Construction of matrigel-free kidney organoid and method for evaluating drug safety by using liver and gall-kidney organoid

Abstract

The invention relates to the technical field of organs, and discloses construction of a matrigel-free kidney organoid and a method for evaluating drug safety by using liver and gall-kidney organoid. By utilizing the culture medium set provided by the invention, the kidney organoid with more complete functions and structures can be constructed through iPSC induction, and matrigel does not need to be adopted in the construction and culture process, so that the construction operation of the organoid is greatly simplified, and the cost is saved. The kidney-like organ constructed by the invention has a basic structure of the kidney, has corresponding response to medicine components which are known in the field and have influence on the kidney, and can be used as a kidney in-vitro model.

Description

Technical Field

[0001] The present invention relates to the technical field of organs, and particularly relates to a method for constructing a matrix-free kidney organoid and evaluating drug safety using hepatobiliary-kidney organoids. Background Art

[0002] With the continuous increase in the incidence and mortality of kidney, urinary, and reproductive diseases, the development of effective in vitro models for in-depth study of their pathogenic mechanisms and drug screening has become increasingly important. Traditional 2D cell models and animal models have shown obvious limitations because they cannot fully simulate the complex microenvironment and cell interactions in the human body. Therefore, promoting the research and development of new in vitro models, especially the application of organoid technology, has become an urgent issue to be solved.

[0003] Organoids are an emerging in vitro model technology. Small in vitro organ models formed by inducing the differentiation of human pluripotent stem cells (hPSCs) can more realistically reproduce the behavior and interactions of cells under physiological conditions. This technology has shown great potential in the modeling, toxicity screening, and drug development of kidney, urinary, and reproductive diseases.

[0004] In the construction and application of organoids, researchers can use this technology to establish kidney organoid models that are closer to human physiological conditions. These models can not only effectively simulate the structure and function of the kidney, but also be used for disease modeling. By observing the performance of organoids under different pathological conditions, it helps researchers deeply understand the disease mechanism.

[0005] However, despite the promising prospects in the construction of kidney organoids, many challenges still exist, including the complexity of the construction method, the diversity of culture medium components, and the non-uniformity of equipment requirements. These factors lead to high costs and inconvenient operations, which are not conducive to their popularization and application in a wider range of fields. Summary of the Invention

[0006] The purpose of the present invention is to overcome the problems existing in the prior art, such as the cumbersome process of constructing kidney organoids, the complex components of the culture medium used, the high culture cost and difficult operation, and the difficulty of the prior art in truly evaluating the toxicity of drugs to the liver and kidney. A method for constructing a matrix-free kidney organoid and evaluating drug safety using hepatobiliary-kidney organoids is provided. The present invention provides a set of culture media composed of multiple culture media with relatively simple components. During the construction process of kidney organoids, the corresponding culture medium is selected according to the construction progress, which is convenient to use, low in cost, and can efficiently construct kidney organoids with multiple cells and stable structures that are easy to observe.

[0007] To achieve the above object, in the first aspect of the present invention, a set of culture media for constructing and culturing kidney organoids is provided. The set of culture media includes the following culture media:

[0008] Medium B: It includes a basal medium and Additive II, wherein the Additive II includes at least one of activin A and hBMP4;

[0009] Medium C: It includes a basal medium and Additive III, wherein the Additive III includes at least one of hBMP4 and CHIR-99021;

[0010] Medium D: It includes a basal medium and Additive IV, wherein the Additive IV includes at least one of retinoic acid, hFGF9, LDN193189, and SB43152;

[0011] Medium E: It includes a basal medium and Additive V, wherein the Additive V includes at least one of retinoic acid, hFGF9, LDN193189, and CHIR-99021;

[0012] Medium F: It includes a basal medium and Additive VI, wherein the Additive VI includes at least one of retinoic acid, hFGF9, LDN193189, CHIR-99021, and hFGF1;

[0013] Medium G: It includes a basal medium and Additive VII, wherein the Additive VII includes at least one of retinoic acid, hFGF9, LDN193189, CHIR-99021, hFGF1, and neurotrophic factor;

[0014] Medium H: It includes a basal medium and Additive VIII, wherein the Additive VIII includes at least one of retinoic acid, LDN193189, CHIR-99021, hFGF1, and neurotrophic factor;

[0015] Medium I: It includes a basal medium and Additive IX, wherein the Additive IX includes at least one of FBS, retinoic acid, LDN193189, hFGF1, neurotrophic factor, R-spondin 1, and hFGF7;

[0016] The basal medium is selected from DMEM / F12 medium containing added components, and the added components include serum-free additive, non-essential amino acids, β-mercaptoethanol, insulin, hTRF, and selenious acid.

[0017] The second aspect of the present invention provides a method for constructing kidney organoids, and the method includes inducing embryoid bodies to form kidney organoids by using the medium set of the first aspect of the present invention.

[0018] The third aspect of the present invention provides a kidney organoid obtained by constructing the method described in the second aspect of the present invention, and the application of the kidney organoid in at least one of drug screening, disease modeling, and toxicological detection.

[0019] The fourth aspect of the present invention provides the application of the culture medium described in the first aspect of the present invention, and / or the method described in the second aspect of the present invention in at least one of improving the success rate of kidney organoid construction, shortening the maturation time of organoids, extending the culture time of kidney organoids, reducing the cost of kidney organoid construction, and increasing the throughput of kidney organoid construction.

[0020] The fifth aspect of the present invention provides a method for detecting drug safety, the method includes contacting a hepatobiliary organoid and a kidney organoid with a drug to be tested respectively, and comprehensively judging the safety of the drug based on the responses of the hepatobiliary organoid and the kidney organoid to the drug, wherein, the kidney organoid is a kidney organoid obtained by constructing the method described in the second aspect of the present invention.

[0021] Through the above technical solutions, the present invention has at least the following beneficial effects:

[0022] (1) The culture medium provided by the present invention has simple components and can be selected according to different culture stages, effectively simplifying the preparation operation of the culture medium for kidney organoid culture while reducing costs, and is suitable for large-scale application.

[0023] (2) The culture medium and method provided by the present invention can effectively induce and obtain kidney organoids that can observe the structure under bright field in a short time, and it has a good response to drugs, and is suitable for in vitro models for research work such as the study of the mechanisms of kidney, urinary, and reproductive diseases and drug screening.

[0024] (3) The method provided by the present invention can form kidney organoids by cell free gravitational sedimentation aggregation and self-assembly without using exogenous additives such as Matrigel, which is convenient for fixing and moving organoids during the experiment, suitable for high-throughput screening, and not using Matrigel can also avoid the adverse effects of steps such as collagenase digestion on the activity of organoids during the experiment, and effectively reduce the batch-to-batch differences of organoids and the cell number gap between individual organoids, making the quality of the kidney organoids obtained by the method provided by the present invention higher. Description of the Drawings

[0025] Figure 1 It is a schematic flow chart of the kidney organoid constructed in Example 1 and the morphological diagrams of the kidney organoids at different stages during this process;

[0026] Figure 2 It is the overall immunofluorescence staining result diagram of the kidney organoid constructed in Example 1;

[0027] Figure 3 It is the HE staining result diagram of the kidney organoids constructed in Example 1;

[0028] Figure 4 It is the Masson staining result diagram of the kidney organoids constructed in Example 1;

[0029] Figure 5 It is the PAS staining result diagram of the kidney organoids constructed in Example 1;

[0030] Figure 6 It is the indocyanine green uptake result diagram of the kidney organoids constructed in Example 1;

[0031] Figure 7 It is the immunohistochemical result diagram of the kidney organoids constructed in Example 1;

[0032] Figure 8 It is the characterization result diagram of the kidney organoids constructed in Example 2;

[0033] Figure 9 It is the cisplatin toxicity response result diagram of the kidney organoids constructed in Example 1;

[0034] Figure 10 It is the rosuvastatin toxicity response result diagram of the kidney organoids constructed in Example 1;

[0035] Figure 11 It is the overall immunofluorescence staining result diagram of the kidney organoids constructed in Example 1 after 97 days of continuous culture. Detailed implementation manners

[0036] The endpoints and any values within the ranges disclosed herein 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 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.

[0037] The inventors of the present invention ingeniously discovered in the research that during the construction of kidney organoids, by using a culture medium containing different small molecule additives in combination with the differentiation situation of the organoids, the construction process of kidney organoids can be effectively simplified, the culture conditions and equipment can be optimized, thereby reducing costs and improving the convenience of constructing kidney organoids, providing a basis for the wide application of organoids in disease modeling, toxicity screening and drug research and development.

[0038] The first aspect of the present invention provides a set of culture media for constructing and culturing kidney organoids, and the set of culture media includes the following culture media:

[0039] Medium B: It includes a basal medium and Additive II. Among them, the Additive II includes at least one of activin A and hBMP4;

[0040] Medium C: It includes a basal medium and Additive III. Among them, the Additive III includes at least one of hBMP4 and CHIR-99021;

[0041] Medium D: It includes a basal medium and Additive IV. Among them, the Additive IV includes at least one of retinoic acid, hFGF9, LDN193189, and SB43152;

[0042] Medium E: It includes a basal medium and Additive V. Among them, the Additive V includes at least one of retinoic acid, hFGF9, LDN193189, and CHIR-99021;

[0043] Medium F: It includes a basal medium and Additive VI. Among them, the Additive VI includes at least one of retinoic acid, hFGF9, LDN193189, CHIR-99021, and hFGF1;

[0044] Medium G: It includes a basal medium and Additive VII. Among them, the Additive VII includes at least one of retinoic acid, hFGF9, LDN193189, CHIR-99021, hFGF1, and neurotrophic factor;

[0045] Medium H: It includes a basal medium and Additive VIII. Among them, the Additive VIII includes at least one of retinoic acid, LDN193189, CHIR-99021, hFGF1, and neurotrophic factor;

[0046] Medium I: It includes a basal medium and Additive IX. Among them, the Additive IX includes at least one of FBS, retinoic acid, LDN193189, hFGF1, neurotrophic factor, R-spondin 1, and hFGF7;

[0047] The basal medium is selected from DMEM / F12 medium containing added components, and the added components include serum-free additive, non-essential amino acids, β-mercaptoethanol, insulin, hTRF, and selenious acid.

[0048] The proteins used in the culture medium set provided by the present invention, such as hBMP4, hFGF9, hFGF1, hFGF7, hTRF, etc., are all human proteins. In their abbreviated forms, "h" represents human, followed by the protein name. For example, hBMP4 means human BMP4 protein (bone morphogenetic protein 4). The protein names and their abbreviated names used in the present invention have their conventional meanings in the art, and those skilled in the art can know their specific sequence, structure, function and other characteristics according to the prior art.

[0049] In the present invention, the DMEM / F12 culture medium is a culture medium with a determined formula that can be obtained through commercial channels. Those skilled in the art can purchase it through commercial channels or prepare it by themselves according to the disclosed formula.

[0050] Activin A is a multifunctional cytokine and belongs to the TGF-β family.

[0051] hBMP4 (human bone morphogenetic protein 4) is a pleiotropic ligand protein, belongs to the TGF-β family, participates in the vascular system circulation, and can activate the receptors on vascular cells.

[0052] CHIR-99021 is a GSK-3α / β inhibitor with a CAS number of 252917-06-9, and its structural formula is shown as formula (1) below.

[0053]

[0054] Retinoic acid is a metabolite of vitamin A, also known as vitamin A acid, tretinoin, etc., and plays an important role in cell growth, differentiation and organogenesis.

[0055] FGF is the abbreviation of fibroblast growth factor, which is the general term for a protein family that can bind to fibroblast growth factor receptors, and it includes multiple different proteins such as FGF1 to FGF21.

[0056] LDN193189 is a selective BMP type I receptor inhibitor with a CAS number of 1062368-24-4, and its structural formula is shown as formula (2) below.

[0057]

[0058] SB43152 is a TGF-β receptor kinase inhibitor with a CAS number of 301836-41-9, and its structural formula is shown as formula (3) below.

[0059]

[0060] In the present invention, the neurotrophic factor is glial cell line-derived neurotrophic factor (GDNF), a neurotrophic factor belonging to the GDNF family ligands, which can promote the survival of dopamine neurons.

[0061] FBS is the abbreviation of fetal bovine serum, which is a source of rich proteins and growth factors that support cell growth.

[0062] R-spondin 1 is a secreted protein that activates Wnt signaling.

[0063] Serum-free additives are cell culture additives that do not contain serum components. For example, B27 additive can provide various nutrients and cytokines for the culture medium.

[0064] Non-essential amino acids, namely NEAA, are a commercially available and composition-defined compound amino acid preparation, mainly including 7 non-essential amino acids required for cell culture. The non-essential amino acids used in the present invention can be directly obtained through commercial purchase or prepared by oneself according to its formula.

[0065] β-Mercaptoethanol can provide a source of sulfhydryl groups, with a CAS number of 60-24-2, and its structural formula is shown as formula (4) below.

[0066]

[0067] In the culture medium set of the present invention, insulin, human transferrin (hTRF), and selenious acid added to the basal medium can be directly added or provided by ITS reagent. ITS reagent is a commercially available and formula-defined culture medium additive, and its main components are insulin, hTRF, and selenious acid (salt).

[0068] According to the present invention, preferably, the amount of the basal medium is such that in each culture medium of the culture medium set: the final concentration of the serum-free additive is not less than 1×, preferably 1× - 2×; the final concentration of the non-essential amino acids is not less than 0.1% by weight, preferably 0.1 - 3% by weight; the final concentration of β-mercaptoethanol is not less than 1×10 -6 % (v / v), preferably 1×10 -6 -1×10 -4 % (v / v); the final concentration of insulin is not less than 0.3% by weight, preferably 0.3 - 1.5% by weight; the final concentration of hTRF is not less than 0.15% by weight, preferably 0.15 - 0.85% by weight; the final concentration of selenious acid is not less than 0.0015% by weight, preferably 0.0015 - 0.0075% by weight.

[0069] According to the present invention, preferably, in culture medium B, the dosage of additive II is such that in culture medium B: the final concentration of activin A is not less than 5 ng / mL, preferably 5 - 20 ng / mL; the final concentration of hBMP4 is not less than 0.5 ng / mL, preferably 0.5 - 2 ng / mL.

[0070] According to the present invention, preferably, in culture medium C, the dosage of additive III is such that in culture medium C: the final concentration of hBMP4 is not less than 0.5 ng / mL, preferably 0.5 - 2 ng / mL; the final concentration of CHIR-99021 is not less than 5 μM, preferably 5 - 30 μM.

[0071] According to the present invention, preferably, in culture medium D, the dosage of additive IV is such that in culture medium D: the final concentration of retinoic acid is not less than 0.05 μM, preferably 0.05 - 1 μM; the final concentration of hFGF9 is not less than 20 ng / mL, preferably 20 - 300 ng / mL; the final concentration of LDN193189 is not less than 10 nM, preferably 10 - 200 nM; the final concentration of SB43152 is not less than 10 nM, preferably 10 - 200 nM.

[0072] According to the present invention, preferably, in culture medium E, the dosage of additive V is such that in culture medium E: the final concentration of retinoic acid is not less than 0.05 μM, preferably 0.05 - 1 μM; the final concentration of LDN193189 is not less than 3 nM, preferably 3 - 20 nM; the final concentration of CHIR-99021 is not less than 0.3 μM, preferably 0.3 - 5 μM.

[0073] According to the present invention, preferably, in culture medium F, the dosage of additive VI is such that in culture medium F: the final concentration of retinoic acid is not less than 0.05 μM, preferably 0.05 - 1 μM; the final concentration of hFGF9 is not less than 2 ng / mL, preferably 2 - 30 ng / mL; the final concentration of LDN193189 is not less than 3 nM, preferably 3 - 20 nM; the final concentration of CHIR-99021 is not less than 0.3 μM, preferably 0.3 - 5 μM; the final concentration of hFGF1 is not less than 20 ng / mL, preferably 20 - 300 ng / mL.

[0074] According to the present invention, preferably, in culture medium G, the dosage of additive VII is such that in culture medium G: the final concentration of retinoic acid is not less than 0.05 μM, preferably 0.05 - 1 μM; the final concentration of hFGF9 is not less than 2 ng / mL, preferably 2 - 30 ng / mL; the final concentration of LDN193189 is not less than 3 nM, preferably 3 - 20 nM; the final concentration of CHIR-99021 is not less than 0.3 μM, preferably 0.3 - 5 μM; the final concentration of hFGF1 is not less than 20 ng / mL, preferably 20 - 300 ng / mL; the final concentration of neurotrophic factor is not less than 0.2 ng / mL, preferably 0.2 - 3 ng / mL.

[0075] According to the present invention, preferably, in culture medium H, the dosage of additive VIII is such that in culture medium H: the final concentration of retinoic acid is not less than 0.05 μM, preferably 0.05 - 1 μM; the final concentration of LDN193189 is not less than 3 nM, preferably 3 - 20 nM; the final concentration of CHIR-99021 is not less than 0.3 μM, preferably 0.3 - 5 μM; the final concentration of hFGF1 is not less than 20 ng / mL, preferably 20 - 300 ng / ml; the final concentration of neurotrophic factor is not less than 0.4 ng / mL, preferably 0.4 - 6 ng / mL.

[0076] According to the present invention, preferably, in culture medium I, the dosage of additive IX is such that in culture medium I: the final concentration of retinoic acid is not less than 0.05 μM, preferably 0.05 - 1 μM; the final concentration of FBS is not less than 5% by volume, preferably 5 - 20% by volume; the final concentration of LDN193189 is not less than 3 nM, preferably 3 - 20 nM; the final concentration of CHIR-99021 is not less than 0.3 μM, preferably 0.3 - 5 μM; the final concentration of hFGF1 is not less than 20 ng / mL, preferably 20 - 300 ng / mL; the final concentration of neurotrophic factor is not less than 0.4 ng / mL, preferably 0.4 - 6 ng / mL; the final concentration of RSPO1 is not less than 30 ng / mL, preferably 30 - 200 ng / mL; the final concentration of hFGF7 is not less than 5 ng / mL, preferably 5 - 50 ng / mL.

[0077] In some specific embodiments of the present invention, preferably, the culture medium set further includes culture medium A: including basal medium A and additive I, wherein, basal medium A is mTeSR1 medium, and additive I includes Y-27632.

[0078] In the present invention, mTeSR1 medium is a commercially available medium with a determined formula. Those skilled in the art can purchase it through commercial channels or prepare it according to the disclosed formula by themselves.

[0079] Y-27632 is a RHO / ROCK pathway inhibitor with a CAS number of 146986-50-7, and its structural formula is shown as formula (5) below.

[0080]

[0081] Further preferably, in medium A, the dosage of additive I is such that in medium A: the final concentration of Y-27632 is not less than 5 μM, preferably 5 - 20 μM.

[0082] According to the present invention, preferably, the medium further contains antibiotics.

[0083] Further preferably, the antibiotic is a compound broad-spectrum antibiotic. In the present invention, a variety of different antibiotics that can be used for culturing in vitro cultures such as cells and organoids (such as penicillin, streptomycin, etc.) can be used in combination, or a commercially available compound antibiotic product (such as etc.) can be used.

[0084] In the present invention, there is no particular limitation on the dosage of the antibiotic, as long as it can ensure that the process of organoid construction and culture is not affected by contamination of miscellaneous bacteria. Those skilled in the art can determine its dosage according to the actually selected antibiotic, or refer to the instruction manual of the commercially available antibiotic product to determine the dosage.

[0085] The second aspect of the present invention provides a method for constructing kidney organoids, which includes inducing embryoid bodies to form kidney organoids by using the medium set of the first aspect of the present invention.

[0086] According to the present invention, preferably, the method includes:

[0087] (2-1) Differentiation I of kidney organoids: Inoculating embryoid bodies into medium B for secondary culture to obtain a first-stage culture;

[0088] (2-2) Differentiation II of kidney organoids: Inoculating the first-stage culture obtained in step (2-1) into medium C for tertiary culture to obtain a second-stage culture;

[0089] (2-3) Differentiation III of kidney organoids: Inoculating the second-stage culture obtained in step (2-2) into medium D for quaternary culture to obtain a third-stage culture;

[0090] (2-4) Differentiation IV of kidney organoids: Inoculating the third-stage culture obtained in step (2-3) into medium E for fifth culture to obtain a fourth-stage culture;

[0091] (2-5) Differentiation of kidney organoids V: Inoculate the fourth-stage culture obtained in step (2-4) into medium F for the sixth culture to obtain a fifth-stage culture;

[0092] (2-6) Differentiation of kidney organoids VI: Inoculate the fifth-stage culture obtained in step (2-5) into medium G for the seventh culture to obtain a sixth-stage culture;

[0093] (2-7) Differentiation of kidney organoids VII: Inoculate the sixth-stage culture obtained in step (2-6) into medium H for the eighth culture to obtain a seventh-stage culture;

[0094] (2-8) Differentiation of kidney organoids VIII: Inoculate the seventh-stage culture obtained in step (2-7) into medium I for the ninth culture to obtain kidney organoids.

[0095] In the present invention, the kidney organoids prepared by the above method can be cultured and maintained for a long time, and the corresponding functions of the kidney organoids can be maintained.

[0096] In the present invention, there are no particular limitations on the source and acquisition method of the embryoid bodies. They can be obtained by conventional embryoid body preparation methods in the art or through commercial purchase. For example, embryoid bodies can be formed by inducing the culture of stem cells.

[0097] According to the present invention, preferably, the method further includes step (1) of obtaining embryoid bodies, preferably including inoculating stem cells into medium A for the first culture to obtain embryoid bodies.

[0098] In the present invention, the stem cells can be pluripotent stem cells that can be used for organoid construction in the art. For example, they can be pluripotent stem cells obtained by inducing culture according to the prior art or directly through commercial purchase, or pluripotent stem cells prepared by oneself according to the prior art in the art. There are also no particular limitations on the type of pluripotent stem cells in the present invention, and induced pluripotent stem cells (iPSCs) can be selected.

[0099] According to the present invention, preferably, in step (2-1), the time of the second culture is 20-30 h.

[0100] According to the present invention, preferably, in step (2-2), the time of the third culture is 40-50 h.

[0101] According to the present invention, preferably, in step (2-3), the time of the fourth culture is 40-50 h.

[0102] According to the present invention, preferably, in step (2-4), the time of the fifth culture is 40-50 h.

[0103] According to the present invention, preferably, in step (2-5), the time of the sixth culture is 40-50 h.

[0104] According to the present invention, preferably, in step (2-6), the time of the seventh culture is 40-50 h.

[0105] According to the present invention, preferably, in step (2-7), the time of the eighth culture is 40-50 h.

[0106] According to the present invention, preferably, in step (2-8), the time of the ninth culture is 40-50 h.

[0107] According to the present invention, preferably, in step (3), the time of the long-term culture is 2-3 months.

[0108] According to the present invention, preferably, in step (1), the culture time of the first culture is 24-36 h.

[0109] More preferably, during the first culture, the culture medium A is replaced every 12-24 h.

[0110] According to the present invention, preferably, the method further includes removing the culture medium and washing the culture before each step. Since the culture media used in each step contain different additives, the purpose of this washing treatment is mainly to remove the residual culture medium on the cells.

[0111] According to the present invention, preferably, the method does not include the treatment of embedding the cultures obtained in each step with Matrigel. When constructing organoids in the art, it is usually necessary to embed the cultures with Matrigel to provide nutrients and support to maintain the morphology of the cultures. However, the presence of Matrigel makes the operation of organoid construction more complex, and the Matrigel needs to be replaced regularly during the culture process. The temperature change and mechanical force during the replacement process will damage the cultures, and at the same time, it will affect the response of the cultures to drugs, resulting in certain experimental errors. In addition, the use of Matrigel also greatly increases the cost of organoid culture. Through a large number of studies, the inventors of the present invention found that when using the method and culture medium provided by the present invention for the construction and culture of kidney organoids, there is no need to use Matrigel, and the cultures can be directly placed in the culture medium to complete, so that the present invention can better protect the cultures and maintain the culture for a longer time. Using the method and culture medium of the present invention can reduce the culture cost and promote the response of the cultures to drugs.

[0112] According to the present invention, preferably, the method further includes step (3) of maintaining the culture of kidney organoids, preferably including long-term culturing of the obtained kidney organoids using culture medium I.

[0113] The third aspect of the present invention provides a kidney organoid constructed by the method described in the second aspect of the present invention, and the application of the kidney organoid in at least one of drug screening, disease modeling, and toxicological detection.

[0114] In the present invention, the kidney organoid at least includes glomerular podocytes, ureteral cells, renal tubular cells, cortical cells, medullary cells, and endothelial cells. The above cells can be detected and confirmed by conventional technical means in the art. For example, immunofluorescence can be used to detect each cell marker in the obtained kidney organoid, so as to determine that the kidney organoid contains the above cells.

[0115] The kidney organoid obtained by the method provided by the present invention is composed of multiple cells, has the basic structure of the kidney, and has corresponding responses to drug components known in the art to have an impact on the kidney. Therefore, the kidney organoid provided by the present invention can be used as an in vitro model for applications such as screening of drugs for the kidney, disease modeling, and toxicological detection.

[0116] The fourth aspect of the present invention provides the application of the culture medium described in the first aspect of the present invention, and / or the method described in the second aspect of the present invention in at least one of improving the success rate of kidney organoid construction, shortening the maturation time of organoids, prolonging the culture time of kidney organoids, reducing the cost of kidney organoid construction, and increasing the throughput of kidney organoid construction.

[0117] In the prior art, the culture time of the constructed kidney organoids is usually more than 25 days, and the components of the culture medium used are relatively complex, requiring complex operations, and the overall construction cost is relatively high (for example, reference can be made to Minoru Takasato, et al., Generating kidney organoids from human pluripotent stem cells; Navin Gupta, et al., Modeling injury and repair in kidney organoids reveals that homologous recombination governs tubular intrinsic repair; Ryuji Morizane, et al., Nephron organoids derived from human pluripotent stem cells model kidney development and injury; Minoru Takasato, et al., Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis; Aneta Przepiorski, et al., A simple bioreactor-based method to generate kidney organoids from pluripotent stem cells, etc.). However, through experiments, the use of the culture medium set and construction method of the present invention can obtain kidney organoids with complete functions and structures in 16 days. The components of the culture medium at each culture stage are clear, and through the cooperation of the culture medium and the culture process, the purposes of shortening the culture time, increasing the culture throughput, simplifying the operation, reducing the cost and the complexity of the experimental protocol are achieved.

[0118] In the process of research, the inventors found that current research on liver-kidney organoids derived from stem cells in this field mainly focuses on the application effects of microfluidic methods and materials, and the cell experiments used mainly involve cell lines. There is also little research on their application in three-dimensional culture, and there is rarely any research on using liver-kidney organoids for toxicity screening. Through long-term research, the inventors discovered that by connecting two types of organoids in series and seeking a method to jointly evaluate drug toxicity, the organoid model can reflect the metabolism and biological responses of drugs in real liver and kidney tissues. Compared with using 2D cells (lines) or single types of organoids as in vitro models, using a combination of liver-kidney organoids provides a more reliable basis for drug safety assessment. By testing the toxicity response of drugs in organoids, the impact of drugs on human liver and kidneys can be predicted more accurately, thereby increasing the success rate of drug development.

[0119] The fifth aspect of the present invention provides a method for detecting drug safety, which includes contacting a hepatobiliary organoid and a kidney organoid with a drug to be tested respectively, and comprehensively judging the safety of the drug based on the responses of the hepatobiliary organoid and the kidney organoid to the drug. Among them, the kidney organoid is the kidney organoid constructed by the method described in the second aspect of the present invention.

[0120] In the present invention, by administering drugs to the kidney organoid and the hepatobiliary organoid respectively, the toxic effects of the drugs in the liver and kidney organoids after administration can be evaluated separately. Through comparison, the toxic manifestations of the combined action of the liver and kidneys can be more clearly understood, and the risk of combined action can be identified. Real-time monitoring technology can be used to continuously observe the dynamic changes of the two types of organoids during drug administration, such as cell viability, inflammatory responses, and drug concentration changes. These data can be used to judge the individual effects of the drugs on the liver and kidneys. Compared with the complex dynamics in vivo, organoid experiments provide more interpretable data and more reliable organ responses, providing basic data for the combined action of organs.

[0121] In the present invention, there are no special limitations on the hepatobiliary organoid, which can be obtained through commercial channels or prepared according to existing technologies. For example, the hepatobiliary organoid can be constructed by referring to the method in CN202410872877.3.

[0122] The present invention will be described in detail below through examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention, and not to limit the present invention.

[0123] In the following examples, rosuvastatin was purchased from MCE, catalog number 147098-20-2; mTeSR1 medium was purchased from STEMCELL Technologies, catalog number 8580; induced pluripotent stem cells iPSC were purchased from Beijing Saibei Biotechnology Co., Ltd.; Y27632 was purchased from MCE, catalog number HY-10071; activin A was purchased from MCE, catalog number HY-P70311; hBMP4 was purchased from MCE, catalog number HY-P7007; CHIR-99021 was purchased from MCE, catalog number HY-10182; retinoic acid was purchased from MCE, catalog number HY-14649; hFGF9 was purchased from MCE, catalog number HY-P7177; hFGF7 was purchased from MCE, catalog number HY-P70597; hFGF1 was purchased from MCE, catalog number HY-P7001; LDN193189 was purchased from MCE, catalog number HY-12071; SB43152 was purchased from MCE, catalog number HY-10431; GDNF was purchased from MCE, catalog number HY-P7182; FBS was purchased from Gibco, product number A5669701; R-spondin1 was purchased from MCE, catalog number HY-P7114; B27 additive was purchased from Gibco, catalog number 12526260; NEAA was purchased from MCE, catalog number HY-K3011; β-mercaptoethanol was purchased from Aladdin Biochemical Technology Co., Ltd., catalog number M301574; ITS reagent was purchased from Yuanpei Biotechnology Co., Ltd., catalog number E431234.

[0124] Other reagents or materials not specifically mentioned were all commercially available products purchased from regular chemical or biological reagent / material suppliers, and the purity of the reagents was all analytical pure.

[0125] Preparation Example 1

[0126] Weigh or measure the additives according to the final concentrations in Table 1 and add them to the basal medium to prepare a set of media consisting of Media A, B, C, D, E, F, G, and H. Among them, each medium contains 0.2% by weight of antibiotics (the type is purchased from InvivoGen, product number ant-pm-05).

[0127] Table 1

[0128]

[0129]

[0130] Example 1

[0131] This example is used to illustrate the effect of the method provided by the present invention in constructing kidney organoids.

[0132] (1) Embryoid body culture: Wash the pre-cultured iPSCs three times with pre-warmed DPBS, digest and separate them into single-cell suspensions, and prepare a cell suspension of 8000 cells / 150 μL with Medium A. Then, add this cell suspension to a 96-well ultra-low attachment plate (U-bottom) (150 μL per well).

[0133] After inoculation, centrifuge the culture plate at 300 g for 5 min at 4°C, and then place it in a 37°C incubator (5% CO2) for the first culture for 48 h. Observe the morphological changes of the cells every 24 h during this period. As Figure 1 shown in DAY1, it can be seen that spherical cell aggregates can be formed after 24 h of culture. After 48 h, remove the medium in the culture plate, add 200 μL / well of Medium A, and continue to culture for 24 h to obtain embryoid bodies.

[0134] (2) Differentiation of kidney organoids

[0135] (2-1) Remove the medium in the culture plate from step (1), add 200 μL / well of Medium B for the second culture, and place it in a 37°C incubator (5% CO2) for 48 h to obtain the first-stage culture (refer to the morphology in Figure 1 DAY3).

[0136] (2-2) Remove the medium in the culture plate from step (2-1), add 200 μL / well of Medium C for the third culture, and place it in a 37°C incubator (5% CO2) for 24 h to obtain the second-stage culture (refer to the morphology in Figure 1 DAY4).

[0137] (2-3) Remove the medium in the culture plate from step (2-2), add 200 μL / well of Medium D for the fourth culture, and place it in a 37°C incubator (5% CO2) for 48 h to obtain the third-stage culture (refer to the morphology in Figure 1 DAY6).

[0138] (2-4) Remove the medium in the culture plate from step (2-3), add 200 μL / well of Medium E for the fifth culture, and place it in a 37°C incubator (5% CO2) for 48 h to obtain the fourth-stage culture (refer to the morphology in Figure 1 DAY8).

[0139] (2-5) Remove the medium in the culture plate from step (2-4), add 200 μL / well of Medium F for the sixth culture, and place it in a 37°C incubator (5% CO2) for 48 h to obtain the fifth-stage culture (refer to the morphology inFigure 1 in DAY10 of

[0140] (2 - 6) Remove the culture medium in the culture plate of step (2 - 5), add 200 μL / well of culture medium G for the seventh culture, and place it in a 37 °C incubator (5% CO2) for 48 h to obtain the culture of the sixth stage (for morphological reference Figure 1 in DAY12 of

[0141] (2 - 7) Remove the culture medium in the culture plate of step (2 - 6), add 200 μL / well of culture medium H for the eighth culture, and place it in a 37 °C incubator (5% CO2) for 48 h to obtain the culture of the seventh stage (for morphological reference Figure 1 in DAY14 of

[0142] (2 - 8) Remove the culture medium in the culture plate of step (2 - 7), add 200 μL / well of culture medium I for the ninth culture, and place it in a 37 °C incubator (5% CO2) for 48 h to obtain kidney organoids (for morphological reference Figure 1 in DAY16 of

[0143] (3) Long - term culture of kidney organoids: Replace the medium every 48 h with culture medium I, and long - term culture of kidney organoids can be achieved.

[0144] (II) Detection of the structure and function of kidney organoids

[0145] (1) Use the whole - mount immunofluorescence staining method to detect the cell - specific markers in the kidney organoids obtained in Example 1. Figure 2 The staining results of the kidney organoids constructed in Example 1 are shown.

[0146] In Figure 2-1 , the blue is DAPI staining, clearly showing the distribution and arrangement of cells. The red CD31 staining indicates the presence of vascular endothelial cells. CD31 (PECAM - 1) is a commonly used vascular marker. This result shows that the kidney organoids cultured by this protocol not only exhibit kidney characteristics in cell structure but also show angiogenesis ability. The expression of CD31 endows the kidney organoids with the potential to form and maintain vascular structures.

[0147] In Figure 2-2 , the blue DAPI staining results mark the position of the cell nuclei. The red MEIS1 staining indicates that the kidney organoids can express MEIS1. MEIS1 is an important transcription factor related to kidney development and differentiation, and is usually closely related to the formation and maintenance of the renal cortex and medulla. The expression of MEIS1 is an important indicator for evaluating whether kidney organoids have mature functional characteristics.

[0148] In Figure 2-3 , the blue DAPI staining results indicate the distribution positions of cell nuclei, which are used to observe the distribution and morphological characteristics of cells. The red E-Cadherin staining shows that the kidney organoids cultured under this protocol can express E-Cadherin, which is a key cell adhesion protein widely present in epithelial cells and helps maintain the integrity and stability of epithelial tissues.

[0149] In Figure 2-4 , the blue DAPI staining results identify the positions of cell nuclei. The red Nephrin staining shows that the kidney organoids cultured under this protocol can express Nephrin. Nephrin is a glomerulus-specific cell membrane protein mainly present in the podocytes of glomeruli.

[0150] In Figure 2-5 , the DAPI staining marks the positions of cell nuclei, reflects the distribution and aggregation of cells, and provides the spatial architecture of kidney organoids. The green CK8 staining indicates that there are epithelial cells in the kidney organoids cultured under this protocol. CK8 is a classic epithelial cell marker, especially in the renal tubular tissue.

[0151] (2) HE, Masson, and PAS stainings were performed on the kidney organoids. Figure 3 The HE staining diagram is shown in Figure 3 . It can be seen that there is a radial cortical-medullary-like structure. Figure 4 The Masson staining diagram is shown in Figure 4 . It can be seen that collagen fibers (blue) are widely distributed, with occasional muscle fibers (red).

[0152] Figure 5 The PAS staining diagram is shown in Figure 5 . It can be seen that the cell distribution represented by cell nuclei (blue) is present, with occasional glycogen distribution (red).

[0153] (3) Indocyanine green staining was performed on the kidney organoids. Figure 6 The staining results are shown. Indocyanine green is a commonly used renal function marker, and its uptake and excretion in the body can reflect the filtration and excretion functions of the kidneys. It can be seen from Figure 6 that the kidney organoids constructed by the present invention can effectively take up indocyanine green, indicating that they have good renal function and can simulate the physiological characteristics of real kidneys in terms of filtration and excretion. After removing indocyanine green and replacing it with normal medium, it gradually returns to the normal state, which further indicates the activity and viability of renal cells in the kidney organoids.

[0154] (4) Stain the kidney organoids with E-Cadherin, MEISI, and Nephrin, Figure 7 The staining results are shown. In the figure, (1) is the staining result of E-Cadherin, indicating the presence of ureter and collecting duct structures with E-Cadherin expression in the kidney organoids; (2) indicates the presence of relevant structures of the cortex and medulla in the kidney organoids; (3) indicates the presence of glomerulus-related structures in the kidney organoids.

[0155] The above test results show that the kidney organoids constructed by the present invention possess the basic structures of the kidney.

[0156] (III) Cultivation success rate of kidney organoids

[0157] By observing the development of kidney organoids under a bright-field microscope, it was found that within 30 days of cultivation, the average construction success rate of kidney organoids on each ultra-low attachment plate (with at least three repeated experiments) exceeded 95% (construction success rate = number of wells successfully constructing kidney organoids on a single culture plate / number of wells inoculated with iPSC on a single culture plate × 100%).

[0158] (IV) Long-term cultivation effect of kidney organoids

[0159] After 97 days of continuous cultivation, the kidney organoids did not show obvious apoptosis, the cell morphology was good, and the overall color was slightly dull. This indicates that during long-term cultivation, the kidney organoids provided by the present invention can effectively absorb nutrients and maintain their own growth and survival. It can be seen through immunofluorescence staining that the cell markers expressed by the kidney organoids after 97 days of continuous cultivation are similar to those of the newly constructed successful kidney organoids (specifically visible Figure 11 ).

[0160] Example 2

[0161] Construct kidney organoids according to the method of Example 1 with the culture medium formula in Table 1. The difference is that the hFGF concentration in additives IV, V, VI, and VII is 5 ng / mL. The characterization results of the kidney organoids constructed with this culture medium are as Figure 8 shown, Figure 8 (1) is the morphological diagram of the kidney organoids, Figure 8 (2) is the morphological diagram of the kidney organoids cultured for 43 days, Figure 8 (3) is the fluorescence staining result diagram. From Figure 8 (1), it can be seen that the kidney organoids failed to complete normal development, the cells were too close, and necrosis already existed inside. They could only survive for 43 days during long-term cultivation. From Figure 8 (3), it can be seen that the absence of the glomerulus-specific protein Nephrin indicates that the kidney organoids do not have the specific functions of the glomerulus.

[0162] Comparative Example 1

[0163] (1) According to the culture medium formula in Table 1, the method of Example 1 was used to construct kidney organoids. The difference was that LDN193189 in Additive IV was omitted. Using this culture medium to construct kidney organoids, the embryoid body development of the kidney organoids was slow, resulting in a decrease in the degree of organoid differentiation.

[0164] (2) According to the culture medium formula in Table 1, the method of Example 1 was used to construct kidney organoids. The difference was that FGF-1 in all additives was omitted. Using this culture medium to construct kidney organoids, the embryoid body development of the kidney organoids was slow and finally could not complete the development.

[0165] (3) According to the culture medium formula in Table 1, the method of Example 1 was used to construct kidney organoids. The difference was that GDNF in Additive VII was omitted. Using this culture medium to construct kidney organoids, the kidney cortex ring structure was not obvious, and the formation rate of kidney organoids decreased.

[0166] (4) According to the culture medium formula in Table 1, the method of Example 1 was used to construct kidney organoids. The difference was that FBS in Additive IX was omitted. Using this culture medium to conduct long-term culture on the constructed kidney organoids, the renin secretion function of the organoids gradually weakened with the prolongation of the culture time during the culture process, and the maintenance time of the long-term culture was also shortened.

[0167] Test Example 1

[0168] This test example is used to illustrate the response of the kidney organoids provided by the present invention to cisplatin.

[0169] Cisplatin was dissolved in DMSO to prepare a stock solution of 40 mM, and the dosing concentrations were set to 0, 5 μM, 20 μM, and 100 μM respectively, and treated for 24 hours. The results were as Figure 9 (1) shown, the appearance of the kidney organoids did not show obvious changes, and the cell viability did not show obvious decline either.

[0170] The dosing concentrations were set to 0, 5 μM, 20 μM, and 100 μM respectively, and treated for 48 hours. The results were as Figure 9 (2) shown, the kidney organoids showed obvious damage and the cell viability decreased significantly.

[0171] The dosing concentrations were set to 0, 5 μM, 20 μM, and 100 μM respectively, and treated for 72 hours. The results were as Figure 9 (3) shown, the kidney organoids were on the verge of fragmentation, showing severe damage.

[0172] Through the toxicity test of cisplatin on the kidney organoids, it can be clearly seen that the kidney organoids used in this experiment have good response ability to drugs.

[0173] The same test was carried out on the kidney organoids after 46 days of culture, and similar test results were obtained, indicating that long-term culture has no effect on the drug response ability of the kidney organoids of the present invention.

[0174] Test Example 2

[0175] This test example is used to illustrate the synergistic response of liver and gall organoids and the kidney organoids provided by the present invention to drugs.

[0176] The liver and gall organoids used in this test example were constructed by referring to the method in CN202410872877.3.

[0177] Rosuvastatin was dissolved in DMSO to prepare a stock solution of 40 mM. The administration concentrations of rosuvastatin were set at 0, 40 μM, 80 μM, and 120 μM respectively, and the kidney organoids and liver and gall organoids were treated for 5 days. The culture medium was changed every day, and the culture supernatant of the organoids was collected on the 5th day. After the treatment, cell apoptosis staining and determination of blood urea nitrogen were carried out on the kidney organoids. For the liver and gall organoids, cell apoptosis staining, Nile red staining, and determination of urea were carried out, and the results are as Figure 10 shown. As can be seen from Figure 10 (1) and (2), after 5 days of rosuvastatin administration, the apoptosis staining of the kidney and liver and gall organoids showed a gradually obvious trend. According to Figure 10 (3) results, after 5 days of rosuvastatin administration, obvious fat accumulation appeared in the liver and gall organoids. Figure 10 (4) and 10(5) results show that the blood urea nitrogen content of the kidney organoids increased significantly after rosuvastatin treatment, while the blood urea nitrogen content of the liver and gall organoids decreased significantly, and this effect is consistent with clinical observations.

[0178] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A culture medium set for constructing and culturing kidney organoids, characterized in that, The culture medium set includes the following culture media: Culture medium B: includes a basal medium and additive II, wherein the additive II includes at least one of activin A and hBMP4; Culture medium C: includes a basal medium and additive III, wherein the additive III includes at least one of hBMP4 and CHIR-99021; Culture medium D: includes a basal medium and additive IV, wherein the additive IV includes at least one of retinoic acid, hFGF9, LDN193189, and SB43152; Culture medium E: includes a basal medium and additive V, wherein the additive V includes at least one of retinoic acid, hFGF9, LDN193189, and CHIR-99021; Culture medium F: includes a basal medium and additive VI, wherein the additive VI includes at least one of retinoic acid, hFGF9, LDN193189, CHIR-99021, and hFGF1; Culture medium G: includes a basal medium and additive VII, wherein the additive VII includes at least one of retinoic acid, hFGF9, LDN193189, CHIR-99021, hFGF1, and neurotrophic factor; Culture medium H: includes a basal medium and additive VIII, wherein the additive VIII includes at least one of retinoic acid, LDN193189, CHIR-99021, hFGF1, and neurotrophic factor; Culture medium I: includes a basal medium and additive IX, wherein the additive IX includes at least one of FBS, retinoic acid, LDN193189, hFGF1, neurotrophic factor, R-spondin 1, and hFGF7; The basal medium is selected from DMEM / F12 medium containing added components, and the added components include serum-free additive, non-essential amino acids, β-mercaptoethanol, insulin, hTRF, and selenious acid.

2. The culture medium according to claim 1, wherein The dosage of the basal medium is such that in each medium of the medium set: the final concentration of the serum-free additive is not less than 1×, preferably 1× - 2×; the final concentration of non-essential amino acids is not less than 0.1% by weight, preferably 0.1 - 3% by weight; the final concentration of β-mercaptoethanol is not less than 1×10 -6 % by volume, preferably 1×10 -6 -1×10 -4 % by volume; the final concentration of insulin is not less than 0.3% by weight, preferably 0.3 - 1.5% by weight; the final concentration of hTRF is not less than 0.15% by weight, preferably 0.15 - 0.85% by weight; the final concentration of selenious acid is not less than 0.0015% by weight, preferably 0.0015 - 0.0075% by weight; And / or, in culture medium B, the dosage of additive II is such that in culture medium B: the final concentration of activin A is not less than 5 ng / mL, preferably 5 - 20 ng / mL; the final concentration of hBMP4 is not less than 0.5 ng / mL, preferably 0.5 - 2 ng / mL; And / or, in culture medium C, the dosage of additive III is such that in culture medium C: the final concentration of hBMP4 is not less than 0.5 ng / mL, preferably 0.5 - 2 ng / mL; the final concentration of CHIR-99021 is not less than 5 μM, preferably 5 - 30 μM; And / or, in culture medium D, the dosage of additive IV is such that in culture medium D: the final concentration of retinoic acid is not less than 0.05 μM, preferably 0.05 - 1 μM; the final concentration of hFGF9 is not less than 20 ng / mL, preferably 20 - 300 ng / mL; the final concentration of LDN193189 is not less than 10 nM, preferably 10 - 200 nM; the final concentration of SB43152 is not less than 10 nM, preferably 10 - 200 nM; And / or, in medium E, the dosage of additive V is such that in medium E: the final concentration of retinoic acid is not less than 0.05 μM, preferably 0.05 - 1 μM; the final concentration of LDN193189 is not less than 3 nM, preferably 3 - 20 nM; the final concentration of CHIR-99021 is not less than 0.3 μM, preferably 0.3 - 5 μM; And / or, in medium F, the dosage of additive VI is such that in medium F: the final concentration of retinoic acid is not less than 0.05 μM, preferably 0.05 - 1 μM; the final concentration of hFGF9 is not less than 2 ng / mL, preferably 2 - 30 ng / mL; the final concentration of LDN193189 is not less than 3 nM, preferably 3 - 20 nM; the final concentration of CHIR-99021 is not less than 0.3 μM, preferably 0.3 - 5 μM; the final concentration of hFGF1 is not less than 20 ng / mL, preferably 20 - 300 ng / mL; And / or, in medium G, the dosage of additive VII is such that in medium G: the final concentration of retinoic acid is not less than 0.05 μM, preferably 0.05 - 1 μM; the final concentration of hFGF9 is not less than 2 ng / mL, preferably 2 - 30 ng / mL; the final concentration of LDN193189 is not less than 3 nM, preferably 3 - 20 nM; the final concentration of CHIR-99021 is not less than 0.3 μM, preferably 0.3 - 5 μM; the final concentration of hFGF1 is not less than 20 ng / mL, preferably 20 - 300 ng / mL; the final concentration of neurotrophic factor is not less than 0.2 ng / mL, preferably 0.2 - 3 ng / mL; And / or, in medium H, the dosage of additive VIII is such that in medium H: the final concentration of retinoic acid is not less than 0.05 μM, preferably 0.05 - 1 μM; the final concentration of LDN193189 is not less than 3 nM, preferably 3 - 20 nM; the final concentration of CHIR-99021 is not less than 0.3 μM, preferably 0.3 - 5 μM; the final concentration of hFGF1 is not less than 20 ng / mL, preferably 20 - 300 ng / ml; the final concentration of neurotrophic factor is not less than 0.4 ng / mL, preferably 0.4 - 6 ng / mL; And / or, in Medium I, the dosage of Additive IX is such that in Medium I: the final concentration of retinoic acid is not less than 0.05 μM, preferably 0.05 - 1 μM; the final concentration of FBS is not less than 5% by volume, preferably 5 - 20% by volume; the final concentration of LDN193189 is not less than 3 nM, preferably 3 - 20 nM; the final concentration of CHIR-99021 is not less than 0.3 μM, preferably 0.3 - 5 μM; the final concentration of hFGF1 is not less than 20 ng / mL, preferably 20 - 300 ng / mL; the final concentration of neurotrophic factor is not less than 0.4 ng / mL, preferably 0.4 - 6 ng / mL; the final concentration of RSPO1 is not less than 30 ng / mL, preferably 30 - 200 ng / mL; the final concentration of hFGF7 is not less than 5 ng / mL, preferably 5 - 50 ng / mL; Preferably, the medium set further includes Medium A: comprising a basal medium A and Additive I, wherein the basal medium A is mTeSR1 medium, and the Additive I includes Y-27632; More preferably, in Medium A, the dosage of Additive I is such that in Medium A: the final concentration of Y-27632 is not less than 5 μM, preferably 5 - 20 μM.

3. The culture medium set according to claim 1 or 2, wherein The medium also contains antibiotics.

4. A method for constructing kidney organoids, characterized in that, The method includes inducing the formation of kidney organoids from embryoid bodies using the medium set according to any one of claims 1 - 3.

5. The method according to claim 4, wherein The method includes: (2-1) Kidney organoid differentiation I: Inoculating the embryoid bodies into Medium B for a second culture to obtain a first-stage culture; (2-2) Kidney organoid differentiation II: Inoculating the first-stage culture obtained in step (2-1) into Medium C for a third culture to obtain a second-stage culture; (2-3) Kidney organoid differentiation III: Inoculating the second-stage culture obtained in step (2-2) into Medium D for a fourth culture to obtain a third-stage culture; (2-4) Kidney organoid differentiation IV: Inoculating the third-stage culture obtained in step (2-3) into Medium E for a fifth culture to obtain a fourth-stage culture; (2-5) Kidney organoid differentiation V: Inoculating the fourth-stage culture obtained in step (2-4) into Medium F for a sixth culture to obtain a fifth-stage culture; (2-6) Kidney organoid differentiation VI: Inoculating the fifth-stage culture obtained in step (2-5) into Medium G for a seventh culture to obtain a sixth-stage culture; (2-7) Kidney organoid differentiation VII: Inoculating the sixth-stage culture obtained in step (2-6) into Medium H for an eighth culture to obtain a seventh-stage culture; (2-8) Kidney organoid differentiation VIII: Inoculating the seventh-stage culture obtained in step (2-7) into Medium I for a ninth culture to obtain kidney organoids; Preferably, the method further includes step (1) of obtaining embryoid bodies, preferably including inoculating stem cells into Medium A for a first culture to obtain embryoid bodies; Preferably, the method further includes step (3) of maintaining and culturing kidney organoids, preferably including long-term culturing of the obtained kidney organoids using Medium I.

6. The method according to claim 5, wherein, In step (2-1), the time of the second culturing is 20-30 h; and / or, in step (2-2), the time of the third culturing is 40-50 h; and / or, in step (2-3), the time of the fourth culturing is 40-50 h; and / or, in step (2-4), the time of the fifth culturing is 40-50 h; and / or, in step (2-5), the time of the sixth culturing is 40-50 h; and / or, in step (2-6), the time of the seventh culturing is 40-50 h; and / or, in step (2-7), the time of the eighth culturing is 40-50 h; and / or, in step (2-8), the time of the ninth culturing is 40-50 h; and / or, in step (3), the time of the long-term culturing is 2-3 months; and / or, in step (1), the culturing time of the first culturing is 24-36 h; Preferably, during the first culturing process, Medium A is replaced every 12-24 h.

7. The method according to claim 5 or 6, wherein The method further includes removing the medium and washing the culture before each step. Preferably, the method does not include the treatment of embedding the culture obtained in each step with Matrigel.

8. A kidney organoid constructed by the method according to any one of claims 4-7, and the application of the kidney organoid in at least one of drug screening, disease modeling, and toxicological detection.

9. The application of the medium according to any one of claims 1-3, and / or the method according to any one of claims 4-7 in at least one of improving the success rate of kidney organoid construction, shortening the maturation time of organoids, extending the culturing time of kidney organoids, reducing the cost of kidney organoid construction, and increasing the throughput of kidney organoid construction.

10. A method for drug safety detection, characterized in that, The method includes bringing a hepatobiliary organoid and a kidney organoid into contact with a test drug respectively, and comprehensively judging the safety of the drug based on the responses of the hepatobiliary organoid and the kidney organoid to the drug, wherein the kidney organoid is a kidney organoid constructed by the method according to any one of claims 4-7.

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