Culture medium and culture method of podocyte fate organoid

Abstract

The invention relates to the technical field of biomedical engineering, in particular to a culture medium and a culture method of podocyte fate organoid. Compared with human induced pluripotent stem cells (hiPSCs), cells separated from human adult kidney tissues are taken as objects to induce and culture podocyte fate organoid, so that the podocyte fate organoid has better gene stability and can better reproduce original tissue phenotypes. Compared with a hiPSCs-derived kidney organ, the culture method of the podocyte fate organ provided by the invention has the advantages that a culture system is simplified; the serum-free culture medium component based on a pure compound combination also greatly improves the safety of a culture product possibly used for clinical biological treatment.

Description

A culture medium and a method for culturing organoids with podocyte fate. Technical Field

[0001] This invention relates to the field of biomedical engineering technology, specifically to a culture medium and a method for culturing organoids with podocyte fate. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Podocytes, also known as visceral epithelial cells of Bowman's capsule, attach to the outer side of the glomerular basement membrane. Together with vascular endothelial cells and the glomerular basement membrane, they form the glomerular blood filtration barrier, playing a crucial role in the kidney's filtration function. Podocyte injury is a disease characterized by abnormalities in the podocytes within the glomeruli of the kidney. Disorders of glucose metabolism, immune system dysfunction, and viral infections can all damage podocytes. Clinically, patients present with massive proteinuria, often accompanied by impaired renal tubular function.

[0004] Obtaining podocytes or podocyte-fate organoids in vitro can be used to study the pathogenesis of podocyte injury, develop therapeutic techniques, and block the occurrence of injury. However, podocytes lack proliferative capacity, making their in vitro culture challenging. Initially, other kidney cell lines were used to study the pathogenesis of podocyte diseases. It wasn't until around the 1970s that a method for isolating primary podocytes from glomeruli in vivo was established, allowing for direct study of podocytes. Because primary cells cannot proliferate or be passaged, the number of cells obtained from each primary culture is limited, restricting the application of primary podocytes. Given these limitations of primary cells, immortalization techniques were used to establish podocyte lines. While podocyte lines overcome the problem of limited podocyte numbers obtained from primary cultures, the expression of podocyte marker proteins in immortalized podocyte lines is unstable, and even phenotypic differences exist between podocyte lines of the same species. Furthermore, attempts have been made to obtain podocyte lines through induced pluripotent stem cell (iPSC) induction culture; however, iPSC-derived organoids exhibit poor genomic stability and pose a risk of tumorigenicity. Therefore, obtaining phenotypically stable podocytes or podocyte fate organoids has positive significance and important role in understanding and treating kidney diseases. Summary of the Invention

[0005] To overcome the above problems, the present invention provides a culture medium and a method for culturing organoids with podocyte fate.

[0006] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: In its first aspect, the present invention provides a method for culturing podocyte fate organoids, comprising the following steps: separating human adult kidney tissue into monocellular cells, resuspending them in a matrix gel to allow the monocellular cells to aggregate for culture, and culturing in cpp medium to obtain podocyte fate organoids; wherein the cpp medium is prepared using Advanced DMEM / F12 medium as the substrate, with the addition of trachomatin, P7C3, and P7C3-A20; the concentration of trachomatin is 0.1~1 μM, the concentration of P7C3 is 1~10 μM, and the concentration of P7C3-A20 is 1~10 μM.

[0007] P7C3 is 3,6-dibromo-A-[(phenylamino)methyl]-9H-carbazole-9-ethanol, belonging to NAMPT activators, CAS number: 301353-96-8.

[0008] P7C3-A20 is 3,6-dibromo-β-fluoro-N-(3-methoxyphenyl)-9H-carbazole-9-propylamine, a derivative of P7C3, CAS number: 1235481-90-9.

[0009] In one or more embodiments, a method for separating human adult kidney tissue into monosodium globules includes: cutting human adult kidney tissue into small pieces, removing blood vessels and connective tissue, adding a DMEM / F12 mixture containing hyaluronidase and collagenase IV, placing it in an incubator for digestion, and then adding DMEM / F12 mixture to terminate digestion; adding erythrocyte lysis buffer to the cells after digestion is terminated, and then adding DMEM / F12 mixture to terminate erythrocyte lysis to obtain the monosodium globules.

[0010] In one or more embodiments, the method of adding matrix gel to resuspend the monosodium cells, causing them to aggregate into clumps for culture, and adding CPP medium to culture to obtain podocyte fate organoids includes: adding matrix gel to resuspend the monosodium cells and then seeding them into a culture plate with droplets of 3-5 mm in diameter; culturing the culture plate in an upright position and then inverted position after seeding to make the seeded cells closer to spheres; adding CPP medium to the culture plate after inverted culture and culturing it in a CO2 incubator, changing the medium every 2-4 days.

[0011] In one or more embodiments, the CPP culture medium further includes antibiotics, antioxidants, glutamine supplements, ROCK kinase inhibitors, growth factors, serum substitutes, and TGF-β inhibitors.

[0012] Preferably, the antibiotic is selected from antibiotics routinely added to the culture medium, such as penicillin, streptomycin, gentamicin, kanamycin, and the primary cell antibiotic Primocin.TM One or more of chloramphenicol; preferably penicillin, streptomycin, and the primary cell antibiotic Primocin. TM .

[0013] Preferably, the antioxidant is acetylcysteine ​​(NAC); NAC can effectively improve the cell's ability to scavenge free radicals and provide protection for the cell.

[0014] Preferably, the glutamine supplement includes one or more of L-glutamine or L-alanyl-L-glutamine (GlutaMAX), and more preferably, the glutamine supplement is L-alanyl-L-glutamine (GlutaMAX).

[0015] Preferably, the ROCK kinase inhibitor includes one or more of K-115 (CAS No.: 887375-67-9), SAR407899 (CAS No.: 923359-38-0), RKI-1447 (CAS No.: 1342278-01-6), and Y-27632 (CAS No.: 146986-50-7), with Y-27632 being the most preferred.

[0016] Preferably, the growth factor includes one or more of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), growth hormone-releasing inhibitory factor (SRIH), nerve growth factor (NGF), and stem cell-regulated growth factor (R-spondin); more preferably, it is a combination of epidermal growth factor (EGF) and stem cell-regulated growth factor (R-spondin).

[0017] Preferably, the serum substitute includes one or more of UltroGRO and B-27, with B-27 being the most preferred.

[0018] Preferably, the TGF-β inhibitor includes one of SB431542 (CAS No.: 301836-41-9), LDN193189 (CAS No.: 1062368-24-4), SB525334 (CAS No.: 356559-20-1), and A83-01 (CAS No.: 909910-43-6); preferably A83-01.

[0019] More preferably, the cpp culture medium comprises: Advanced DMEM / F12, penicillin-streptomycin, and Primocin. TMThe culture medium contains GlutaMAX, B27, acetylcysteine, epidermal growth factor, stem cell regulatory growth factor, Y27632, chrysogenin, P7C3, P7C3-A20, and A83-01. The above culture medium components are non-ionic amphoteric buffers, such as HEPES buffer.

[0020] More preferably, the cpp culture medium comprises: 90-110 units / mL Penicillin-90-110 μg / mL Streptomycin Solution; HEPES buffer, 9-11 mM; GlutaMAX, 0.09-0.11 mg / mL; B-27, 0.09-0.11 mg / mL; Primocin TM 0.09–0.11 mg / mL; NAC, 0.9–1.1 mM; EGF, 40–60 ng / mL; Y27632, 9–11 μM; A83-01, 4–6 μM; R-spondin, 90–110 ng / mL; trachomatin, 0.1–1 μM; P7C3, 1–10 μM; P7C3-A20, 11–10 μM; each component was diluted in Advanced DMEM / F12.

[0021] In a second aspect, the present invention provides a cpp medium for culturing organoids with podocyte fate, the cpp medium being prepared by adding trachomatin, P7C3 and P7C3-A20 to Advanced DMEM / F12 medium as the substrate; the concentration of trachomatin is 0.1~1 μM, the concentration of P7C3 is 1~10 μM, and the concentration of P7C3-A20 is 1~10 μM.

[0022] In one or more embodiments, the CPP culture medium further includes antibiotics, antioxidants, glutamine supplements, ROCK kinase inhibitors, growth factors, serum substitutes, and TGF-β inhibitors.

[0023] Preferably, the antibiotic is selected from antibiotics routinely added to the culture medium, such as penicillin, streptomycin, gentamicin, kanamycin, and the primary cell antibiotic Primocin. TM One or more of chloramphenicol; preferably penicillin, streptomycin, and the primary cell antibiotic Primocin. TM .

[0024] Preferably, the antioxidant is acetylcysteine ​​(NAC); NAC can effectively improve the cell's ability to scavenge free radicals and provide protection for the cell.

[0025] Preferably, the glutamine supplement includes one or more of L-glutamine or -L-alanyl-L-glutamine (GlutaMAX), and more preferably, the glutamine supplement is -L-alanyl-L-glutamine (GlutaMAX).

[0026] Preferably, the ROCK kinase inhibitor includes one or more of K-115 (CAS No.: 887375-67-9), SAR407899 (CAS No.: 923359-38-0), RKI-1447 (CAS No.: 1342278-01-6), and Y-27632 (CAS No.: 146986-50-7), with Y-27632 being the most preferred.

[0027] Preferably, the growth factor includes one or more of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), growth hormone-releasing inhibitory factor (SRIH), nerve growth factor (NGF), and stem cell-regulated growth factor (R-spondin); more preferably, it is a combination of epidermal growth factor (EGF) and stem cell-regulated growth factor (R-spondin).

[0028] Preferably, the serum substitute includes one or more of UltroGRO and B-27, with B-27 being the most preferred.

[0029] Preferably, the TGF-β inhibitor includes one of SB431542 (CAS No.: 301836-41-9), LDN193189 (CAS No.: 1062368-24-4), SB525334 (CAS No.: 356559-20-1), and A83-01 (CAS No.: 909910-43-6); preferably A83-01.

[0030] More preferably, the cpp culture medium comprises: Advanced DMEM / F12, penicillin-streptomycin, and Primocin. TM The culture medium contains GlutaMAX, B27, acetylcysteine, epidermal growth factor, stem cell regulatory growth factor, Y27632, chrysogenin, P7C3, P7C3-A20, and A83-01. The above culture medium components are non-ionic amphoteric buffers, such as HEPES buffer.

[0031] More preferably, the cpp culture medium comprises: 90-110 units / mL Penicillin-90-110 μg / mL Streptomycin Solution; HEPES buffer, 9-11 mM; GlutaMAX, 0.09-0.11 mg / mL; B-27, 0.09-0.11 mg / mL; Primocin TM 0.09–0.11 mg / mL; NAC, 0.9–1.1 mM; EGF, 40–60 ng / mL; Y27632, 9–11 μM; A83-01, 4–6 μM; R-spondin, 90–110 ng / mL; trachomatin, 0.1–1 μM; P7C3, 1–10 μM; P7C3-A20, 11–10 μM; each component was diluted in Advanced DMEM / F12.

[0032] A third aspect of the invention provides the use of trachomatin, P7C3, and P7C3-A20 in the culture of podocyte fate organoids.

[0033] In one or more embodiments, the application includes at least the following: for preparing culture media for podocyte fate organoids.

[0034] Preferably, the podocyte fate organoid culture medium is CPP medium, which further includes antibiotics, antioxidants, glutamine supplements, ROCK kinase inhibitors, growth factors, serum substitutes, and TGF-β inhibitors.

[0035] Preferably, the antibiotic is selected from antibiotics routinely added to the culture medium, such as penicillin, streptomycin, gentamicin, kanamycin, and the primary cell antibiotic Primocin. TM One or more of chloramphenicol; preferably penicillin, streptomycin, and the primary cell antibiotic Primocin. TM .

[0036] Preferably, the antioxidant is acetylcysteine ​​(NAC); NAC can effectively improve the cell's ability to scavenge free radicals and provide protection for the cell.

[0037] Preferably, the glutamine supplement includes one or more of L-glutamine or -L-alanyl-L-glutamine (GlutaMAX), and more preferably, the glutamine supplement is -L-alanyl-L-glutamine (GlutaMAX).

[0038] Preferably, the ROCK kinase inhibitor includes one or more of K-115 (CAS No.: 887375-67-9), SAR407899 (CAS No.: 923359-38-0), RKI-1447 (CAS No.: 1342278-01-6), and Y-27632 (CAS No.: 146986-50-7), with Y-27632 being the most preferred.

[0039] Preferably, the growth factor includes one or more of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), growth hormone-releasing inhibitory factor (SRIH), nerve growth factor (NGF), and stem cell-regulated growth factor (R-spondin); more preferably, it is a combination of epidermal growth factor (EGF) and stem cell-regulated growth factor (R-spondin).

[0040] Preferably, the serum substitute includes one or more of UltroGRO and B-27, with B-27 being the most preferred.

[0041] Preferably, the TGF-β inhibitor includes one of SB431542 (CAS No.: 301836-41-9), LDN193189 (CAS No.: 1062368-24-4), SB525334 (CAS No.: 356559-20-1), and A83-01 (CAS No.: 909910-43-6); preferably A83-01.

[0042] More preferably, the cpp culture medium comprises: Advanced DMEM / F12, penicillin-streptomycin, and Primocin. TM The culture medium contains GlutaMAX, B27, acetylcysteine, epidermal growth factor, stem cell regulatory growth factor, Y27632, chrysogenin, P7C3, P7C3-A20, and A83-01. The above culture medium components are non-ionic amphoteric buffers, such as HEPES buffer.

[0043] More preferably, the cpp culture medium comprises: 90-110 units / mL Penicillin-90-110 μg / mL Streptomycin Solution; HEPES buffer, 9-11 mM; GlutaMAX, 0.09-0.11 mg / mL; B-27, 0.09-0.11 mg / mL; Primocin TM0.09–0.11 mg / mL; NAC, 0.9–1.1 mM; EGF, 40–60 ng / mL; Y27632, 9–11 μM; A83-01, 4–6 μM; R-spondin, 90–110 ng / mL; trachomatin, 0.1–1 μM; P7C3, 1–10 μM; P7C3-A20, 11–10 μM; each component was diluted in Advanced DMEM / F12.

[0044] The beneficial effects of the present invention are as follows: (1) The present invention uses cells isolated from human adult kidney tissue as the object to induce and culture podocyte fate organoids. Compared with human induced pluripotent stem cells (hiPSCs), it has better gene stability and can better reproduce the original tissue phenotype. From the candidate pathways that promote kidney development, differentiation and tissue regeneration, after screening with antagonist or activator small molecule compounds, three positive small molecule compounds were obtained; among them, trachomatin is a specific inhibitor of histone methyltransferase (HMT) SU(VAR)3-9, which can reduce the methylation level of H3K9, break the partial gene silencing mediated by heterochromatin, and make the chromatin open to a certain extent, leading to the induction of cell differentiation towards podocyte fate; P7C3 and P7C3-A20 are structurally similar compounds, and P7C3-A20 is a fluorinated highly active derivative of P7C3. Both of them increase NAD by binding and activating NAMPT. + This level of change leads to alterations in a series of downstream pathways, inducing cells to differentiate into podocytes.

[0045] (2) The podocyte fate organoid culture method provided by the present invention simplifies the culture system compared with the kidney organoids derived from hiPSCs; the culture medium composition based on pure compound combination without serum also greatly improves the safety of the culture products for clinical biotherapy.

[0046] (3) The podocyte fate organoids provided by the present invention are expected to be used in clinical cell therapy, disease simulation, drug toxicity screening, and disease damage mechanism research. Attached Figure Description

[0047] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0048] Figure 1 shows the qPCR sequencing results of the culture products of the active ingredients in the experimental group and the control group, respectively, after adding chaetocin, P7C3, and P7C3-A20. In the figure, a represents P7C3, b represents P7C3-A20, and c represents chaetocin. Figure 2 shows the relationship between the expression levels of podocyte fate markers and the treatment concentrations of chaetocin, P7C3, and P7C3-A20. In the figure, a represents P7C3, b represents P7C3-A20, and c represents... Figure 3 shows the relationship between the expression levels of podocyte fate markers and the time concentrations of chaetocin, P7C3, and P7C3-A20, where a represents P7C3, b represents P7C3-A20, and c represents chaetocin. Figure 4 shows the qPCR sequencing results of changes in stem gene expression in organoids after induction with chaetocin, P7C3, and P7C3-A20, respectively, where a represents P7C3, b represents P7C3-A20, and c represents chaetocin. Figure 5 shows the qPCR sequencing results of the effects of replacing the original chaetocin, P7C3, and P7C3-A20 with similar or opposite functional compounds on podocyte fate induction. In Figure 5, a represents the qPCR sequencing results of the effects of small chemical molecules with similar functions to chaetocin (BIX01294, CM272, UNC0642) on podocyte fate induction. Figure 5 also shows the qPCR sequencing results of small chemical molecules with similar or opposite functions to P7C3 and P7C3-A20 (SBI-797812) and those with opposite functions (KPT-9274). Figure 6 shows the qPCR sequencing results of the effect of small molecules on podocyte fate induction; Figure 7 shows the qPCR sequencing results of the changes in the expression of podocyte fate-related genes in organoids after induction by trachomatin, P7C3 and P7C3-A20 respectively and in combination; Figure 7 shows the immunofluorescence detection results of the induced culture products in a, b and c, where DAPI represents the cell nucleus; β-Catenin represents the cell membrane; Nephrin, PODXL and Synaptopodin are all podocyte marker proteins; Figure 8 shows the scanning electron microscope image of the culture products. Detailed Implementation

[0049] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0050] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0051] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0052] Example 1: Culture of podocyte-determined organoids: Human adult kidney tissue was placed in a 60×16 mm culture dish. Excess blood vessels and connective tissue were removed using sterilized surgical instruments. The tissue was washed three times with 5 mL of DMEM / F12 medium, the DMEM / F12 medium was aspirated, and the tissue was minced. 5 mL of DMEM / F12 medium, 50 μL of hyaluronidase, and 50 μL of collagenase IV were added, and the mixture was thoroughly mixed by pipetting. The mixture was then incubated at 37 ℃ in a 5% CO2 incubator for 40 min, with thorough pipetting every 10 min. 5 mL of DMEM / F12 medium was added to terminate the digestion. The cells were filtered through a 70 μm filter into a 50 mL centrifuge tube and centrifuged (900 rpm, 4 ℃, 5 min). The supernatant was removed. 5 mL of erythrocyte lysis buffer was added, and the mixture was thoroughly mixed by pipetting. The cells were then incubated in a 37 ℃ water bath for 10 min to lyse the erythrocytes. 5 mL of DMEM / F12 medium was added to terminate the erythrocyte lysis, and the mixture was thoroughly mixed by pipetting. The cells were then transferred to a 15% CO2 incubator. Centrifuge in mL centrifuge tubes (900 rpm, 4 ℃, 5 min), discard supernatant; add 3-4 mL DMEM / F12 medium to resuspend cells, mix well by pipetting, centrifuge, discard supernatant, repeat 2-3 times; mix DMEM / F12 medium with Matrigel at a volume ratio of 1:3, resuspend cell pellet, adjust cell concentration, mix well by pipetting, and drop the cell mixture into 6-well plates of cell suspension culture in round droplet form, with droplet diameter of about 4 mm and appropriate spacing between droplets; incubate the cell plate upright in a 37 ℃, 5% CO2 incubator for 5 min, then invert it in a 37 ℃, 5% CO2 incubator for 40 min, add 2 mL RGF medium to each well, and incubate in a 37 ℃, 5% CO2 incubator; change the medium every 2-3 days using CPP medium.

[0053] The specific components and concentrations of the CPP culture medium are as follows: 100 units / mL Penicillin-100 μg / mL Streptomycin Solution; HEPES buffer, 10 mM; GlutaMAX, 1×, 0.10 mg / mL; B-27, 1×, 0.10 mg / mL; Primocin TM 0.10 mg / mL; NAC, 1 mM; EGF, 50 ng / mL; Y27632, 10 μM; A83-01, 5 μM; R-spondin, 100 ng / mL; trachomatin, 0.1~1 μM; P7C3, 1~10 μM; P7C3-A20, 11~10 μM; each component was diluted in Advanced DMEM / F12.

[0054] Example 2: In this example, qPCR sequencing was performed on the culture products of three small molecule compounds, and the results are shown in Figure 1. qRT-PCR was used to detect the expression of podocyte fate-related markers in organoids cultured for 7 days at the mRNA level. It was found that the addition of chaetocin, P7C3, and P7C3-A20 to the basal medium increased the overall mRNA expression level of podocyte fate-related markers compared to the control group (basal medium with an equal amount of the small molecule solubilizer dimethyl sulfoxide DMSO). Furthermore, within a certain range, the expression level of podocyte fate markers was positively correlated with the treatment concentration and treatment time of chaetocin, P7C3, and P7C3-A20, as shown in Figures 2 and 3.

[0055] In addition, treatment with chaetocin, P7C3, and P7C3-A20 did not significantly alter / reduce the stemness characteristics of the organoids themselves (stemness genes: CD24; PROM1; CD44), as shown in Figure 4.

[0056] Considering the functions of trachomatin, P7C3, and P7C3-A20, trachomatin can significantly inhibit H3K9me2, and both P7C3 and P7C3-A20 are important activators of NAMPT. Replacing the original trachomatin, P7C3, and P7C3-A20 with homologous or anti-homocytic compounds (trachomatin homologs: BIX 01294, CM272, UNC0642; P7C3 and P7C3-A20 homologs: SBI-797812; P7C3 and P7C3-A20; anti-homocytic compound: KPT-9274) yielded the results shown in Figure 5. The results indicate that compounds with the same function as trachomatin, P7C3, and P7C3-A20 also exhibit certain podocyte fate organoid induction effects, while compounds with the opposite function to trachomatin, P7C3, and P7C3-A20 do not possess such induction effects.

[0057] In this invention, the simultaneous presence of trachomatin, P7C3, and P7C3-A20 significantly enhances the induction effect on podocyte fate organoids compared to their individual presence, as shown in Figure 6.

[0058] The immunofluorescence detection results of the induced culture products are shown in Figure 7. The culture products after culturing with the above-mentioned active ingredients showed abundant expression of podocyte fate markers. Furthermore, transmission electron microscopy revealed that the culture products after culturing with the above-mentioned active ingredients exhibited abundant microstructural features related to podocyte fate, such as primary foot processes, secondary foot processes, and gap septa, as shown in Figure 8.

[0059] Adult-derived podocyte fate organoids can be used for early screening of nephrotoxicity by doxorubicin and antibiotics, and can facilitate the elucidation of mechanisms and personalized medication for rare podocyte diseases. Simultaneously, as an expandable and transplantable human podocyte bank, they will provide readily available components for microfluidic glomerular chips and bioartificial kidneys, ultimately integrating damage repair and regenerative transplantation. In short, adult-derived podocyte fate organoids can become a core hub for new drug development, toxicological evaluation, and cell therapy.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for culturing organoids with podocyte fate, characterized in that, The procedure includes the following steps: separating human adult kidney tissue into monocellular cells, resuspending them in matrix gel to allow the monocellular cells to aggregate and culture, and then culturing them in CPP medium to obtain podocyte-fate organoids; wherein, the CPP medium is prepared by adding trachomatis, P7C3 and P7C3-A20 to Advanced DMEM / F12 medium as the substrate; the concentration of trachomatis is 0.1~1 μM, the concentration of P7C3 is 1~10 μM, and the concentration of P7C3-A20 is 1~10 μM.

2. The cultivation method as described in claim 1, characterized in that, A method for separating human adult kidney tissue into monosodium cytokines includes: cutting human adult kidney tissue into small pieces, removing blood vessels and connective tissue, adding a DMEM / F12 mixture containing hyaluronidase and collagenase IV, placing it in an incubator for digestion, and then adding DMEM / F12 mixture to terminate the digestion; adding erythrocyte lysis buffer to the cells after digestion is terminated, and then adding DMEM / F12 mixture to terminate erythrocyte lysis to obtain the monosodium cytokines.

3. The cultivation method as described in claim 1, characterized in that, The method for obtaining podocyte fate organoids by adding matrix gel to resuspend the monosodium cells and culturing them in CPP medium includes: adding matrix gel to resuspend the monosodium cells and then seeding them into culture plates with droplets of 3-5 mm in diameter; culturing the culture plates in an upright position and then inverted position to make the seeded cells more spherical; adding CPP medium to the culture plates after inverted culture and culturing them in a CO2 incubator, changing the medium every 2-4 days.

4. The cultivation method as described in claim 1, characterized in that, CPP culture medium also includes antibiotics, antioxidants, glutamine supplements, ROCK kinase inhibitors, growth factors, serum substitutes, and TGF-β inhibitors.

5. The cultivation method as described in claim 4, characterized in that, The antibiotics mentioned are selected from those routinely added to the culture medium, such as penicillin, streptomycin, gentamicin, kanamycin, and the primary cell antibiotic Primocin. TM One or more of chloramphenicol; the antioxidant is acetylcysteine ​​(NAC); the glutamine supplement includes one or more of L-glutamine or L-alanyl-L-glutamine (GlutaMAX); the ROCK kinase inhibitor includes one or more of K-115 (CAS No.: 887375-67-9), SAR407899 (CAS No.: 923359-38-0), RKI-1447 (CAS No.: 1342278-01-6), and Y-27632 (CAS No.: 146986-50-7); the growth factor includes epidermal growth factor (EGF), serum... One or more of platelet-derived growth factor (PDGF), growth hormone-releasing inhibitory factor (SRIH), nerve growth factor (NGF), and stem cell-regulated growth factor (R-spondin); the serum substitute includes one or more of UltroGRO and B-27; the TGF-β inhibitor includes one of SB431542 (CAS No.: 301836-41-9), LDN193189 (CAS No.: 1062368-24-4), SB525334 (CAS No.: 356559-20-1), and A83-01 (CAS No.: 909910-43-6).

6. The cultivation method as described in claim 5, characterized in that, The CPP culture medium comprises: 90–110 units / mL Penicillin-90–110 μg / mL Streptomycin Solution; HEPES buffer, 9–11 mM; GlutaMAX, 0.09–0.11 mg / mL; B-27, 0.09–0.11 mg / mL; Primocin TM 0.09–0.11 mg / mL; NAC, 0.9–1.1 mM; EGF, 40–60 ng / mL; Y27632, 9–11 μM; A83-01, 4–6 μM; R-spondin, 90–110 ng / mL; trachomatin, 0.1–1 μM; P7C3, 1–10 μM; P7C3-A20, 11–10 μM; each component was diluted in Advanced DMEM / F12.

7. The CPP culture medium in the culture method according to any one of claims 1 to 6, characterized in that, CPP medium was prepared using Advanced DMEM / F12 medium as the matrix, with the addition of chitin, P7C3 and P7C3-A20; the concentration of chitin was 0.1~1 μM, the concentration of P7C3 was 1~10 μM, and the concentration of P7C3-A20 was 1~10 μM.

8. The CPP culture medium as described in claim 7, characterized in that, The CPP culture medium comprises: 90–110 units / mL Penicillin-90–110 μg / mL Streptomycin Solution; HEPES buffer, 9–11 mM; GlutaMAX, 0.09–0.11 mg / mL; B-27, 0.09–0.11 mg / mL; Primocin TM 0.09–0.11 mg / mL; NAC, 0.9–1.1 mM; EGF, 40–60 ng / mL; Y27632, 9–11 μM; A83-01, 4–6 μM; R-spondin, 90–110 ng / mL; trachomatin, 0.1–1 μM; P7C3, 1–10 μM; P7C3-A20, 11–10 μM; each component was diluted in Advanced DMEM / F12.

9. Application of trachotoxin, P7C3 and P7C3-A20 in the culture of podocyte fate organoids.

10. The application as described in claim 9, characterized in that, The application includes at least the following methods: for preparing culture media for podocyte fate organoids.

Images