Renal tubular organoid culture medium and application, and renal tubular organoid culture method

Abstract

The present invention provides a renal tubular organoid culture medium and its application, as well as a method for culturing renal tubular organoids. The renal tubular organoid culture medium of the present invention has a good supporting and promoting effect on the growth of renal tubular organoids. Typical renal tubular organoids can be obtained in about 7 days, and the consistency and heterogeneity of patient-derived tissue are well preserved in vitro, laying the foundation for further research and application of renal tubules. The culturing method of the renal tubular organoids of the present invention, which is cultured using the renal tubular organoid culture medium of the present invention, can effectively simulate renal tubular tissue and provide a powerful platform for the study of the pathogenesis and pathogenic factors of renal tubular-related diseases and the study of drug toxicity of normal renal tubular tissue. The culturing method of the present invention has simple and clear steps, and the operator has little influence on the culture results, which improves the consistency of the culture results and provides convenient conditions for subsequent large-scale culture.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a renal tubular organoid culture medium and application thereof, and a renal tubular organoid culture method. Background Art

[0002] The kidneys are vital organs in the human body, clearing metabolic products, certain waste products, and toxins from the body, producing urine, and regulating water and electrolyte balance and maintaining acid-base balance. The kidneys also have endocrine functions, producing substances such as renin and erythropoietin. These kidney functions ensure a stable internal environment and enable normal metabolism. The renal tubules and tubular interstitium constitute crucial parts of the kidneys and are the primary sites of injury response, making them susceptible to various insults, including hypoxia, infection, toxins, metabolic disorders, and aging. Accumulating evidence indicates that renal tubular epithelial cells play a crucial role in kidney repair and progression to chronic kidney disease.

[0003] In order to understand tubular-related kidney diseases and translate this knowledge into clinical applications, representative and stable preclinical tubular models are urgently needed. Currently, the most common preclinical models are mouse models and two-dimensional cultured cell lines. However, these two models have many defects that hinder their clinical application. Cell lines cannot reflect the heterogeneity of patients and therefore cannot fundamentally represent the complexity of kidney-related diseases. Although human tumor xenografts can maintain the genetic and histological characteristics of the original kidney disease, the genetic characteristics and growth environment of mice are different from those of kidney disease patients, which largely leads to the limitations of their application.

[0004] Organoids are a newly developed in vitro three-dimensional culture technology. Compared with conventional cell line culture, the tissue structure and function formed by the organoid culture system are more complex. It has the advantages of high fit, stable passage, short culture cycle, and high success rate. It is currently an ideal in vitro culture model. Because it maintains the key characteristics of the parent tissue, organoids can be used for drug screening, disease modeling, etc., contributing to personalized treatment. Therefore, the construction of renal tubular organoids is of great significance for the study of renal tubular-related kidney diseases. However, there is currently no better technical solution for the construction of renal tubular organoids. Summary of the Invention

[0005] In view of this, the present invention provides a culture medium for renal tubular organoids and a method for culturing renal tubular organoids to solve or at least partially solve the technical problems existing in the prior art.

[0006] In a first aspect, the present invention provides a renal tubular organoid culture medium comprising a basal culture medium, L-alanyl-L-glutamine, a pH buffer, an antioxidant, a serum replacement, a human recombinant protein, a TGF-β receptor inhibitor, a growth factor, and a ROCK inhibitor.

[0007] Preferably, the renal tubular organoid culture medium further comprises primary cell antibiotics and penicillin / streptomycin dual antibodies.

[0008] Preferably, the basal medium of the renal tubular organoid culture medium is Advanced DMEM / F12 basal medium;

[0009] and / or, the pH buffer is a HEPES buffer;

[0010] and / or, the antioxidant is N-acetylcysteine;

[0011] and / or, the serum replacement is B-27 supplement;

[0012] and / or, the human recombinant protein is R-Spondin-3;

[0013] and / or, the TGF-β receptor inhibitor is A83-01;

[0014] and / or, the growth factors include EGF and FGF-10;

[0015] And / or, the ROCK inhibitor is Y27632.

[0016] Preferably, in the renal tubular organoid culture medium, the primary cell antibiotic is primocin.

[0017] Preferably, in the renal tubular organoid culture medium, the concentration of L-alanyl-L-glutamine is 1-3 mM, the concentration of the HEPES buffer is 10-25 mM, the concentration of the N-acetylcysteine ​​is 0.5-2 mM, the volume concentration of the B-27supplement is 1-2%, the concentration of the R-Spondin-3 is 200-250 ng / mL, the concentration of the A83-01 is 5-10 μM, the concentration of the EGF is 50-100 ng / mL, the concentration of the FGF-10 is 50-100 ng / mL, the concentration of the Y27632 is 5-10 μM, the concentration of the primocin is 80-120 μg / mL, and the concentration of the penicillin / streptomycin dual antibody is 50-100 IU / mL.

[0018] In a second aspect, the present invention further provides a use of the renal tubular organoid culture medium in culturing renal tubular organoids.

[0019] In a third aspect, the present invention further provides a method for culturing renal tubular organoids, which uses the renal tubular organoid culture medium for culturing.

[0020] Preferably, the method for culturing renal tubular organoids comprises the following steps:

[0021] Obtain kidney tissue samples;

[0022] Kidney tissue samples were washed, digested, and resuspended to obtain kidney cell pellets;

[0023] The renal tubular organoid culture medium was mixed with the renal cell pellet to obtain a cell solution;

[0024] The cell solution was mixed with Matrigel and then inoculated into the well plate. After inoculation, the plate was left to stand until the Matrigel solidified.

[0025] After the Matrigel matrix gel solidifies, renal tubular organoid culture medium is added for culture to obtain renal tubular organoids.

[0026] Preferably, the method for culturing renal tubular organoids comprises washing, digesting, and resuspending a renal tissue sample to obtain a renal cell pellet, and specifically comprises the following steps:

[0027] The kidney tissue samples were minced and washed with DPBS solution containing penicillin / streptomycin double antibody;

[0028] Add trypsin digestion solution to the cleaned kidney tissue sample and digest it in a 37°C, 5% CO2 incubator for 40 to 50 minutes. Then add Advanced DMEM / F12 basal culture medium to terminate the digestion. After centrifugation, discard the supernatant and collect the precipitate to obtain the kidney cell pellet.

[0029] Preferably, the method for culturing renal tubular organoids, if the obtained renal cell precipitate is red, further comprises: adding red blood cell lysate to the precipitate, incubating, centrifuging again, discarding the supernatant, and collecting the precipitate to obtain the renal cell precipitate before mixing the renal tubular organoid culture medium with the renal cell precipitate;

[0030] In the step of mixing the cell solution with Matrigel, the volume ratio of the cell solution to the Matrigel is (1-2):(1-2);

[0031] After the Matrigel matrix gel solidifies, renal tubular organoid culture medium is added for culturing, and the culture is adjusted to: 37°C, 5% CO2 environment;

[0032] After the Matrigel matrix gel solidifies, add the renal tubular organoid culture medium for culture, and replace the renal tubular organoid culture medium every 2 to 3 days.

[0033] The renal tubular organoid culture medium and application, and the renal tubular organoid culture method of the present invention have the following beneficial effects compared to the prior art:

[0034] 1. The renal tubular organoid culture medium of the present invention comprises: a basal culture medium, L-alanyl-L-glutamine, a pH buffer, an antioxidant, a serum replacement, a human recombinant protein, a TGF-β receptor inhibitor, a growth factor, and a ROCK inhibitor. The culture medium of the present invention effectively supports and promotes the growth of renal tubular organoids, and typical renal tubular organoids can be obtained in approximately 7 days. The culture medium effectively retains the consistency and heterogeneity of patient-derived tissue in vitro, laying the foundation for further research and application of renal tubules.

[0035] 2. The culture method of the renal tubular organoids of the present invention adopts the renal tubular organoid culture medium of the present invention for culture, which can effectively simulate renal tubular tissue and provide a powerful platform for the study of the pathogenesis and pathogenic factors of renal tubular-related diseases and the study of drug toxicity of normal renal tubular tissue; the culture method of the present invention has simple and clear steps, and the operator has little influence on the culture results, which improves the consistency of the culture results and provides convenient conditions for subsequent large-scale culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0037] Figure 1 This is a morphological image of the renal tubular organoid cultured in the renal tubular organoid culture medium on day 7 in Example 2 of the present invention under an optical microscope; wherein the magnification is 100 times;

[0038] Figure 2 This is a morphological image of the renal tubular organoids stably cultured in the renal tubular organoid culture medium on the 60th day under an optical microscope in Example 2 of the present invention; wherein the magnification is 200 times;

[0039] Figure 3This is a diagram showing the results of HE staining and immunohistochemistry of renal tubular organoids in Example 2 of the present invention;

[0040] Figures 4-5 This is the transmission electron microscopy result of the renal tubular organoid in Example 2 of the present invention. DETAILED DESCRIPTION

[0041] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0042] For a better understanding of the present invention and not to limit the scope of the present invention, all numbers used in this application to express amounts, percentages, and other numerical values ​​should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may vary depending on the desired properties to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods.

[0043] It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of the embodiments. In addition, in the description of this application, the term "including" means "including but not limited to". Various embodiments of the present invention may be presented in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be understood as a rigid limitation on the scope of the invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0044] The embodiments of the present application provide a renal tubular organoid culture medium, comprising: a basal culture medium, L-alanyl-L-glutamine, a pH buffer, an antioxidant, a serum replacement, a human recombinant protein, a TGF-β receptor inhibitor, a growth factor, and a ROCK inhibitor.

[0045] The culture medium of the present invention has a good supporting and promoting effect on the growth of renal tubular organoids. Typical renal tubular organoids can be obtained in about 7 days. The consistency and heterogeneity of patient-derived tissues are well preserved in vitro, laying the foundation for further research and application of renal tubules.

[0046] In some embodiments, the culture medium for renal tubular organoids further includes primary cell antibiotics and penicillin / streptomycin.

[0047] In some embodiments, the basal culture medium is DMEM / F12 basal culture medium, specifically Advanced DMEM / F12.

[0048] In some embodiments, the pH buffer is HEPES buffer.

[0049] In some embodiments, the antioxidant is N-acetylcysteine.

[0050] In some embodiments, the serum replacement is a B-27 supplement.

[0051] In some embodiments, the human recombinant protein is R-Spondin-3.

[0052] In some embodiments, the TGF-β receptor inhibitor is A83-01.

[0053] In some embodiments, the growth factors include EGF, FGF-10.

[0054] In some embodiments, the ROCK inhibitor is Y27632.

[0055] In some embodiments, the primary cell antibiotic is primocin.

[0056] Specifically, Advanced DMEM / F12 basal culture medium is used as a solvent, and L-alanyl-L-glutamine, pH buffer, antioxidant, serum replacement, human recombinant protein, TGF-β receptor inhibitor, growth factor, ROCK inhibitor, and penicillin / streptomycin double antibody are added to the basal culture medium to obtain the renal tubular organoid culture medium of the present application.

[0057] The renal tubular organoid culture medium of the present invention comprises L-alanyl-L-glutamine, HEPES, an antioxidant N-acetylcysteine, and a serum substitute as basic nutrients; wherein L-alanyl-L-glutamine (GlutaMax) is a cell culture additive that can improve cell viability and growth and prevent glutamine degradation and ammonia accumulation during long-term culture; HEPES is a zwitterionic organic chemical buffer commonly used in cell culture, N-acetylcysteine ​​is an antioxidant, and B-27 is a serum substitute; human recombinant proteins are R-Spondin-3 and A83-01 as signaling pathway factors; R-Spondin-3 is a positive regulator of the wnt / β-catenin signaling pathway, and studies have shown that it is crucial for renal homeostasis and repair; A83-01 is an effective inhibitor of TGF-β type I receptor ALK5 / ALK4 / ALK7, which can prevent growth arrest and epithelial-mesenchymal transition. The culture medium of the renal tubular organoids of the present invention contains two factors, EGF and FGF-10. EGF (epidermal growth factor) is an effective growth factor that can stimulate the proliferation of various epidermal and epithelial cells. FGF-10 (fibroblast growth factor) plays an important role in the development of the liver and kidneys and can promote the proliferation of epithelial cells. The inhibitor Y27632 is a highly effective inhibitor of human ROCK-1 and ROCK-2 isozymes, which can prevent cell apoptosis and improve the survival rate of organoids during passage. The renal tubular organoid culture medium also includes antibacterial ingredients primocin and penicillin / streptomycin double resistance, wherein primocin is an antibiotic for protecting primary cells from microbial contamination, and has a killing effect on bacteria, mycoplasma and fungi. Penicillin / streptomycin double resistance has an effective combined antibacterial effect on Gram-positive bacteria and Gram-negative bacteria, and can be used to prevent cell culture from being contaminated by bacteria. Further ensure that the culture medium can be conducive to the culture of renal tubular organoids.

[0058] In some embodiments, the concentration of L-alanyl-L-glutamine is 1-3 mM, the concentration of HEPES buffer is 10-25 mM, the concentration of N-acetylcysteine ​​is 0.5-2 mM, the volume concentration of B-27supplement is 1-2%, the concentration of R-Spondin-3 is 200-250 ng / mL, the concentration of A83-01 is 5-10 μM, the concentration of EGF is 50-100 ng / mL, the concentration of FGF-10 is 50-100 ng / mL, the concentration of Y27632 is 5-10 μM, the concentration of primocin is 80-120 μg / mL, and the concentration of penicillin / streptomycin dual antibody is 50-100 IU / mL.

[0059] Based on the same inventive concept, the present invention also provides a use of the above-mentioned renal tubular organoid culture medium in culturing renal tubular organoids.

[0060] Based on the same inventive concept, the present invention also provides a method for culturing renal tubular organoids, which uses the above-mentioned renal tubular organoid culture medium for culturing.

[0061] In some embodiments, the method for culturing renal tubular organoids comprises the following steps:

[0062] S1. Obtain kidney tissue samples;

[0063] S2. Wash, digest, and resuspend the kidney tissue sample to obtain a kidney cell pellet;

[0064] S3, mixing the renal tubular organoid culture medium with the renal cell pellet to obtain a cell solution;

[0065] S4. Mix the cell solution with Matrigel and then inoculate into the well plate. After inoculation, let it stand until the Matrigel solidifies.

[0066] S5. After the Matrigel matrix gel solidifies, add renal tubular organoid culture medium for culturing to obtain renal tubular organoids.

[0067] In some embodiments, step S1 is specifically as follows: a 4.5×2×1 cm tumor is found in the renal pelvis of a renal urothelial carcinoma specimen (confirmed by pathology) that has been surgically removed. A 3 mm diameter renal pelvis is cut out of the gross kidney specimen away from the tumor using a sterile scalpel. 3 Normal kidney tissue of about 100 grams is collected to obtain kidney tissue samples.

[0068] In some embodiments, step S2 specifically includes the following steps:

[0069] S21. Mince the kidney tissue sample and wash it with DPBS solution containing penicillin / streptomycin.

[0070] S22. Add 3-7 mL of trypsin digestion solution to the cleaned kidney tissue sample and digest in a 37°C, 5% CO2 incubator for 40-50 min. Then add Advanced DMEM / F12 basal culture medium to terminate the digestion. After centrifugation, discard the supernatant and collect the precipitate to obtain the kidney cell pellet.

[0071] In some embodiments, if the obtained renal cell precipitate is red, before mixing the renal tubular organoid culture medium with the renal cell precipitate, the process further includes: adding 1 to 2 mL of red blood cell lysis buffer to the precipitate, incubating, centrifuging again, discarding the supernatant, and collecting the precipitate to obtain the renal cell precipitate; if, after digestion and resuspension, the obtained renal cell precipitate is not red, proceed directly to step S3.

[0072] In some embodiments, in the step of mixing the cell solution with Matrigel, the volume ratio of the cell solution to the Matrigel is (1-2):(1-2), preferably, the volume ratio is 1:1.

[0073] In some embodiments, after the Matrigel is solidified, renal tubular organoid culture medium is added for culturing, and the culture is adjusted to: 37° C. and 5% CO 2 environment.

[0074] In some embodiments, after the Matrigel matrix gel solidifies, renal tubular organoid culture medium is added for culturing, and new renal tubular organoid culture medium is replaced every 2 to 3 days. After culturing for 7 days, renal tubular organoids are obtained.

[0075] In some embodiments, in step S22, centrifugation is performed again, the supernatant is discarded, and the precipitate is collected to obtain the kidney cell precipitate, wherein the centrifugation conditions are: 4°C, 300g, and centrifugation for 5 minutes.

[0076] In some embodiments, 1-2 mL of red blood cell lysis buffer is added to the precipitate, incubated, centrifuged again, the supernatant is discarded, and the precipitate is collected to obtain a kidney cell precipitate, wherein the centrifugation conditions are: 300g, centrifugation for 5 minutes.

[0077] The culture method of the renal tubular organoids of the present invention can effectively simulate renal tubular tissue and provide a powerful platform for the study of the pathogenesis and pathogenic factors of renal tubular diseases and the study of drug toxicity of normal renal tubular tissue. The culture method of the present invention has simple and clear steps, and the operator has little influence on the culture results, which improves the consistency of the culture results and provides convenient conditions for subsequent large-scale culture.

[0078] The following further describes the culture method of the renal tubular organoid culture medium and the renal tubular organoid of the present application with specific examples. This part further illustrates the present invention in conjunction with specific examples, but should not be construed as limiting the present invention. If not otherwise specified, the technical means adopted in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this area.

[0079] Example 1

[0080] The present application provides a renal tubular organoid culture medium, comprising: a basal culture medium, L-alanyl-L-glutamine, a pH buffer, an antioxidant, a serum replacement, a human recombinant protein, a TGF-β receptor inhibitor, a growth factor, a ROCK inhibitor, primary cell antibiotics, and penicillin / streptomycin dual antibodies;

[0081] The basal medium was Advanced DMEM / F12, the pH buffer was HEPES buffer, the antioxidant was N-acetylcysteine, the serum replacement was B-27supplement, the human recombinant protein was R-Spondin-3, the TGF-β receptor inhibitor was A83-01, the growth factors included EGF and FGF-10, the ROCK inhibitor was Y27632, and the primary cell antibiotic was primocin.

[0082] Specifically, the names, suppliers, and final concentrations of the components of the renal tubular organoid culture medium are shown in Table 1 below.

[0083] Table 1 - Name, supplier, and final concentration of each component of renal tubular organoid culture medium

[0084]

[0085]

[0086] Example 2

[0087] The present application also provides a method for culturing renal tubular organoids, wherein the renal tubular organoid culture medium of Example 1 is used for culturing. All operation-related steps are performed in a biosafety cabinet to maintain aseptic operation, and specifically include the following steps:

[0088] S1. Obtaining renal tissue: A 4.5 × 2 × 1 cm tumor was found in the renal pelvis of a surgically resected renal urothelial carcinoma specimen (confirmed by pathology). A 3 mm septum was removed from the gross renal specimen away from the tumor using a sterile scalpel. 3 Normal kidney tissue on the left and right to obtain kidney tissue samples;

[0089] S2. Place the kidney tissue sample from S1 in a culture dish and wash it repeatedly with DPBS (Dublin's Phosphate Buffered Saline) containing 100 IU / mL penicillin / streptomycin to remove blood until the wash solution is clear. Remove any remaining DPBS in the culture dish.

[0090] S3. Add 2 mL of Advanced DMEM / F12 basal medium to the culture dish and cut the red kidney tissue into 1 mm pieces. 3 of fragments;

[0091] S4. Transfer the mesonephric tissue pieces from S3 to a 50 mL centrifuge tube, add 5 mL of trypsin digestion solution to the centrifuge tube, and digest in a shaker at 37°C with a volume concentration of 5% CO2 for 45 min.

[0092] S5. Add 20 mL of Advanced DMEM / F12 basal medium to the centrifuge tube in S4 to terminate the digestion. Grind the digested tissue and cell suspension through a cell strainer with a pore size of 70 μm, then centrifuge at 4°C and 300 g for 5 min. Discard the supernatant to obtain the kidney tissue cell pellet.

[0093] S6. Add 2 mL of red blood cell lysis buffer to the renal tissue cell pellet in S5; pipette up and down, and incubate at room temperature (25°C) for 2 min; after the incubation, centrifuge at 4°C, 300g for 5 min, and discard the supernatant to obtain the cell pellet, which is the renal cell pellet;

[0094] S7, mixing the renal tubular organoid culture medium in Example 1 with the renal cell pellet in S6 to obtain a cell solution, then mixing the cell solution with Matrigel matrix gel at a volume ratio of 1:1, and after mixing, inoculating 50 μL per well into an ultra-low adsorption 24-well plate for gel drop inoculation;

[0095] S8. After inoculation, the mixture was placed in a 37°C incubator for 30 min until the Matrigel gel solidified.

[0096] S9. After the Matrigel gel solidifies, add 500 μL of the renal tubular organoid culture medium described in Example 1, which has been preheated at 37° C., to each well and culture in a cell culture incubator at 37° C. and 5% CO 2 .

[0097] S10. Replace the renal tubular organoid culture medium every 3 days. After 7 days of culture, renal tubular organoids are obtained.

[0098] According to the culture method in Example 2, renal tubular organoids were obtained after 7 days of culture. The morphology of the renal tubular organoids under the microscope was as follows: Figure 1 As shown, a large number of tubular structures can be seen, the luminal morphology is relatively consistent, and circular and regularly distributed cell nuclei can be seen in the luminal space.

[0099] The renal tubular organoids obtained in Example 2 can be passaged for 7 to 14 days and can still grow stably after multiple passages. The morphology of the renal tubular organoids under the microscope after culturing in the renal tubular organoid culture medium in Example 1 for 60 days is as follows: Figure 2 As shown. Figure 2 Tortuous, loop-like tubular structures, tubular branches, and varying numbers of cell nuclei can be seen.

[0100] The renal tubular organoids obtained in Example 2 were embedded and sectioned and subjected to conventional HE staining and immunohistochemical staining to detect the expression of markers including PAX8, CK7, CAM5.2, CD10, and EMA. The results are as follows: Figure 3 shown.

[0101] from Figure 3 As can be seen, HE staining shows that the branched labyrinthine tubular structures fuse with each other to form an organ-like structure, and the cell nucleus is located at the periphery of the tubular lumen; PAX8 is a specific marker for the nucleus of renal epithelial cells, which is diffusely stained and distributed at the base of the renal tubules; CK7, CAM5.2, CD10, and EMA are positive, which are specific markers of epithelial cells, confirming the immunohistochemical specificity of renal tubular epithelial cells.

[0102] The renal tubular organoids in Example 2 were embedded and sliced ​​and then analyzed by transmission electron microscopy. The results are as follows: Figure 4 and Figure 5 As shown. Figures 4-5 Obvious tubular structure can be seen. The epithelial cells are cubic, with round nuclei and nucleoli near the base. Abundant mitochondria are seen in the cytoplasm, lateral processes are seen on the sides of the cells, and there is a brush border on the luminal surface of the epithelial cells.

[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for culturing renal tubular organoids, characterized in that: The following steps are involved: Obtain kidney tissue samples; Kidney tissue samples were washed, digested, and resuspended to obtain kidney cell pellets; The renal tubular organoid culture medium was mixed with the renal cell pellet to obtain a cell solution; The cell solution was mixed with Matrigel and then inoculated into the well plate. After inoculation, the plate was left to stand until the Matrigel solidified. After the Matrigel matrix gel solidifies, renal tubular organoid culture medium is added for culture to obtain renal tubular organoids; The kidney tissue sample is washed, digested, and resuspended to obtain a kidney cell pellet, specifically comprising the following steps: The kidney tissue samples were minced and washed with DPBS solution containing penicillin / streptomycin double antibody; Add trypsin digestion solution to the cleaned kidney tissue sample and digest in a 37°C, 5% CO2 incubator for 40-50 minutes. Then add Advanced DMEM / F12 basal medium to terminate the digestion. After centrifugation, discard the supernatant and collect the precipitate to obtain the kidney cell pellet. The renal tubular organoid culture medium comprises a basal culture medium, L-alanyl-L-glutamine, a pH buffer, an antioxidant, a serum replacement, a human recombinant protein, a TGF-β receptor inhibitor, a growth factor, and a ROCK inhibitor; and also comprises primary cell antibiotics and penicillin / streptomycin dual antibodies; The basal medium is Advanced DMEM / F12 basal medium; the pH buffer is HEPES buffer; the antioxidant is N-acetylcysteine; the serum substitute is B-27supplement; the human recombinant protein is R-Spondin-3; the TGF-β receptor inhibitor is A83-01; the growth factors include EGF and FGF-10; the ROCK inhibitor is Y27632; and the primary cell antibiotic is primocin. The concentration of the L-alanyl-L-glutamine is 1-3 mM, the concentration of the HEPES buffer is 10-25 mM, the concentration of the N-acetylcysteine ​​is 0.5-2 mM, the volume concentration of the B-27supplement is 1-2%, the concentration of the R-Spondin-3 is 200-250 ng / mL, the concentration of the A83-01 is 5-10 μM, the concentration of the EGF is 50-100 ng / mL, the concentration of the FGF-10 is 50-100 ng / mL, the concentration of the Y27632 is 5-10 μM, the concentration of the primocin is 80-120 μg / mL, and the concentration of the penicillin / streptomycin dual antibody is 50-100 IU / mL.

2. The method for culturing renal tubular organoids according to claim 1, wherein: If the obtained renal cell pellet is red, before mixing the renal tubular organoid culture medium with the renal cell pellet, the steps further include: adding red blood cell lysis buffer to the pellet, incubating, centrifuging again, discarding the supernatant, and collecting the pellet to obtain the renal cell pellet; In the step of mixing the cell solution with Matrigel, the volume ratio of the cell solution to the Matrigel is (1-2):(1-2); After the Matrigel matrix gel solidifies, renal tubular organoid culture medium is added for culturing under the following conditions: 37°C, 5% CO2 concentration; After the Matrigel matrix gel solidifies, add the renal tubular organoid culture medium for culture, and replace the renal tubular organoid culture medium every 2 to 3 days.

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