Kidney organoid culture medium and application thereof

Abstract

The invention discloses a kidney organoid culture medium and application thereof, and relates to the field of organoid culture. The culture medium comprises a basic culture medium and specific addition factors, wherein the specific addition factors comprise N-acetylcysteine, nicotinamide, an ALK-5 inhibitor, a p38MAPK inhibitor, EGF (Epidermal Growth Factor) protein and FGF10 (Fibroblast Growth Factor 10) protein. The culture medium is used for culturing kidney organoid, the organoid formation rate is high, the research value is high, and the application prospect is wide.

Description

Technical Field

[0001] The present invention relates to the field of organoid culture, and particularly to a kidney organoid culture medium and its application. Background Art

[0002] An organoid refers to a micro-organ formed by inoculating pluripotent stem cells, adult stem cells, progenitor cells or tumor cells into Matrigel for in vitro culture, through cell self-proliferation, directed differentiation, lineage commitment and self-assembly. Organoids can partially or even completely restore the cell composition and structural function of in vivo organs, and maintain genetic and phenotypic stability during long-term expansion, with a high tissue memory of restoring in vivo tissues and organs and a strong self-assembly ability, thus having broad application prospects.

[0003] When culturing kidney organoids from kidney tissue cells, the organoid formation rate is low, and it is difficult to obtain kidney organoids with multi-lineage characteristics of nephrons and ureteric buds, with a large difference from the biological characteristics of the human kidney. The kidney organoid disease model constructed therefrom cannot truly reproduce the occurrence and development of kidney diseases.

[0004] Therefore, developing a kidney organoid culture medium with a high organoid formation rate and a small difference from the biological characteristics of the human kidney is still a technical problem in this field and urgently needs to be solved. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to some extent. To this end, the present invention provides a kidney organoid culture medium and its application. This culture medium is used for culturing kidney organoids, with a high organoid formation rate, high research value and broad application prospects.

[0006] In the first aspect of the present invention, the present invention proposes a culture medium. According to an embodiment of the present invention, the culture medium includes: a basal medium and a specific additive factor. The specific additive factor includes: N-acetylcysteine, nicotinamide, an ALK-5 inhibitor, a p38 MAPK inhibitor, an EGF protein and an FGF10 protein. The culture medium according to the embodiment of the present invention is used for culturing kidney organoids, with a high organoid formation rate, high research value and broad application prospects.

[0007] According to an embodiment of the present invention, the specific additive factor further includes: an R-Spondin protein. Thereby, it promotes the growth of kidney organoids.

[0008] According to an embodiment of the present invention, the concentration of the R-Spondin protein is 100-900 g / mL; preferably 500 ng / mL. The inventors obtained the above preferred protein concentration through a large number of experiments. Thereby, the kidney organoid formation rate is further increased.

[0009] According to an embodiment of the present invention, the specific additive factor further includes: a GSK-3β inhibitor. The inventors have found through research that the GSK-3β inhibitor plays an important role in obtaining kidney organoids with multi-lineage characteristics of nephrons and ureteric buds.

[0010] According to an embodiment of the present invention, the GSK-3β inhibitor is CHIR99021. The inventors further found through research that this GSK-3β inhibitor has better effects on the formation of nephrons and ureteric bud multi-lineage characteristics in kidney organoids.

[0011] According to an embodiment of the present invention, the concentration of CHIR99021 is 1 - 5 μM; preferably 1 μM. The inventors determined this optimal concentration through a large number of optimization experiments.

[0012] According to an embodiment of the present invention, the ALK-5 inhibitor is A83-01. The inventors found through continuous experiments that when the culture medium contains this additive, it can improve the formation rate of kidney organoids and promote the formation of nephron and ureteric bud multi-lineage characteristics.

[0013] According to an embodiment of the present invention, the concentration of A83-01 is 0.2 - 2 μM; preferably 1 μM. The inventors determined this optimal concentration through a large number of optimization experiments.

[0014] According to an embodiment of the present invention, the p38 MAPK inhibitor is SB202190. The inventors found through continuous experiments that when the culture medium contains this additive, it can improve the formation rate of kidney organoids and promote the formation of nephron and ureteric bud multi-lineage characteristics.

[0015] According to an embodiment of the present invention, the concentration of SB202190 is 0.2 - 2 μM; preferably 1 μM. The inventors determined this optimal concentration through a large number of optimization experiments.

[0016] According to an embodiment of the present invention, the concentration of N-acetylcysteine is 0.5 - 2.5 mM; preferably 1.25 mM. The inventors determined this optimal concentration through a large number of optimization experiments. When the N-acetylcysteine in the culture medium is within this concentration range, it can achieve cell expansion and long-term subculture of organoids.

[0017] According to an embodiment of the present invention, the concentration of nicotinamide is 5 - 15 mM; preferably 10 mM. The inventors determined this optimal concentration through a large number of optimization experiments. When the nicotinamide in the culture medium is within this concentration range, it can achieve cell expansion and long-term subculture of organoids.

[0018] According to an embodiment of the present invention, the concentration of the EGF protein is 10-100 ng / mL; preferably 50 ng / mL. Through a large number of optimization experiments, the inventor determined this optimal concentration. When the EGF protein in the culture medium is within this concentration range, cell expansion and long-term subculture of the organoids can be achieved.

[0019] According to an embodiment of the present invention, the concentration of the FGF10 protein is 10-100 ng / mL; preferably 100 ng / mL. Through a large number of optimization experiments, the inventor determined this optimal concentration. When the FGF10 protein in the culture medium is within this concentration range, cell expansion and long-term subculture of the organoids can be achieved.

[0020] According to an embodiment of the present invention, the basal medium is DMEM / F12 serum-free medium. When the basal medium is DMEM / F12 serum-free medium, cell expansion and long-term subculture of the organoids can be achieved.

[0021] According to an embodiment of the present invention, the basal medium further comprises: a hydrogen ion buffer, penicillin, and streptomycin.

[0022] According to an embodiment of the present invention, the hydrogen ion buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

[0023] According to an embodiment of the present invention, the concentration of the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid is 5-15 mM; preferably 10 mM. Through a large number of optimization experiments, the inventor determined this optimal concentration. When the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid in the culture medium is within this concentration range, cell expansion and long-term subculture of the organoids can be achieved.

[0024] According to an embodiment of the present invention, the concentration of penicillin is 20-70 U / mL, and the concentration of streptomycin is 0.02-0.07 mg / mL. Through a large number of optimization experiments, the inventor determined this optimal concentration.

[0025] According to an embodiment of the present invention, the basal medium further comprises: a B27 additive.

[0026] According to an embodiment of the present invention, the concentration of the B27 additive is 1-2% by volume. Through a large number of optimization experiments, the inventor determined this optimal concentration.

[0027] In a second aspect of the present invention, the present invention provides a kidney organoid culture medium. According to an embodiment of the present invention, the kidney organoid culture medium comprises: a basal medium and a specific additive factor, and the specific additive factor comprises: N-acetylcysteine, nicotinamide, an ALK-5 inhibitor, a p38 MAPK inhibitor, EGF protein, FGF10 protein, R-Spondin protein, a GSK-3β inhibitor; wherein, the ALK-5 inhibitor is A83-01, the p38 MAPK inhibitor is SB202190, and the GSK-3β inhibitor is CHIR99021; the basal culture medium comprises: DMEM / F12 serum-reduced medium, a hydrogen ion buffer, penicillin, streptomycin, and a B27 additive. The culture medium according to the embodiment of the present invention is used for culturing kidney organoids, and the obtained organoids have a high formation rate and are kidney organoids with multi-lineage characteristics of nephrons and ureteric buds, and have a small difference in biological characteristics from human kidneys, and are used for constructing a kidney organoid library with a high number of passages.

[0028] According to an embodiment of the present invention, the hydrogen ion buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

[0029] According to an embodiment of the present invention, based on the total volume of the kidney organoid culture medium, the kidney organoid culture medium comprises the basal medium and the specific additive factor at the following concentrations: 98% to 99% by volume of the DMEM / F12 serum-reduced medium, 5 to 15 mM of the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 20 to 70 U / mL of the penicillin, 0.02 to 0.07 mg / mL of the streptomycin, 1% to 2% by volume of the B27 additive, 0.5 to 2.5 mM of the N-acetylcysteine, 5 to 15 mM of the nicotinamide, 0.2 to 2 μM of the A83-01, 0.2 to 2 μM of the SB202190, 1 to 5 μM of the CHIR99021, 10 to 100 ng / mL of the EGF protein, 10 to 100 ng / mL of the FGF10 protein, and 100 to 900 μg / mL of the R-Spondin protein. When the components in the culture medium are within the above concentration ranges, cell expansion, organoid formation, and long-term subculture of kidney organoids can be achieved.

[0030] According to an embodiment of the present invention, based on the total volume of the kidney organoid culture medium, the kidney organoid culture medium comprises the following concentrations of the basal medium and specific additive factors: 99% by volume of the DMEM / F12 serum-free medium, 10 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 50 U / mL of penicillin, 0.05 mg / mL of streptomycin, 1% by volume of the B27 additive, 1.25 mM of N-acetylcysteine, 10 mM of nicotinamide, 1 μM of A83-01, 1 μM of SB202190, 1 μM of CHIR99021, 50 ng / mL of EGF protein, 100 ng / mL of FGF10 protein, and 500 ng / mL of R-Spondin protein. When the components in the culture medium are within the above-mentioned preferred concentration ranges, cell expansion, organoid formation, and long-term subculture of kidney organoids can be achieved.

[0031] In a third aspect of the present invention, the present invention provides the use of the foregoing culture medium in constructing kidney organoids or kidney organoid disease models.

[0032] Those skilled in the art can understand that the characteristics and advantages described above for the culture medium also apply to this use and will not be elaborated herein.

[0033] In a fourth aspect of the present invention, the present invention provides a method for constructing kidney organoids. According to an embodiment of the present invention, the method includes: culturing kidney tissue cells in the foregoing culture medium to obtain the kidney organoids.

[0034] Those skilled in the art can understand that the characteristics and advantages described above for the culture medium also apply to this method and will not be elaborated herein.

[0035] In a fifth aspect of the present invention, the present invention provides a method for constructing a kidney organoid disease model. The method includes:

[0036] Obtaining kidney organoids according to the foregoing method; subjecting the kidney organoids to gene editing treatment to obtain the kidney organoid disease model.

[0037] Those skilled in the art can understand that the characteristics and advantages described above for the culture medium also apply to this use and will not be elaborated herein. The obtained kidney organoid disease model has a high consistency in histopathological structure and cellular and molecular characteristics with the animal from which the cells for kidney organoid culture are derived. Thus, it can provide a more accurate research model for the occurrence, development, and treatment of human-related diseases, as well as drug development.

[0038] According to an embodiment of the present invention, the gene editing treatment includes: gene silencing or gene knockout treatment or gene overexpression treatment.

[0039] According to an embodiment of the present invention, the gene includes: a disease-related gene. In some specific embodiments, the dosage form-related gene is a pathogenic gene or a disease marker gene.

[0040] According to an embodiment of the present invention, the gene silencing or gene knockout treatment includes: digesting the kidney organoids to obtain single kidney organoid cells, introducing a nucleic acid with gene silencing or gene knockout activity or a nucleic acid construct containing the same into the single kidney organoid cells to obtain single kidney organoid cells after gene silencing or gene knockout, wherein the nucleic acid targets the disease-related gene; culturing the single kidney organoid cells after gene silencing or gene knockout to obtain the kidney organoid disease model.

[0041] According to an embodiment of the present invention, the single kidney organoid cells after gene silencing or gene knockout are cultured in the aforementioned medium to obtain the kidney organoid disease model.

[0042] According to an embodiment of the present invention, the nucleic acid includes: at least one of shRNA, siRNA, and sgRNA.

[0043] According to an embodiment of the present invention, the disease is nephroblastoma.

[0044] According to an embodiment of the present invention, the gene editing treatment is gene silencing treatment, and the disease-related gene is at least one of WTX, P53, WT1, and Trim28.

[0045] According to an embodiment of the present invention, the disease-related gene is WTX and / or P53.

[0046] According to an embodiment of the present invention, the nucleic acid is shRNA.

[0047] According to an embodiment of the present invention, the shRNA has the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence having at least 90% sequence homology thereto and having the activity of silencing the WTX gene.

[0048] According to an embodiment of the present invention, the shRNA has the nucleotide sequence shown in SEQ ID NO: 2 or a nucleotide sequence having at least 90% sequence homology thereto and having the activity of silencing the P53 gene.

[0049] According to an embodiment of the present invention, the shRNA has a nucleotide sequence as shown in SEQ ID NO: 3 or a nucleotide sequence having at least 90% sequence homology thereto and having the activity of silencing the WT1 gene.

[0050] According to an embodiment of the present invention, the shRNA has a nucleotide sequence as shown in SEQ ID NO: 4 or a nucleotide sequence having at least 90% sequence homology thereto and having the activity of silencing the Trim28 gene.

[0051] According to an embodiment of the present invention, the nucleic acid construct is a viral vector.

[0052] According to an embodiment of the present invention, the viral vector is a non-pathogenic viral vector.

[0053] According to an embodiment of the present invention, the non-pathogenic viral vector is selected from one of a retroviral vector, a poxvirus vector, a herpes simplex virus I vector, a lentivirus vector, an adenovirus vector, and an adeno-associated virus vector.

[0054] According to an embodiment of the present invention, the non-pathogenic viral vector is a lentiviral vector.

[0055] According to an embodiment of the present invention, the infection titer of the lentiviral vector is 10 7 ~10 9 TU / mL.

[0056] In the sixth aspect of the present invention, the present invention provides a method for constructing an animal disease model. According to an embodiment of the present invention, the method includes: obtaining a kidney organoid disease model according to the foregoing method, and transplanting the kidney organoid disease model into the animal to obtain the animal disease model.

[0057] Those skilled in the art can understand that the features and advantages described above for the culture medium also apply to this method and will not be repeated here. The animal disease model obtained thereby can more accurately simulate the occurrence and development process of the disease in the human body, which is beneficial to disease treatment and drug development.

[0058] According to an embodiment of the present invention, the animals include: mice, rats, guinea pigs, rabbits, cats, dogs, monkeys, pigs, or sheep.

[0059] In the seventh aspect of the present invention, a method for constructing a kidney organoid library or a kidney organoid disease model library is proposed. The method includes: obtaining kidney organoids according to the foregoing method or obtaining a kidney organoid disease model according to the foregoing method, subculturing the kidney organoids or the kidney organoid disease model to obtain subcultured kidney organoids or a kidney organoid disease model, and cryopreserving the subcultured kidney organoids or the kidney organoid disease model to obtain the kidney organoid library or the kidney organoid disease model library.

[0060] Those skilled in the art can understand that the features and advantages described above for the culture medium also apply to this method and will not be elaborated here.

[0061] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0062] 1. The culture medium of the present invention is suitable for culturing kidney organoids, and the organoid formation rate is high.

[0063] 2. The culture medium of the present invention is conducive to the formation of kidney organoids with multi-lineage characteristics of nephrons and ureteric buds.

[0064] 3. The culture medium of the present invention is particularly suitable for constructing kidney organoids from human kidney tissue cells. Further, by means of gene editing, a kidney organoid disease model can be constructed. According to the embodiments of the present invention, through gene silencing technology, a human nephroblastoma organoid occurrence model has been successfully constructed. This nephroblastoma organoid has the same genetic epigenetics and disease occurrence characteristics as real nephroblastoma tissue, and can be used as an effective model for the research, drug development, and treatment of nephroblastoma (especially infantile nephroblastoma), with high application value.

[0065] The additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0067] Figure 1 It is the bright-field inverted microscope image of kidney organoids after culturing human kidney tissue cells in M1 - M9 culture medium for 6 days (D6) and 12 days (D12) in Example 1 of the present invention, scale bar: 200μm;

[0068] Figure 2 It is the result graph of the corresponding organoid formation rate of kidney organoids cultured in M1 - M9 culture medium in Example 1 of the present invention;

[0069] Figure 3 This is the mRNA expression quantification result diagram of the kidney organoids obtained by culturing in M8 - M9 medium in Example 1 of the present invention;

[0070] Figure 4 This is the bright - field inverted microscope image of the human kidney organoids from the 1st to the 5th passage in Example 2 of the present invention, scale bar: 200μm;

[0071] Figure 5 This is the mRNA expression quantification result diagram of the kidney organoids obtained by culturing in M9 medium and passaged 5 times in Example 2 of the present invention;

[0072] Figure 6 This is the bright - field image of four human nephroblastoma occurrence models and human kidney organoids obtained by culturing after lentiviral transfection of shRNA to knockdown WT1, WTX, Trim28, and P53 genes respectively in Example 3 of the present invention. Among them, Control represents human kidney organoids without gene - editing treatment, WT1 - KD represents the human nephroblastoma occurrence model with WT1 gene knockdown, WTX - KD represents the human nephroblastoma occurrence model with WTX gene knockdown, Trim28 - KD represents the human nephroblastoma occurrence model with Trim28 gene knockdown, and P53 - KD represents the human nephroblastoma occurrence model with P53 gene knockdown;

[0073] Figure 7 This is the statistical result diagram of the organoid amplification area of four human nephroblastoma occurrence models and human kidney organoids obtained by culturing after lentiviral transfection of shRNA to knockdown WT1, WTX, Trim28, and P53 genes respectively in Example 3 of the present invention. Among them, Control represents human kidney organoids without gene - editing treatment, WT1 - KD represents the human nephroblastoma occurrence model with WT1 gene knockdown, WTX - KD represents the human nephroblastoma occurrence model with WTX gene knockdown, Trim28 - KD represents the human nephroblastoma occurrence model with Trim28 gene knockdown, and P53 - KD represents the human nephroblastoma occurrence model with P53 gene knockdown;

[0074] Figure 8 This is the diagram of the growth of tumor - like masses in the kidneys of mice after orthotopic transplantation of the nephroblastoma occurrence model mice in Example 4 of the present invention;

[0075] Figure 9 This is the result diagram of the source - specific analysis of the tumor - like masses in mice after orthotopic transplantation of the nephroblastoma occurrence model mice in Example 4 of the present invention. Detailed implementation manners

[0076] Embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as a limitation of the present invention.

[0077] It should be noted that the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, the meaning of "a plurality" is two or more.

[0078] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0079] Terms and Definitions

[0080] To make it easier to understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0081] In this document, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified by the present invention, but does not exclude other aspects.

[0082] In this document, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may or may not occur, and this description includes the cases where the events or conditions occur and the cases where the events or conditions do not occur.

[0083] In this document, the term "FBS" is an abbreviation for "Fetal Bovine Serum" and is equivalent to "fetal bovine serum".

[0084] In this document, the term "serum-free medium of DMEM / F12" is Advanced DMEM / F12.

[0085] In this document, the reagent "penicillin-streptomycin double antibody" is a penicillin-streptomycin mixture of 100-fold concentration stock solution. The recommended working concentration of penicillin in cell culture is 100 U / mL, and the working concentration of streptomycin is 0.1 mg / mL. The penicillin-streptomycin mixture of 100-fold concentration stock solution can be diluted 100-fold.

[0086] In this text, the term "nucleic acid" is equivalent to "polynucleotide", "isolated nucleic acid", and can be obtained by synthetic methods.

[0087] Methods for introducing genes into cells and expressing genes in cells are known in the art. Vectors can be easily introduced into host cells by any method in the art, such as mammalian, bacterial, yeast, or insect cells. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0088] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and so on. Biological methods for introducing polynucleotides of interest into host cells include using DNA and RNA vectors. Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including water-in-oil emulsions, micelles, mixed micelles, and liposomes.

[0089] Biological methods for introducing polynucleotides into host cells include using viral vectors, especially retroviral vectors. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. Many virus-based systems have been developed for gene transfer into mammalian cells. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells in vivo or ex vivo. Many retroviral systems are known in the art. Lentiviruses are a genus under the Retroviridae family. Lentiviral vectors are a more complex type of retroviral vector. Reagents for lentiviral packaging are well-known in the art. For example, conventional lentiviral vector systems include pRsv-REV, pMDlg-pRRE, pMD2G, and the target interference plasmid.

[0090] Details of the sequences involved in the present invention are shown in Table 1.

[0091] Table 1 Description of Nucleotide Sequences

[0092]

[0093] The solutions of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. The manufacturers and catalog numbers of the main reagents used are shown in Table 2. Reagents not included in Table 2 and instruments without specified manufacturers are all conventional products that can be obtained commercially.

[0094] Table 2 Major reagent manufacturers and catalog numbers

[0095]

[0096] It should be noted that in the following examples, the "plasmid" and "vector" have the same meaning and can be used interchangeably.

[0097] Example 1: Optimization and screening of kidney organoid medium

[0098] In this example, based on a large number of previous kidney organoid medium developments, the inventors further optimized and screened 9 alternative kidney organoid media. The specific method is as follows:

[0099] 1. Medium preparation

[0100] Prepare M1, M2, M3, M4, M5, M6, M7, M8, and M9 kidney organoid media according to Table 3 respectively.

[0101] Table 3 Component table of M1 - M9 kidney organoid media

[0102]

[0103] 2. Obtaining human kidney tissue samples

[0104] Soak fresh surgically resected kidney specimens in tissue preservation solution and transfer them from the hospital to the laboratory for organoid culture - related experiments.

[0105] Before conducting relevant experiments such as taking samples from surgically resected kidney specimens and culturing kidney organoids, informed consent from the patients was obtained. The patients voluntarily participated in the experiment and signed the informed consent form.

[0106] 3. Isolation of human kidney tissue cells and culture of kidney organoids

[0107] (1) Tissue sample cleaning treatment: Use forceps to hold the kidney tissue and place it in a 1×DPBS (containing penicillin at a final concentration of 50 U / mL and streptomycin at 0.05 mg / mL) cleaning solution, 10 mL each time, and clean 3 times;

[0108] (2) Transfer the cleaned kidney tissue to a sterile culture dish. After removing as much non - epithelial tissue as possible (such as muscle or fat), use a sterilized tissue scissors to cut the tissue into pieces approximately 0.5 - 2 mm 3 in size;

[0109] (3) Wash the chopped kidney tissue again with 1×DPBS (containing penicillin at a final concentration of 50 U / mL and streptomycin at 0.05 mg / mL), then add tissue digestion solution (the formula is shown in Table 4), and perform enzymatic hydrolysis on a shaker at 37°C for 1 to 1.5 hours at 100 rpm. Use pipettes of different sizes (from large to small, 10 mL, 5 mL, and 1 mL) to blow the tissue digestion suspension until the kidney tissue is visibly dispersed. After sufficient digestion, a tissue digestion suspension is obtained;

[0110] Table 4 Tissue digestion solution formula

[0111] Additive reagent Working concentration Advanced DMEM / F12 1× Small molecule compound Y-27632 10 μM Collagenase I 400 U / mL DNase I 10 U / mL

[0112] (4) adding fetal bovine serum at a final concentration of 10% to the digested tissue suspension to protect the cells;

[0113] (5) The tissue digestion suspension was passed through a 100 μm cell sieve to obtain a single-cell suspension, and the cells were washed twice with a basal culture medium (Advanced DMEM / F12) (mixed and centrifuged at 150 g for 3 min to discard the supernatant). For tissue blocks that did not pass through the filter, steps (3) to (5) were collected and repeated. If the obtained single-cell suspension contained a large number of red blood cells, red blood cell lysis solution (hypotonic ammonium chloride solution) could be added to the precipitate before washing to lyse the red blood cells.

[0114] (7) taking a small amount of the washed cell suspension for live cell detection and counting, and adding matrigel at low temperature according to a cell density of 100,000 to 500,000 cells / mL and mixing on ice to obtain matrigel containing cells;

[0115] (8) Place the mixed matrix gel containing cells on ice and inoculate 10 μL / well in a 48-well plate (or 30 μL / well in a 24-well plate);

[0116] (9) Place the 48-well plate (or 24-well plate) seeded with cells in a 37°C incubator and incubate for 20 minutes to solidify the matrix gel;

[0117] (10) After the matrix gel is solidified, slowly add the above-mentioned M1-M9 culture medium (500 μL / well for 24-well plate and 250 μL / well for 48-well plate) along the wall of the cell culture well plate for P0 culture. Replace the culture medium every 3-4 days, culture for about one week, and then continue to culture for 12 days. Take pictures to record the growth of kidney organoids ( Figure 1 ).

[0118] The results showed that after culturing kidney tissue cells in the M4-M9 medium of this example for 6 days, human kidney organoids with complete morphology could be obtained ( Figure 1);The organoid formation rate was statistically analyzed on the 12th day of culture, and it was found that the organoid formation rate of M7 - M9 culture media exceeded 400%. Unexpectedly, the organoid formation rate of M9 culture media was close to 800%( Figure 2 ).

[0119] The organoid formation rate is the proportion of kidney tissue cells separated that successfully differentiate into kidney organoid spheroids. Specifically, it is obtained by statistically analyzing the number and diameter of organoids in each well through software analysis.

[0120] Furthermore, through gene expression profiling analysis, the inventors examined whether the kidney organoids obtained after culturing kidney tissue cells in M8 - M9 culture media with the highest organoid formation rate for 12 days have multi - lineage characteristics of nephrons and ureteric buds.

[0121] Gene expression profiling analyzed the mRNA expression abundances of genes EYA1, GATA3, ABCC4, SLC4A4, SLC12A1, SLC41A3, and NR3C2 in kidney organoid samples. Among them, EYA1 - PC is a key gene for progenitor cell development, GATA3 - UB is a key gene for kidney ureteric bud development, ABCC4 - Proximal is a key gene for kidney proximal tubule development, SLC4A4 - Proximal is a key gene for kidney proximal tubule development, SLC12A1 - LoH is a key gene for nephron loop of Henle development, SLC41A3 - distal is a key gene for kidney distal tubule development, and NR3C2 - CD is a key gene for kidney collecting duct development.

[0122] The gene expression profiling analysis method is as follows: By comparing the expression abundances of key genes for kidney development in organoids under the culture conditions of M8 and M9 culture media, the higher the mRNA transcription level, the higher the expression level of the corresponding gene - encoded protein, and the more perfect the renal function development.

[0123] The results showed that the kidney organoids obtained after culturing kidney tissue cells in M9 culture media for 12 days have multi - lineage characteristics of nephrons and ureteric buds( Figure 3 ).

[0124] Example 2: Construction of a kidney organoid library

[0125] Referring to Example 1, in a 48 - well plate or a 24 - well plate, kidney organoids obtained by culturing kidney tissue cells in M9 culture media for 12 days were used, and then sub - cultured. Organoids with good growth status were selected, and after cryopreservation treatment, they were stored in liquid nitrogen for a long time, thereby constructing a kidney organoid library.

[0126] The operation steps of sub - culturing are as follows:[[]]

[0127] (1) Aspirate the culture medium in the 48-well plate, add pre-cooled 1×DPBS at a volume of 1 mL per well, and let it stand on ice for 5 min;

[0128] (2) Pipette the Matrigel containing organoids in the 48-well plate with a 1 mL pipette tip to detach the Matrigel;

[0129] (3) Transfer the detached Matrigel containing organoids to a 15 mL centrifuge tube, wash the 48-well plate with pre-cooled 1×PBS, collect the washing solution and combine it into the above centrifuge tube;

[0130] (4) Place the 15 mL centrifuge tube on ice and let it stand for 15 min to soften the Matrigel and obtain an organoid suspension;

[0131] (5) Pipette the organoid suspension obtained in step (4) 15 times with a 10 mL pipette to separate the organoids from the Matrigel;

[0132] (6) Centrifuge the organoid suspension treated in step (5) at 4°C and 250 g for 5 min, discard the supernatant and add 10 mL of pre-cooled 1×DPBS to resuspend the organoids and count them;

[0133] (7) Centrifuge again at 4°C and 250 g for 5 min, discard the supernatant and let it stand on ice for 5 min;

[0134] (8) Aspirate the refluxed liquid (residual 1×DPBS) and Matrigel;

[0135] (9) Resuspend the organoids by adding fresh Matrigel at a ratio of 8000 - 10000 cells / 10 μL of Matrigel;

[0136] (10) Drop the organoid-Matrigel suspension at 30 μL per well in the center of the wells of a 24-well plate;

[0137] (11) Invert the 24-well plate and place it in an incubator to solidify the Matrigel for 20 min;

[0138] (12) After the Matrigel solidifies, add 500 μL of pre-warmed M9 medium at 37°C to each well and culture the cells under standard cell culture conditions (cell incubator, 37°C, 5% CO2);

[0139] (13) Replace the M9 medium every 3 - 4 days and culture for 6 - 12 days.

[0140] The operation steps for cryopreservation are as follows:

[0141] (1) Prepare a cell cryopreservation programmable cooling box and equilibrate the temperature at room temperature;

[0142] (2) Select the culture wells with good organoid growth status, discard the culture medium, gently scrape off the mixture of Matrigel and organoids with a micropipette tip, transfer it to a centrifuge tube, pipette to mix well, add 1 mL of 1× DPBS to wash the organoids 1 - 2 times, centrifuge the organoids at 200 g for 5 minutes after thoroughly removing the residual Matrigel, and remove the supernatant to obtain the organoids to be cryopreserved;

[0143] (3) Add 500 μL - 1000 μL of pre-cooled organoid cryopreservation solution to the organoids to be cryopreserved, pipette to mix well and quickly transfer it to a cryogenic storage tube;

[0144] (4) Place the cryogenic storage tube in a cell cryopreservation program cooling box, then quickly place the cell cryopreservation program cooling box in a -80 °C ultra-low temperature freezer, and transfer the storage tube to liquid nitrogen for long-term storage the next day.

[0145] To further verify the effectiveness of the kidney organoid library, the inventor further performed resuscitation treatment and subculture on the kidney organoids cryopreserved in liquid nitrogen.

[0146] The operation steps of the resuscitation treatment are as follows:

[0147] (1) Prepare M9 medium, basal medium (Advanced DMEM / F12) and related reagents in advance, turn on the water bath and set the temperature to 37 °C, and preheat the basal medium. Take out the organoid cryopreservation tube from the liquid nitrogen tank, quickly place the cryopreservation tube in a 37 °C water bath, and shake it from time to time to make it melt as soon as possible. Stop the water bath before the ice completely melts, transfer the organoid cryopreservation suspension to a 15 mL centrifuge tube, and slowly add 5 times the volume of preheated basal medium at 37 °C to dilute the cryopreservation suspension;

[0148] (2) Centrifuge the mixed suspension of the organoids obtained in step (1) and the cryopreservation suspension diluted with preheated basal medium at 37 °C at 300 g for 3 minutes, thoroughly remove the supernatant, add 1 mL of basal medium to resuspend the organoids and transfer them to a 1.5 mL centrifuge tube for centrifugation, and repeat the washing 1 - 2 times to remove the residual cryopreservation solution;

[0149] (3) Centrifuge at 300 g for 3 minutes to remove the supernatant, add an appropriate amount of Matrigel to resuspend the organoid precipitate and mix well on ice, and place it on ice after mixing;

[0150] (4) Aspirate the mixed suspension of the extracellular matrix and cells and transfer it to a cell culture well plate. For example, add 20 - 30 μL of the mixed suspension to each well of a 24-well cell culture plate, and the mixed suspension must be dropped to the bottom of the culture well;

[0151] (5) Place the 24-well cell culture plate in a 37 °C, 5% carbon dioxide cell culture incubator and let it stand for 15 minutes. After confirming that the extracellular matrix has completely solidified, add M9 medium;

[0152] (6) Place the cell culture plate in a 37 °C, 5% CO2 cell culture incubator for culturing. Observe and take pictures every 1 - 2 days, and change the M9 medium every 2 - 4 days. Culture for 6 - 12 days to obtain kidney organoids.

[0153] Generally, under an inverted microscope, cells with a bright and smooth surface are living cells, while cells that are dull, opaque, and have a rough surface are dead cells or cells with low viability. As the culture time extends, the living cells will gradually grow into 3D organoids.

[0154] The inverted microscope photos of the kidney organoids obtained by the inventor after 5 consecutive resuscitation treatments and passage cultures are as Figure 4 shown.

[0155] The results show that: the kidney organoids obtained by passage culture with M9 medium have a complete morphology and a high organoid formation rate; gene expression profile analysis of the kidney organoids after the 5th passage culture shows that they have multi-lineage characteristics of nephrons and ureteric buds ( Figure 5 ).

[0156] The above results show that the kidney organoids obtained by passage culture of the M9 medium in Example 1 have multi-lineage characteristics of nephrons and ureteric buds, with a complete morphology and a high organoid formation rate.

[0157] Therefore, the M9 medium can be used to construct a kidney organoid library and has a high number of passages.

[0158] Example 3: Construction of a Wilms tumor occurrence model (kidney organoid disease model)

[0159] In this example, gene silencing treatment was performed on WT1, WTX, Trim28, and P53 of kidney cells through gene silencing technology. Then, the gene-silenced kidney cells were cultured into organoids using the M9 medium in Example 1, and the growth status of the organoids was further investigated.

[0160] This example was set up with 5 groups in total, including: a control group, a WT1-KD group, a WTX-KD group, a Trim28-KD group, and a P53-KD group. Among them, the control group infected human kidney cells with lentivirus expressing empty vector, and the WT1-KD group, WTX-KD group, Trim28-KD group, and P5-KD group infected human kidney cells with lentivirus of the corresponding shRNA respectively.

[0161] The specific experimental method is as follows:

[0162] Preparation of lentivirus containing shRNA and infection of human kidney cells:

[0163] (1) The shRNAs of the WT1-KD group, WTX-KD group, Trim28-KD group and P53-KD group were designed on the siDirect website and outsourced for synthesis respectively; the synthesized shRNAs were cloned into the pLKO.1puro vector to obtain the core plasmids. Among them,

[0164] The shRNA targeting the WTX gene has the nucleotide sequence shown in SEQ ID NO:1,

[0165] 5’-ATTGCTGGTGAACTCTACCAG-3’(SEQ ID NO:1);

[0166] The shRNA targeting the P53 gene has the nucleotide sequence shown in SEQ ID NO:2,

[0167] 5’-GACTCCAGTGGTAATCTAC-3’(SEQ ID NO:2);

[0168] Among them, the shRNA targeting the WT1 gene has the nucleotide sequence shown in SEQ ID NO:3,

[0169] 5’-ATGCTTGAATGAGTGGTTGGG-3’(SEQ ID NO:3);

[0170] The shRNA targeting the Trim28 gene has the nucleotide sequence shown in SEQ ID NO:4,

[0171] 5’-TAAGCACAGGTTTGGTCTCAG-3’(SEQ ID NO:4).

[0172] (2) Resuscitate a vial of 293T cells in good condition and culture them in DMEM medium (containing 10% fetal bovine serum) until the cell density reaches 70% - 90% at the time of transfection;

[0173] (3) 1 - 2 hours before transfection, replace the cell medium with DMEM (product number: 12800017, company: Gibco) medium containing 10% fetal bovine serum (product number: F8318, company: Sigma) without antibiotics;

[0174] (4) Take 5 - 8 μg of DNA (starting amount 5 μg), add dilution solution (enzyme - free water) to a total volume of 100 μL, mix gently, and let it stand at room temperature. The core plasmid∶lentiviral packaging plasmid psPAX2 (hereinafter referred to as "PH1")∶lentiviral packaging plasmid pMD2.G (hereinafter referred to as "PH2") are added to a 10 - cm culture dish at a mass ratio of 7∶5∶2. Add 7 μg of the core plasmid, 5 μg of PH1, and 2 μg of PH2 respectively, and then complete plasmid packaging through 293T cells;

[0175] (5) Take 4 μL of eukaryotic transfection reagent (VigoFect) (starting amount 2 μL), add it to dilution solution (enzyme - free water) to a total volume of 100 μL, mix gently, and let it stand at room temperature for 5 minutes. Dropwise add the diluted VigoFect into the diluted DNA solution, mix gently. Let the obtained transfection working solution stand at room temperature for 15 minutes, then gently and evenly drop it into 293T cells, and finally gently shake and mix. Place the 293T cells in an incubator for culture;

[0176] (6) After 16 - 20 hours of transfection, discard the culture medium, replace it with fresh DMEM medium (containing FBS), continue transfection for 36 hours, collect the virus supernatant, centrifuge at 4℃ and 1000 rpm for 10 minutes, and take the supernatant and aliquot it into 1.5 - mL centrifuge tubes. Temporarily store at 4℃ or store long - term at - 80℃;

[0177] (7) After collecting the virus, refer to the method of Example 1, digest the kidney organoids cultured with M9 medium for 5 - 10 minutes, filter through a 40 - μm filter to obtain a single - cell suspension, and place it on ice for later use.

[0178] Prepare the infection medium: Add 100 μL of virus solution and transfection reagent polybrene (10 μg / mL) to M9 medium to obtain a mixed solution with a total volume of 250 μL.

[0179] Virus infection: After resuspending the cell pellet of the single - cell suspension and mixing evenly, add it to a 48 - well plate, seal the well plate with a sealing film. Transfer it to a well - plate centrifuge, centrifuge at 32℃ and 600g for 60 minutes, and then transfer it to a 37℃ incubator for continued culture for 5 - 6 hours. Then centrifuge the kidney cells after 5 - 6 hours of infection at room temperature, centrifuge at 1000 rpm for 3 minutes, and discard the supernatant.

[0180] Organoid culture: Resuspend the cells with 30 μL of Matrigel, and spread the transfected cells onto a 24 - well plate, add 500 μL of M9 medium, and culture in a 37℃, 5% CO2 cell incubator. Change the medium every 3 - 4 days. After 7 days of culture, take microscope photos to observe the growth status of organoids in each group ( Figure 6 )), and use imageJ to statistically calculate the amplified area of the organoids (Figure 7 )。

[0181] Results showed that nephroblastoma organoids were cultured in each experimental group. Among them, the amplification area of the organoids in the WTX-KD group was significantly higher than that in the control group ( Figure 7 ), and the amplification effect was most obvious in the P53-KD group ( Figure 6 ). The diameter of the organoids in the WTX-KD group was significantly better than that in the WT1-KD group, Trim28-KD group, P53-KD group, and control group ( Figure 6 )。

[0182] Example 4: Establishment of a xenograft animal model of nephroblastoma

[0183] To further verify whether the nephroblastoma organoids obtained by gene silencing treatment in Example 3 have tumor characteristics, the inventors orthotopically transplanted the nephroblastoma organoids in the WTX-KD group and P53-KD group of Example 3 under the renal capsule of NSG mice and performed tissue identification and analysis. The specific operation steps are as follows:

[0184] Sixty severely immunodeficient mice of the NOD-Prkdcscid Il2rgem1 / Smoc (M-NSG) strain, 4-7 weeks old and weighing 18-24 g, were selected for the experiment, including a control group, a WTX-KD group, and a P53-KD group, with 20 mice in each group. The treatment for the control group mice was to inject kidney organoids infected with empty lentivirus under the renal capsule of NSG mice. The mice in the WTX-KD group and P53-KD group were subjected to nephroblastoma transplantation, and the kidney organoids infected with shRNA lentivirus in the WTX-KD group and P53-KD group were respectively injected under the renal capsule of NSG mice. The treatment methods are as follows;

[0185] (1) Before inoculation, collect organoids (about 2×10 5 cells), wash and centrifuge them with the culture medium to remove all supernatants and resuspend them in 15 μL of Matrigel, and place them on ice for later use;

[0186] (2) Prepare 1% sodium pentobarbital, inject anesthetic into the abdominal cavity of the mice at a dose of 40 mg / kg. After the mice are anesthetized and breathing smoothly, use sterilized surgical instruments to cut open the abdominal cavity of the mice and fully expose the kidneys;

[0187] (3) Inject the Matrigel and organoid mixture under the renal capsule of the mice, suture the wound, and inject antibiotics into the mice to prevent infection;

[0188] (4) After continuing to raise the experimental mice for 30 days, dissect and observe the tumorigenicity, and analyze the origin of the grafts in the WTX-KD group and P53-KD group mice through immunofluorescence staining experiments using human-derived maker Numa (Figure 9 )。

[0189] The results showed that: (1) In the mice transplanted with the kidney organoids of the WTX-KD group and the P53-KD group in Example 3, obvious cancer lesions appeared in their kidneys, while only local tissue amplification occurred at the injection site in the control group. Figure 8 )。(2) The results of the immunofluorescence staining experiment showed that the grafts in the mice of the WTX-KD group and the P53-KD group both expressed the human maker Numa. Figure 9 ) This indicates that the kidney organoids of the WTX-KD shRNA and P53-KD shRNA lentiviruses successfully mimicked the characteristics of human nephroblastoma.

[0190] The above results showed that the nephroblastoma organoids of Example 3 have tumor characteristics and can be used as an effective model for studying the pathogenesis and development mechanism of nephroblastoma, screening related drugs, and researching treatment regimens.

[0191] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A culture medium, characterized in that, Comprising: A basal medium and specific addition factors, wherein the specific addition factors include: N-acetylcysteine, nicotinamide, an ALK-5 inhibitor, a p38 MAPK inhibitor, EGF protein, and FGF10 protein.

2. The culture medium according to claim 1, characterized in that, The specific addition factors further include: R-Spondin protein; Furthermore, the concentration of the R-Spondin protein is 100 - 900 ng / mL; preferably 500 ng / mL; Optionally, the specific addition factors further include: a GSK-3β inhibitor; Furthermore, the GSK-3β inhibitor is CHIR99021; Even further, the concentration of the CHIR99021 is 1 - 5 μM; preferably 1 μM.

3. The culture medium according to claim 1, wherein The ALK-5 inhibitor is A83-01; Furthermore, the concentration of the A83-01 is 0.2 - 2 μM; preferably 1 μM; Optionally, the p38 MAPK inhibitor is SB202190; Furthermore, the concentration of the SB202190 is 0.2 - 2 μM; preferably 1 μM; Optionally, the concentration of the N-acetylcysteine is 0.5 - 2.5 mM; preferably 1.25 mM; Optionally, the concentration of the nicotinamide is 5 - 15 mM; preferably 10 mM; Optionally, the concentration of the EGF protein is 10 - 100 ng / mL; preferably 50 ng / mL; Optionally, the concentration of the FGF10 protein is 10 - 100 ng / mL; preferably 100 ng / mL.

4. The culture medium according to claim 1, characterized in that, The basal medium is a DMEM / F12 serum-free medium; Optionally, the basal medium further includes: a hydrogen ion buffer, penicillin, and streptomycin; Optionally, the hydrogen ion buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Optionally, the concentration of the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid is 5 - 15 mM; preferably 10 mM; Optionally, the concentration of the penicillin is 20 - 70 U / mL, and the concentration of the streptomycin is 0.02 - 0.07 mg / mL; Optionally, the basal medium further includes: a B27 additive; Optionally, the concentration of the B27 additive is 1 - 2% volume percentage.

5. A kidney organoid culture medium, characterized in that, Comprising: A basal medium and specific addition factors, wherein the specific addition factors include: N-acetylcysteine, nicotinamide, an ALK-5 inhibitor, a p38 MAPK inhibitor, EGF protein, FGF10 protein, R-Spondin protein, a GSK-3β inhibitor; Wherein, the ALK-5 inhibitor is A83-01, the p38 MAPK inhibitor is SB202190, and the GSK-3β inhibitor is CHIR99021; The basal culture includes: a DMEM / F12 serum-free medium, a hydrogen ion buffer, penicillin, streptomycin, and a B27 additive; Optionally, the hydrogen ion buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Optionally, based on the total volume of the kidney organoid medium, the kidney organoid medium includes the basal medium and specific addition factors at the following concentrations: 98% to 99% by volume of the DMEM / F12 serum-reduced medium, 5 to 15 mM of the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 20 to 70 U / mL of the penicillin, 0.1 to 0.2 mg / mL of the streptomycin, 1% to 2% by volume of the B27 additive, 0.5 to 2.5 mM of the N-acetylcysteine, 5 to 15 mM of the nicotinamide, 0.2 to 2 μM of the A83-01, 0.2 to 2 μM of the SB202190, 1 to 5 μM of the CHIR99021, 10 to 100 ng / mL of the EGF protein, 10 to 100 ng / mL of the FGF10 protein, 100 to 900 μg / mL of the R-Spondin protein; Furthermore, based on the total volume of the kidney organoid medium, the kidney organoid medium comprises the following concentrations of the basal medium and specific additive factors: 99% by volume of the DMEM / F12 serum-reduced medium, 10 mM of the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 50 U / mL of the penicillin, 0.05 mg / mL of the streptomycin, 1% by volume of the B27 additive, 1.25 mM of the N-acetylcysteine, 10 mM of the nicotinamide, 1 μM of the A83-01, 1 μM of the SB202190, 1 μM of the CHIR99021, 50 ng / mL of the EGF protein, 100 ng / mL of the FGF10 protein, 500 μg / mL of the R-Spondin protein.

6. Use of the medium according to any one of claims 1 to 5 in constructing a kidney organoid or a kidney organoid disease model.

7. A method for constructing kidney organoids, characterized in that, Comprising: Culturing kidney tissue cells in the medium according to any one of claims 1 to 5 to obtain the kidney organoid.

8. A method for constructing a kidney organoid disease model, characterized in that, Comprising: Obtaining a kidney organoid according to the method of claim 7; Performing gene editing on the kidney organoid to obtain the kidney organoid disease model; Optionally, the gene editing includes: gene silencing or gene knockout or gene overexpression; Optionally, the gene includes: a disease-related gene; Optionally, the gene silencing or gene knockout includes: Digesting the kidney organoid to obtain single kidney organoid cells, Introducing a nucleic acid having gene silencing or gene knockout activity or a nucleic acid construct containing the same into the single kidney organoid cells to obtain single kidney organoid cells after gene silencing or gene knockout, the nucleic acid targeting the disease-related gene; Culturing the single kidney organoid cells after gene silencing or gene knockout to obtain the kidney organoid disease model; Optionally, culturing the single kidney organoid cells after gene silencing or gene knockout in the medium according to any one of claims 1 to 5 to obtain the kidney organoid disease model; Optionally, the nucleic acid comprises at least one of shRNA, siRNA, and sgRNA; Optionally, the disease is Wilms tumor; Optionally, the gene editing treatment is a gene silencing treatment, and the disease-related gene is at least one of WTX, P53, WT1, and Trim28; Optionally, the disease-related gene is WTX and / or P53; Optionally, the nucleic acid is shRNA; Optionally, the shRNA has the nucleotide sequence shown in SEQ ID NO:1 or a nucleotide sequence having at least 90% sequence homology thereto and having the activity of silencing the WTX gene; Optionally, the shRNA has the nucleotide sequence shown in SEQ ID NO:2 or a nucleotide sequence having at least 90% sequence homology thereto and having the activity of silencing the P53 gene; Optionally, the shRNA has the nucleotide sequence shown in SEQ ID NO:3 or a nucleotide sequence having at least 90% sequence homology thereto and having the activity of silencing the WT1 gene; Optionally, the shRNA has the nucleotide sequence shown in SEQ ID NO:4 or a nucleotide sequence having at least 90% sequence homology thereto and having the activity of silencing the Trim28 gene; Optionally, the nucleic acid construct is a viral vector; Optionally, the viral vector is a non-pathogenic viral vector; Optionally, the non-pathogenic viral vector is selected from one of retroviral vectors, poxviral vectors, herpes simplex virus I vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors; Optionally, the non-pathogenic viral vector is a lentiviral vector; Optionally, the lentiviral vector infection titer is 10 7 ~10 9 TU / mL.

9. A method for constructing an animal disease model, characterized in that, Comprising: The kidney organoid disease model obtained by the method according to claim 8, Transplanting the kidney organoid disease model into the animal body to obtain the animal disease model; Optionally, the animal includes: mouse, rat, guinea pig, rabbit, cat, dog, monkey, pig, or sheep.

10. A method for constructing a kidney organoid library or a kidney organoid disease model library, characterized in that, Comprising: The kidney organoid obtained by the method according to claim 7 or the kidney organoid disease model obtained by the method according to claim 8, Subculturing the kidney organoid or the kidney organoid disease model to obtain the subcultured kidney organoid or the kidney organoid disease model, Cryopreserving the subcultured kidney organoid or the kidney organoid disease model to obtain the kidney organoid bank or the kidney organoid disease model bank.

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