidney organoid to obtain second bright field three-dimensional imaging data; Obtaining an area change of the kidney organoid according to the first bright field three-dimensional imaging data and the second bright field three-dimensional imaging data; Determine the estimated toxicity of the target drug based on the area changes of kidney organoids.

[0009] Furthermore, the estimated toxicity of the target drug is determined based on the area change of the kidney organoid, including: If the area change of the kidney organoid is ≤-35%, the target drug is estimated to be nephrotoxic; If -35% < area change of kidney organoids ≤ 25%, the target drug is estimated to have mild nephrotoxicity; If the area change of the kidney organoid is 25% ≤, the target drug is estimated to have no nephrotoxicity.

[0010] Furthermore, based on the estimated toxicity and the cell activity inhibition rate, molecular pathology detection is performed on the kidney organoid to determine the actual toxicity of the target drug, including: If the target drug is estimated to be nephrotoxic and the renal organoid cell activity inhibition rate after administration is greater than 60%, the target drug is determined to be toxic. If the target drug is estimated to be nephrotoxic and the renal organoid cell activity inhibition rate after administration is ≤60%, the target drug is estimated to be toxic, and molecular pathology testing is performed on the renal organoid to determine the true toxicity of the target drug; If the target drug is estimated to have mild nephrotoxicity and the renal organoid cell activity inhibition rate after administration is greater than 60%, the target drug is estimated to be toxic, and molecular pathology testing is performed on the renal organoid to determine the true toxicity of the target drug; if the target drug is estimated to have mild nephrotoxicity and the renal organoid cell activity inhibition rate after administration is ≤60%, the target drug is estimated to be non-toxic, and molecular pathology testing is performed on the renal organoid to determine the true toxicity of the target drug; If the target drug is estimated to have no nephrotoxicity and the inhibition rate of kidney organoid cell activity after administration is greater than 60%, the target drug is estimated to be toxic, and molecular pathology testing is performed on the kidney organoid to determine the true toxicity of the target drug. If the target drug is estimated to have no nephrotoxicity and the inhibition rate of kidney organoid cell activity after administration is ≤60%, the target drug is determined to be non-toxic.

[0011] Furthermore, based on the estimated toxicity and the cell activity inhibition rate, molecular pathology detection is performed on the kidney organoid to determine the actual toxicity of the target drug, including: The renal organoids after drug administration were labeled with fluorescent probes to determine the changes in oxidative stress in the renal organoids after drug administration; Obtaining the creatinine content of the kidney organoids after administration, and determining changes in creatinine secretion of the kidney organoids after administration; Obtain the content of human kidney injury molecule 1 in kidney organoids after administration, and determine the changes in the release of kidney injury factors in kidney organoids after administration; Based on the estimated toxicity and the cell activity inhibition rate, combined with the changes in oxidative stress, creatinine secretion and renal injury factor release in the kidney organoids after administration, the actual toxicity of the target drug is determined.

[0012] Furthermore, the method of using fluorescent probes to label kidney organoids after administration to determine changes in oxidative stress in kidney organoids after administration includes: The relative oxidative stress ratio of kidney organoids after drug administration was calculated using the following formula: in: It represents the relative fluorescence value of kidney organoids after treatment with target drugs, and the relative oxidative stress ratio is reflected by the relative fluorescence value; Represents the fluorescence value of kidney organoids after treatment with target drugs, represents the fluorescence value of kidney organoids without target drug treatment; Based on the relative oxidative stress ratio, the changes in oxidative stress in renal organoids after drug administration were obtained.

[0013] Furthermore, obtaining the creatinine content of the kidney organoid after administration and determining the change in creatinine secretion of the kidney organoid after administration include: The relative creatinine-protein content ratio of kidney organoids after administration was calculated using the following formula: in: Represents the relative creatinine protein content of kidney organoids after treatment with the target drug. The relative creatinine protein content ratio is reflected by the relative creatinine protein content. represents the amount of creatinine protein in kidney organoids after treatment with the target drug, represents the amount of creatinine protein in kidney organoids not treated with target drugs; Based on the relative creatinine-protein content ratio, the changes in creatinine secretion of kidney organoids after administration were obtained.

[0014] Furthermore, obtaining the human kidney injury molecule 1 content of the kidney organoid after administration and determining the changes in the release of kidney injury factors in the kidney organoid after administration include: The relative renal injury molecular ratio of kidney organoids after administration was calculated using the following formula: in: It indicates the relative ratio of Kim protein in kidney organoids after treatment with target drug. The relative renal injury molecular ratio is reflected by the relative ratio of Kim protein. Represents the amount of Kim protein in kidney organoids after treatment with the target drug, represents the amount of Kim protein in kidney organoids not treated with target drugs; Based on the relative renal injury molecule ratio, the changes in the release of renal injury factors in the renal organoids after administration were obtained.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention preliminarily estimates the toxicity of the target drug based on the morphological differences of kidney organoids before and after administration, and then obtains the cell activity inhibition rate of the kidney organoids. Based on the preliminarily estimated toxicity and cell activity inhibition rate, the kidney organoids are subjected to molecular pathology testing to ultimately determine the toxicity of the target drug. Through differential gradient classification and comparative testing, the toxicity of the target drug can be accurately detected, which solves the problem of low reliability and low accuracy of current drug renal toxicity testing.

[0016] 2. The present invention uses kidney organoids as drug receptors, which can simulate kidney function from the molecular level to the cellular, tissue, and organ levels, which is closer to the real environment in the body, thereby more accurately reflecting the effects of drugs on the kidneys and evaluating the potential toxicity of drugs to the kidneys.

[0017] 3. After preliminarily estimating the toxicity of the target drug, the present invention combines the cell activity inhibition rate and the changes in oxidative stress, creatinine secretion, and renal injury factor release of kidney organoids after administration, and uses differential gradient classification and comparative detection at the molecular level to further determine the renal toxicity of the target drug. The test results are accurate and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a flow chart of a drug toxicity detection method based on kidney organoids provided in an embodiment of the present invention; Figure 2 It is a schematic diagram of the detection results of a drug toxicity detection method based on kidney organoids provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention. Example

[0020] like Figure 1 As shown, a drug toxicity detection method based on kidney organoids comprises: Treat kidney organoids that have been cultured from pluripotent stem cells with targeted drugs; Based on the morphological differences of kidney organoids before and after administration, the estimated toxicity of the target drug is determined, including: Performing bright-field three-dimensional imaging on the kidney organoid before drug administration to obtain first bright-field three-dimensional imaging data; After the administration of the kidney organoid, after the preset administration cycle ends, performing bright field three-dimensional imaging on the administered kidney organoid to obtain second bright field three-dimensional imaging data; Obtaining an area change of the kidney organoid according to the first bright field three-dimensional imaging data and the second bright field three-dimensional imaging data; Based on the changes in the area of ​​kidney organoids, the estimated toxicity of the target drug was determined, specifically: If the area change of the kidney organoid is ≤-35%, the target drug is estimated to be nephrotoxic; If -35% < area change of kidney organoids ≤ 25%, the target drug is estimated to have mild nephrotoxicity; If the area change of the kidney organoid is 25% ≤, the target drug is estimated to have no nephrotoxicity.

[0021] Perform cell viability test on kidney organoids to obtain the cell activity inhibition rate of kidney organoids, including: Cell viability was detected using the CellTiter-Glo® Luminescent Cell Viability Assay Kit. The number of live cells was detected by quantitative determination of ATP. The cell activity inhibition rate of kidney organoids was calculated using the following formula:

[0022] Represents the cell activity inhibition rate of kidney organoids, Represents the cell viability value of kidney organoids after treatment with target drugs, represents the fluorescence value of kidney organoids without target drug treatment; Based on the estimated toxicity and cell activity inhibition rate, molecular pathology testing is performed on kidney organoids to determine the true toxicity of the target drug, including: If the cell bioactivity inhibition rate is >60% and the target drug is estimated to have mild or no nephrotoxicity, molecular pathology testing will be performed on the kidney organoids to determine the true toxicity of the target drug; If the cell bioactivity inhibition rate is ≤60% and the target drug is estimated to be nephrotoxic, molecular pathology testing will be performed on the kidney organoids to determine the true toxicity of the target drug; If the cell biological activity inhibition rate is >60% and the target drug is estimated to be nephrotoxic, the target drug is determined to be nephrotoxic; The renal organoids after administration were labeled with fluorescent probes to determine the changes in oxidative stress of the renal organoids after administration. Specifically, the relative oxidative stress ratio of the renal organoids after administration was calculated using the following formula: in: It represents the relative fluorescence value of kidney organoids after treatment with target drugs, and the relative oxidative stress ratio is reflected by the relative fluorescence value; Represents the fluorescence value of kidney organoids after treatment with target drugs, represents the fluorescence value of kidney organoids without target drug treatment; Obtain the creatinine content of the kidney organoids after administration and determine the changes in creatinine secretion of the kidney organoids after administration, including: The relative creatinine-protein content ratio of kidney organoids after administration was calculated using the following formula: in: Represents the relative creatinine protein content of kidney organoids after treatment with the target drug. The relative creatinine protein content ratio is reflected by the relative creatinine protein content. represents the amount of creatinine protein in kidney organoids after treatment with the target drug, represents the amount of creatinine protein in kidney organoids not treated with target drugs; Obtain the content of human kidney injury molecule 1 in kidney organoids after administration, and determine the changes in the release of kidney injury factors in kidney organoids after administration, including: The relative renal injury molecular ratio of kidney organoids after administration was calculated using the following formula: in: It indicates the relative ratio of Kim protein amount in kidney organoids after treatment with target drug. The relative renal injury molecular ratio is reflected by the relative ratio of Kim protein amount. Represents the amount of Kim protein in kidney organoids after treatment with the target drug, represents the amount of Kim protein in kidney organoids not treated with target drugs; Based on the estimated toxicity and cell activity inhibition rate, combined with the changes in oxidative stress, creatinine secretion, and renal injury factor release in kidney organoids after administration, the true toxicity of the target drug was determined. Specifically: like Figure 2 As shown, this example tests the nephrotoxicity of drugs A, B, C, D, E, and F; If the target drug is estimated to be nephrotoxic and the renal organoid cell activity inhibition rate after administration is ≤60%, then the target drug is estimated to be toxic: If the changes in oxidative stress, creatinine secretion, and renal injury factor release of the renal organoids after administration meet 2-3 of the following preset judgment conditions, the target drug is determined to have nephrotoxicity; if 0-1 of the following preset judgment conditions is met, the target drug is determined to have low nephrotoxicity; If the target drug is estimated to have mild nephrotoxicity and the renal organoid cell activity inhibition rate after administration is greater than 60%, then the target drug is estimated to be toxic: If the changes in oxidative stress, creatinine secretion, and renal injury factor release of the renal organoids after administration meet 2-3 of the following preset judgment conditions, the target drug is determined to have nephrotoxicity; if it meets 1 of the following preset judgment conditions, the target drug is determined to have low nephrotoxicity; if it meets 0 of the following preset judgment conditions, the target drug is determined to have no nephrotoxicity; If the target drug is estimated to have mild nephrotoxicity and the renal organoid cell activity inhibition rate after administration is ≤60%, then the target drug is estimated to be non-toxic: If the changes in oxidative stress, creatinine secretion, and renal injury factor release of the renal organoids after administration meet 2-3 of the following preset judgment conditions, the target drug is determined to have nephrotoxicity; if it meets 1 of the following preset judgment conditions, the target drug is determined to have low nephrotoxicity; if it meets 0 of the following preset judgment conditions, the target drug is determined to have no nephrotoxicity; If the target drug is estimated to be non-nephrotoxic and the renal organoid cell activity inhibition rate after administration is >60%, the target drug is estimated to be toxic: If the changes in oxidative stress, creatinine secretion, and renal injury factor release of the renal organoids after administration meet 2-3 of the following preset judgment conditions, the target drug is determined to have nephrotoxicity; if it meets 1 of the following preset judgment conditions, the target drug is determined to have low nephrotoxicity; if it meets 0 of the following preset judgment conditions, the target drug is determined to have no nephrotoxicity; The preset judgment conditions include: relative oxidative stress ratio>2, relative creatinine-protein content ratio>2, and relative renal injury molecule ratio>2.

[0023] Embodiment 2 like Figure 1 As shown, a drug toxicity detection method based on kidney organoids comprises: The kidney organoids that have been cultured in advance based on pluripotent stem cells are treated with a target drug, wherein the culture method of the pre-cultured kidney organoids comprises the following steps: (1) Prepare human pluripotent stem cells into a single cell suspension, specifically: (1.1) Inoculate human pluripotent stem cells into 6-well plates coated with lactose dehydrogenase-free virus-low growth factor basement membrane matrix and add feeder-free culture medium containing FGF factor; (1.2) Subculture the cells in step (1.1) every 5-7 days, and replace the feeder-free medium containing fresh FGF every 2-3 days; (1.3) When the cells in step (1.2) show a round colony morphology with clear boundaries and the cell density has expanded to 80-90%, add Accutase cell digestion solution and resuspend in medium containing Y27632; (1.4) Obtain a single cell suspension; (2) Inoculate the single cell suspension onto a 24-well plate coated with a lactose dehydrogenase-free virus-enhanced low-growth factor basement membrane matrix and culture for 3 days; (3) Transfer the cell suspension in step (2) to a culture medium containing L-glutamine, CHIR99021, Dorsomorphin and Noggin protein factors, and culture the medium every 2-3 days until the cell morphology changes into loose dense cell clusters; (4) Resuspend the dense cell clusters in step (3) and inoculate them into a 96-well plate for static culture for 2-3 days; (5) converting the culture medium in step (4) into a culture medium containing L-glutamine and fibroblast growth factor-9, and culturing for 3-5 days; (6) Obtain cultured kidney organoids.

[0024] Based on the morphological differences of kidney organoids before and after administration, the estimated toxicity of the target drug is determined, including: Performing bright-field three-dimensional imaging on the kidney organoid before drug administration to obtain first bright-field three-dimensional imaging data; After the administration of the kidney organoid, after the preset administration cycle ends, performing bright field three-dimensional imaging on the administered kidney organoid to obtain second bright field three-dimensional imaging data; Obtaining an area change of the kidney organoid according to the first bright field three-dimensional imaging data and the second bright field three-dimensional imaging data; Based on the changes in the area of ​​kidney organoids, the estimated toxicity of the target drug was determined, specifically: If the area change of the kidney organoid is ≤-35%, the target drug is estimated to be nephrotoxic; If -35% < area change of kidney organoids ≤ 25%, the target drug is estimated to have mild nephrotoxicity; If the area change of the kidney organoid is 25% ≤, the target drug is estimated to have no nephrotoxicity.

[0025] Perform cell viability test on kidney organoids to obtain the cell activity inhibition rate of kidney organoids, including: Cell viability was detected using the CellTiter-Glo® Luminescent Cell Viability Assay Kit. The number of live cells was detected by quantitative determination of ATP. The cell activity inhibition rate of kidney organoids was calculated using the following formula:

[0026] Represents the cell activity inhibition rate of kidney organoids, Represents the cell viability value of kidney organoids after treatment with target drugs, represents the fluorescence value of kidney organoids without target drug treatment; Based on the estimated toxicity and cell activity inhibition rate, molecular pathology testing is performed on kidney organoids to determine the true toxicity of the target drug, including: If the cell bioactivity inhibition rate is >60% and the target drug is estimated to have mild or no nephrotoxicity, molecular pathology testing will be performed on the kidney organoids to determine the true toxicity of the target drug; If the cell bioactivity inhibition rate is ≤60% and the target drug is estimated to be nephrotoxic, molecular pathology testing will be performed on the kidney organoids to determine the true toxicity of the target drug; If the cell biological activity inhibition rate is >60% and the target drug is estimated to be nephrotoxic, the target drug is determined to be nephrotoxic; The renal organoids after administration were labeled with fluorescent probes to determine the changes in oxidative stress of the renal organoids after administration. Specifically, the relative oxidative stress ratio of the renal organoids after administration was calculated using the following formula:

[0027] in: It represents the relative fluorescence value of kidney organoids after treatment with target drugs, and the relative oxidative stress ratio is reflected by the relative fluorescence value; Represents the fluorescence value of kidney organoids after treatment with target drugs, represents the fluorescence value of kidney organoids without target drug treatment; Obtain the creatinine content of the kidney organoids after administration and determine the changes in creatinine secretion of the kidney organoids after administration, including: The relative creatinine-protein content ratio of kidney organoids after administration was calculated using the following formula:

[0028] in: Represents the relative creatinine protein content of kidney organoids after treatment with the target drug. The relative creatinine protein content ratio is reflected by the relative creatinine protein content. represents the amount of creatinine protein in kidney organoids after treatment with the target drug, represents the amount of creatinine protein in kidney organoids not treated with target drugs; Obtain the content of human kidney injury molecule 1 in kidney organoids after administration, and determine the changes in the release of kidney injury factors in kidney organoids after administration, including: The relative renal injury molecular ratio of kidney organoids after administration was calculated using the following formula: in: It indicates the relative ratio of Kim protein in kidney organoids after treatment with target drug. The relative renal injury molecular ratio is reflected by the relative ratio of Kim protein. Represents the amount of Kim protein in kidney organoids after treatment with the target drug, represents the amount of Kim protein in kidney organoids not treated with target drugs; Based on the estimated toxicity and cell activity inhibition rate, combined with the changes in oxidative stress, creatinine secretion, and renal injury factor release in kidney organoids after administration, the true toxicity of the target drug was determined. Specifically: like Figure 2 As shown, this example tests the nephrotoxicity of drugs A, B, C, D, E, and F; If the target drug is estimated to be nephrotoxic and the renal organoid cell activity inhibition rate after administration is ≤60%, then the target drug is estimated to be toxic: If the changes in oxidative stress, creatinine secretion, and renal injury factor release of the renal organoids after administration meet 2-3 of the following preset judgment conditions, the target drug is determined to have nephrotoxicity; if 0-1 of the following preset judgment conditions is met, the target drug is determined to have low nephrotoxicity; If the target drug is estimated to have mild nephrotoxicity and the renal organoid cell activity inhibition rate after administration is greater than 60%, then the target drug is estimated to be toxic: If the changes in oxidative stress, creatinine secretion, and renal injury factor release of the renal organoids after administration meet 2-3 of the following preset judgment conditions, the target drug is determined to have nephrotoxicity; if it meets 1 of the following preset judgment conditions, the target drug is determined to have low nephrotoxicity; if it meets 0 of the following preset judgment conditions, the target drug is determined to have no nephrotoxicity; If the target drug is estimated to have mild nephrotoxicity and the renal organoid cell activity inhibition rate after administration is ≤60%, then the target drug is estimated to be non-toxic: If the changes in oxidative stress, creatinine secretion, and renal injury factor release of the renal organoids after administration meet 2-3 of the following preset judgment conditions, the target drug is determined to have nephrotoxicity; if it meets 1 of the following preset judgment conditions, the target drug is determined to have low nephrotoxicity; if it meets 0 of the following preset judgment conditions, the target drug is determined to have no nephrotoxicity; If the target drug is estimated to be non-nephrotoxic and the renal organoid cell activity inhibition rate after administration is >60%, the target drug is estimated to be toxic: If the changes in oxidative stress, creatinine secretion, and renal injury factor release of the renal organoids after administration meet 2-3 of the following preset judgment conditions, the target drug is determined to have nephrotoxicity; if it meets 1 of the following preset judgment conditions, the target drug is determined to have low nephrotoxicity; if it meets 0 of the following preset judgment conditions, the target drug is determined to have no nephrotoxicity; The preset judgment conditions include: relative oxidative stress ratio>2, relative creatinine-protein content ratio>2, and relative renal injury molecule ratio>2.

[0029] In the description of the present invention, it is to be understood that the terms "first", "second", etc. are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first", "second", etc. may explicitly or implicitly include one or more of the features. In the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0030] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for drug toxicity detection based on kidney organoids, characterized in that: include: Treat kidney organoids that have been cultured from pluripotent stem cells with targeted drugs; Determine the estimated toxicity of the target drug based on the morphological differences of kidney organoids before and after administration; Conduct cell viability tests on kidney organoids to obtain the cell activity inhibition rate of kidney organoids; Based on the estimated toxicity and the cell activity inhibition rate, molecular pathology testing is performed on the kidney organoids to determine the actual toxicity of the target drug.

2. The drug toxicity detection method based on kidney organoids according to claim 1, characterized in that: The method for culturing kidney organoids pre-cultured based on pluripotent stem cells comprises the following steps: (1) Preparing pluripotent stem cells into single-cell suspension; (2) Inoculate the single cell suspension onto a 24-well plate coated with a lactose dehydrogenase-free virus-enhanced low-growth factor basement membrane matrix and culture for 3 days; (3) Transfer the cell suspension in step (2) to a culture medium containing L-glutamine, CHIR99021, Dorsomorphin and Noggin protein factors, and culture the medium every 2-3 days until the cell morphology changes into loose dense cell clusters; (4) Resuspend the dense cell clusters in step (3) and inoculate them into a 96-well plate for static culture for 2-3 days; (5) converting the culture medium in step (4) into a culture medium containing L-glutamine and fibroblast growth factor-9, and culturing for 3-5 days; (6) Obtain cultured kidney organoids.

3. The method for drug toxicity detection based on kidney organoids according to claim 2, characterized in that: The method of preparing the pluripotent stem cells into a single cell suspension comprises the following steps: (1.1) Inoculate pluripotent stem cells into 6-well plates coated with lactose dehydrogenase-free virus-low growth factor basement membrane matrix and add feeder-free culture medium containing FGF factor; (1.2) Subculture the cells in step (1.1) every 5-7 days, and replace the feeder-free medium containing fresh FGF every 2-3 days; (1.3) When the cells in step (1.2) show a round colony morphology with clear boundaries and the cell density has expanded to 80-90%, add Accutase cell digestion solution and resuspend in medium containing Y27632; (1.4) to obtain a single cell suspension.

4. The drug toxicity detection method based on kidney organoids according to claim 1, characterized in that: The estimated toxicity of the target drug is determined based on the morphological differences of kidney organoids before and after administration, including: Performing bright-field three-dimensional imaging on the kidney organoid before drug administration to obtain first bright-field three-dimensional imaging data; After the preset drug administration cycle of the kidney organoid ends, performing bright field three-dimensional imaging of the drug-administered kidney organoid to obtain second bright field three-dimensional imaging data; Obtaining an area change of the kidney organoid according to the first bright field three-dimensional imaging data and the second bright field three-dimensional imaging data; Determine the estimated toxicity of the target drug based on the area changes of kidney organoids.

5. The method for drug toxicity detection based on kidney organoids according to claim 4, characterized in that: The estimated toxicity of the target drug is determined based on the area changes of the kidney organoids, including: If the area change of the kidney organoid is ≤-35%, the target drug is estimated to be nephrotoxic; If -35% < area change of kidney organoids ≤ 25%, the target drug is estimated to have mild nephrotoxicity; If the area change of the kidney organoid is 25% ≤, the target drug is estimated to have no nephrotoxicity.

6. The method for drug toxicity detection based on kidney organoids according to claim 5, characterized in that: Based on the estimated toxicity and the cell activity inhibition rate, molecular pathology detection is performed on the kidney organoid to determine the actual toxicity of the target drug, including: If the target drug is estimated to be nephrotoxic and the renal organoid cell activity inhibition rate after administration is greater than 60%, the target drug is determined to be toxic. If the target drug is estimated to be nephrotoxic and the renal organoid cell activity inhibition rate after administration is ≤60%, the target drug is estimated to be toxic, and molecular pathology testing is performed on the renal organoid to determine the true toxicity of the target drug; If the target drug is estimated to have mild nephrotoxicity and the renal organoid cell activity inhibition rate after administration is greater than 60%, the target drug is estimated to be toxic, and molecular pathology testing is performed on the renal organoid to determine the true toxicity of the target drug; if the target drug is estimated to have mild nephrotoxicity and the renal organoid cell activity inhibition rate after administration is ≤60%, the target drug is estimated to be non-toxic, and molecular pathology testing is performed on the renal organoid to determine the true toxicity of the target drug; If the target drug is estimated to have no nephrotoxicity and the inhibition rate of kidney organoid cell activity after administration is greater than 60%, the target drug is estimated to be toxic, and molecular pathology testing is performed on the kidney organoid to determine the true toxicity of the target drug. If the target drug is estimated to have no nephrotoxicity and the inhibition rate of kidney organoid cell activity after administration is ≤60%, the target drug is determined to be non-toxic.

7. The method for drug toxicity detection based on kidney organoids according to claim 1, characterized in that: Based on the estimated toxicity and the cell activity inhibition rate, molecular pathology detection is performed on the kidney organoid to determine the actual toxicity of the target drug, including: The renal organoids after drug administration were labeled with fluorescent probes to determine the changes in oxidative stress in the renal organoids after drug administration; Obtaining the creatinine content of the kidney organoids after administration, and determining changes in creatinine secretion of the kidney organoids after administration; Obtain the content of human kidney injury molecule 1 in kidney organoids after administration, and determine the changes in the release of kidney injury factors in kidney organoids after administration; Based on the estimated toxicity and the cell activity inhibition rate, combined with the changes in oxidative stress, creatinine secretion and renal injury factor release in the kidney organoids after administration, the actual toxicity of the target drug is determined.

8. The method for drug toxicity detection based on kidney organoids according to claim 7, characterized in that: The method of using fluorescent probes to label kidney organoids after administration to determine changes in oxidative stress in kidney organoids after administration includes: The relative oxidative stress ratio of kidney organoids after drug administration was calculated using the following formula: in: It represents the relative fluorescence value of kidney organoids after treatment with target drugs, and the relative oxidative stress ratio is reflected by the relative fluorescence value; Represents the fluorescence value of kidney organoids after treatment with target drugs, represents the fluorescence value of kidney organoids without target drug treatment; Based on the relative oxidative stress ratio, the changes in oxidative stress in renal organoids after drug administration were obtained.

9. The method for drug toxicity detection based on kidney organoids according to claim 7, characterized in that: The obtaining of the creatinine content of the kidney organoid after administration and determining the change in creatinine secretion of the kidney organoid after administration include: The relative creatinine-protein content ratio of kidney organoids after administration was calculated using the following formula: in: Represents the relative creatinine protein content of kidney organoids after treatment with the target drug. The relative creatinine protein content ratio is reflected by the relative creatinine protein content. represents the amount of creatinine protein in kidney organoids after treatment with the target drug, represents the amount of creatinine protein in kidney organoids not treated with target drugs; Based on the relative creatinine-protein content ratio, the changes in creatinine secretion of kidney organoids after administration were obtained.

10. The method for drug toxicity detection based on kidney organoids according to claim 7, characterized in that: The obtaining of the human kidney injury molecule 1 content of the kidney organoid after administration and determining the changes in the release of kidney injury factors in the kidney organoid after administration include: The relative renal injury molecular ratio of kidney organoids after administration was calculated using the following formula: in: It indicates the relative ratio of Kim protein in kidney organoids after treatment with target drug. The relative renal injury molecular ratio is reflected by the relative ratio of Kim protein. Represents the amount of Kim protein in kidney organoids after treatment with the target drug, represents the amount of Kim protein in kidney organoids not treated with target drugs; Based on the relative renal injury molecule ratio, the changes in the release of renal injury factors in the renal organoids after administration were obtained.

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