A culture medium, a method for culturing polycystic kidney organoids and applications thereof

Abstract

The present invention relates to a culture medium, a method for culturing polycystic kidney organoids and an application thereof. The present invention uses the renal pathological cyst wall tissue of ADPKD patients as the cell source, and constructs 3D organoids of polycystic kidney through RGF culture medium. The 3D organoids have better genetic stability and can better simulate the pathological characteristics of ADPKD in vivo. In order to enhance the vesiculation state of the above 3D organoids and better simulate the physiological state of diseased tissues, the present invention introduces Forskolin and applies the organoids to the construction of a drug screening model. After screening, GSK2193874 can significantly inhibit the vesicle growth of polycystic kidney organoids, suggesting its potential application in the preparation of therapeutic drugs for polycystic kidney disease. The above culture method is applied to the in vitro culture of ADPKD polycystic kidney organoids, and is expected to be used for disease mechanism research, drug screening and clinical cell therapy after correction, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of polycystic kidney tissue organoid culture, and specifically relates to the application of Forskolin in a polycystic kidney disease model, a culture medium, and a method and application for culturing polycystic kidney organoids. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the overall understanding of the present invention, and it is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Polycystic kidney disease (PKD) is the most common monogenic genetic disease globally, including autosomal recessive polycystic kidney disease (ARPKD) and autosomal dominant polycystic kidney disease (ADPKD). ARPKD accounts for 5% of PKD, with a low survival rate during embryogenesis. ADPKD accounts for 95% of PKD and occurs in adulthood, and it is a systemic disease characterized by the formation of cysts in duct organs. Although genetic studies have shown that 85% of ADPKD cases are due to mutations in the PKD1 gene and 15% are due to mutations in the PKD2 gene. However, the pathogenesis of ADPKD remains unclear, and there is a lack of specific therapeutic drugs for polycystic kidney disease globally. The dilemma in clinical treatment mechanism research and drug discovery lies in the lack of a good model that can accurately simulate ADPKD.

[0004] Currently, in vitro models of polycystic kidney mainly include animal models and organoid models. Rodent models are common in animal models. Rodent models of polycystic kidney mainly include: ① Spontaneous genetic polycystic kidney models such as cpk, pcy, jck, bpk polycystic kidney mouse models and Han:SPRD-cy, PCK polycystic kidney rat models, etc. The pathogenesis of such models is essentially different from that of human polycystic kidney. ② Chemically induced polycystic kidney models, such as rat polycystic kidney models induced by DPT, NDGA, etc. Such models induce polycystic phenotypes through drugs and cannot truly simulate the essential characteristics of ADPKD genetic diseases. ③ PKD1 and PKD2 gene knockout polycystic kidney mouse models, but the progression of polycystic kidney lesions in heterozygous mice is slow, and almost all homozygous mice die in infancy and cannot be used for mechanism research. Therefore, current animal models for simulating the pathogenesis of polycystic kidney are lackluster. Organoids simulating ADPKD currently mainly focus on induction models derived from hiPSCs. They are obtained by reprogramming peripheral blood mononuclear cells from polycystic kidney patients into hiPSCs retaining disease characteristics or knocking out PKD1 or PKD2 genes in ordinary hiPSCs through gene editing technology. However, they have the following disadvantages: (1) The induction and differentiation efficiency is unstable and the batch effect is significant; (2) There are off-target effects such as non-renal cells and a potential risk of teratoma; (3) The cells are immature and more resemble ARPKD rather than ADPKD, and are not suitable for simulating adult-onset diseases such as polycystic kidney.

[0005] In view of the above research status, the present invention believes that in order to improve the current clinical lack of specific therapeutic drugs for ADPKD, it is necessary to provide an organoid that can stably maintain the physiological structure of ADPKD and its application as a drug screening model. Summary of the Invention

[0006] The present invention is designed to use cells isolated from renal cyst tissues of ADPKD patients as a source, and induce them into a polycystic kidney disease model that can stably express disease morphology and has good proliferation ability through in vitro culture.

[0007] To achieve this goal, the present invention first provides an RGF medium for culturing cells isolated from renal cyst tissues of ADPKD patients into polycystic kidney 3D organoids; further, the present invention also designs to further induce and culture the above polycystic kidney 3D organoids, and construct a polycystic kidney disease model by introducing Forskolin, so that it can fully present the cyst morphology of polycystic kidney disease and be closer to the physiological structure of in vivo pathological tissues.

[0008] The present invention uses the above polycystic kidney disease model as a drug screening model. By screening 20 small molecules, it is found that GSK2193874 has a significant effect of inhibiting vesicle dilation among many small molecules. This screening result also confirms the superiority of the above drug screening model and the feasibility of Forskolin in the culture of polycystic kidney 3D organoid models.

[0009] Based on the above technical effects, the present invention specifically provides the following technical solutions:

[0010] In a first aspect, a method for in vitro culture of polycystic kidney 3D organoids is provided. The method includes the following steps: separating the renal pathological cyst wall tissue of autosomal dominant polycystic kidney disease patients into single cells, resuspending with Matrigel, aggregating the single cells into clusters for culture, and adding RGF medium for culture to obtain polycystic kidney 3D organoids; the RGF medium is based on Advanced DMEM / F12 medium and at least includes the following components: antibiotics, antioxidants, glutamine supplements, ROCK kinase inhibitors, growth factors, serum substitutes, TGF-β inhibitors, and retinoic acid.

[0011] In the above culture method, the separation method of the single cells can adopt conventional methods in the art. A feasible implementation method is as follows: cleaning and cutting the cyst wall tissue into pieces, adding a DMEM / F12 mixture containing hyaluronidase and collagenase IV to a culture box for digestion for a period of time, and adding a DMEM / F12 mixture to terminate digestion; adding erythrocyte lysate to the cells after terminating digestion, lysing red blood cells in a water bath, and adding a DMEM / F12 mixture to terminate erythrocyte lysis to obtain the single cells.

[0012] The components of the RGF medium are as follows: 90 - 110 units / ml Penicillin - 90 - 110 μg / ml Streptomycin Solution; HEPES buffer, 9 - 11 mM; GlutaMAX, 1x; B27, 1x; Primocin, 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; Retinoic Acid, 1 - 3 μM; GDNF, 90 - 110 ng / ml; FGF20, 90 - 110 ng / ml; each component is diluted in Advanced DMEM / F12.

[0013] In one implementation mode verified by the present invention, after the single cells are resuspended in Matrigel, they are inoculated in the culture plate as droplets. The diameter of the inoculated droplets is 3-5 mm. After inoculation, the culture plate is cultured upright and then inverted for a period of time to make the inoculated cells closer to spheres. Then, the above-mentioned RGF medium is added to the culture plate after the inverted culture is completed, and it is cultured in a CO2 incubator, and the medium is changed every 2-4 days.

[0014] Adult somatic cells have better genetic stability. The above-mentioned culture method uses the isolated tissues of patients with autosomal dominant polycystic kidney disease (ADPKD) as the source, and single cells are isolated and cultured into polycystic kidney 3D organoids. This culture method can significantly improve cell viability, promote the growth, amplification of single cells and self-organization into highly complex 3D organoid structures, and can produce polycystic vacuole morphology, which well simulates the biological activity of the pathological tissues of patients, providing broad application prospects for disease mechanism research, drug screening and clinical cell therapy after correction, etc.

[0015] In order to further accelerate the disease progression, make the vesicles larger and more conform to the polycystic kidney state in vivo, and become a polycystic kidney disease model, increasing the persuasiveness of its use as a drug screening model, the present invention introduces a cAMP agonist into the above-mentioned RGF medium to enhance the vesiculation effect of the above-mentioned polycystic kidney 3D organoids. According to the screening results of the cAMP agonist of the present invention, Forskolin has a relatively significant promoting effect on the above-mentioned adult somatic cells and can be added to the RGF medium to promote the co-growth of polycystic kidney organoids after digestion into single cells for drug screening.

[0016] In a second aspect, the application of Forskolin in the culture of polycystic kidney disease models is provided.

[0017] Forskolin (also known as FORSKOLIN, alias Coleonol) mentioned in the above application is a natural product extracted from Coleus forskohlii, CAS number: 66575-29-9.

[0018] The above application at least includes the following methods:

[0019] (1) Using Forskolin to prepare a medium for polycystic kidney disease models;

[0020] (2) Using Forskolin for in vitro culture of polycystic kidney 3D organoids to construct a polycystic kidney disease model.

[0021] The medium mentioned in the above aspect (1) includes the above-mentioned RGF medium and also includes a certain dose of Forskolin.

[0022] In a specific embodiment, the components of the culture medium are as follows: 90 - 110 units / ml Penicillin - 90 - 110 μg / ml Streptomycin Solution; HEPES buffer, 9 - 11 mM; GlutaMAX, 1x; B27, 1x; Primocin, 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; Retinoic Acid, 1 - 3 μM; GDNF, 90 - 110 ng / ml; FGF20, 90 - 110 ng / ml; Forskolin, 10 uM; each component is diluted in Advanced DMEM / F12.

[0023] In the third aspect, a method for constructing a polycystic kidney disease model is provided, and the method includes: re-digesting the 3D organoids of polycystic kidney constructed by the in vitro culture method in the first aspect into single cells, and culturing them with the above polycystic kidney disease model culture medium.

[0024] In a preferred embodiment, the culture time is 7 days.

[0025] According to the verification of the present invention, the polycystic kidney disease model obtained by the above culture method can well simulate the physiological activity of cyst tissues in real polycystic kidney disease, and can be used as a disease model in the field of drug screening. During the process of verifying the feasibility of the above polycystic kidney disease model as a drug screening model, the present invention also screened GSK2193874, which has significant inhibitory activity on polycystic kidney tissues and is expected to be applied to the clinical treatment of polycystic kidney and the corresponding drug development.

[0026] In the fourth aspect, the application of GSK2193874 in the preparation of drugs for treating polycystic kidney disease is provided.

[0027] The beneficial effects of the above one or more technical solutions are as follows:

[0028] 1. Considering the genetic stability of adult cells, the present invention designs to use the cells isolated from the differentiated and mature polycystic kidney cyst wall tissue as the object to induce and culture the 3D organoids of polycystic kidney; in existing research, the organoid culture technology with adult cells as the culture object is relatively blank and there is little research content for reference. The inventor designs to introduce a cAMP agonist in the culture of polycystic kidney 3D organoids to improve the viability of single cells in the cyst wall tissue. After screening, Forskolin can improve the viability of single cells in cyst tissues and can significantly promote the cell co-growth after the digestion of PKD polycystic kidney organoids into single cells for drug screening.

[0029] 2. The present invention also adopts an in vitro culture medium (RGF medium) and an in vitro culture method for polycystic kidney 3D organoids. This medium is a low growth factor type medium with less growth factors and no serum, effectively reducing the risk of culturing tissue contamination and improving the stability and safety of the application of organoid tissues.

[0030] 3. The present invention extracts the cyst wall tissue of polycystic kidney patients for organoid culture based on adult tissues, establishing the first patient-derived ADPKD kidney organoid model that highly mimics the adult disease onset process, providing the possibility for disease development and polycystic kidney drug screening, and being the first international organoid model based on the adult kidney tissues of polycystic kidney patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0032] Figure 1 It is the screening result of the active ingredients of the PKD organoid culture medium in the examples;

[0033] Figure 1 A is the statistical result of the change in the diameter of vesicles generated after culturing with cAMP agonists at different gradient concentrations;

[0034] Figure 1 B is the statistical result of the change in the diameter of vesicles generated after culturing with cAMP agonists at the same concentration;

[0035] Figure 2 It is the pattern diagram of the gene sequencing identification result of the polycystic kidney organoid culture products of two different patients in the examples;

[0036] Figure 3 It is the bright field comparison diagram of the growth morphology of polycystic kidney organoids and normal kidney organoids in the examples;

[0037] Figure 3 A are the effect diagrams of culturing normal kidney organoids for 0 - 4 days from left to right;

[0038] Figure 3 B are the effect diagrams of culturing polycystic kidney organoids for 0 - 4 days from left to right;

[0039] Figure 4 It is the morphology comparison diagram of the culture products and in vivo PKD tissues in the examples;

[0040] Figure 4 A is the magnetic resonance imaging of this polycystic kidney patient;

[0041] Figure 4 B is the gross pathology diagram;

[0042] Figure 4 C is the HE staining image;

[0043] Figure 4 D is the differential interference contrast microscopy image of polycystic kidney organoids after the second passage;

[0044] Figure 4 E is the differential interference contrast microscopy image of polycystic kidney organoids after the fourth passage;

[0045] Figure 4 F is the HE staining image of this polycystic kidney organoid;

[0046] Figure 5 It is the transmission electron microscopy image of the culture product;

[0047] Figure 5 A is the representative transmission electron microscopy image of PKD organoids (Scale bars, 1μm);

[0048] Figure 5 B is the enlarged view of a part of the representative transmission electron microscopy image of PKD organoids;

[0049] Figure 5 C is the representative transmission electron microscopy image of UB organoids (Scale bars, 1μm);

[0050] Figure 5 D is the enlarged view of a part of the representative transmission electron microscopy image of UB organoids;

[0051] N, nucleus; TJ, tight junction; BB, brush border; Mi, mitochondria; BM, basal membrane; ER, endoplasmic reticulum; Ci, cilia; red arrows mark autophagosome;

[0052] Figure 6 It is the Upset diagram of PKD organoids derived from adult tissues, human PKD tissues, mouse PKD tissues, embryos and PKD cells in the present invention;

[0053] Figure 7 It is the drug screening strategy diagram in the embodiment;

[0054] Figure 8 It is the inhibitory effect diagram of 20 small molecules on the vesicle expansion of PKD organoids;

[0055] Figure 9Inhibitory effect diagram of GSK2193874 on the vesicle dilation of PKD organoids;

[0056] Figure 9 A is the light microscope change diagram of PKD organoids intervened by GSK2193874;

[0057] Figure 9 B is the time change curve of the inhibitory effect of GSK2193874 on the vesicle dilation of PKD organoids. Detailed implementation manners

[0058] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0059] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, 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.

[0060] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0061] Example 1

[0062] In this example, an RGF culture medium and a culture method for culturing polycystic kidney disease organoids are provided. Single cells are isolated from the ex vivo tissues of polycystic kidney disease patients and cultured using the RGF culture medium. The components of the RGF culture medium are as follows: 100 units / ml Penicillin - 100 μg / ml Streptomycin Solution; HEPES buffer, 10 mM; GlutaMAX, 1×; B27, 1×; Primocin, 0.1 mg / ml; NAC, 1 mM; EGF, 50 ng / ml; Y27632, 10 μM; A83 - 01, 5 μM; R-spondin, 100 ng / ml; Retinoic Acid, 2 μM; GDNF, 100 ng / ml; FGF20, 100 ng / ml; each component is diluted in Advanced DMEM / F12.

[0063] The single-cell isolation steps are as follows: Take the polycystic kidney cyst wall tissue resected from ADPKD patients and place it in a 60×16 mm culture dish. Use sterilized surgical scissors to remove as much redundant blood vessels and connective tissues as possible. Wash it three times with 5 ml of DMEM / F12 mixture, suck off the DMEM / F12 mixture, cut the tissue into pieces, add 5 ml of DMEM / F12 mixture, 50 μl of hyaluronidase, and 50 μl of collagenase IV, pipette and mix well, and place it in a 37°C, 5% CO2 incubator for digestion for 60 min, and pipette the digest thoroughly every 5 - 10 min; add 5 ml of DMEM / F12 mixture to terminate digestion, filter the cells through a 70 μm filter into a 50 ml centrifuge tube, centrifuge (900 r, 4°C, 5 min), remove the supernatant, add 5 ml of red blood cell lysate, pipette and mix well, place it in a 37°C water bath for 10 min to lyse red blood cells, add 5 ml of DMEM / F12 mixture to terminate red blood cell lysis, pipette and mix well, transfer the cells to a 15 ml centrifuge tube, centrifuge (900 rpm, 4°C, 5 min), discard the supernatant; add 3 - 4 ml of DMEM / F12 mixture to resuspend the cells, pipette and mix well, centrifuge, discard the supernatant, and repeat 2 - 3 times; mix the DMEM / F12 mixture and Matrigel in a ratio of 1:3, resuspend the cell pellet, adjust the cell concentration, pipette and mix well, and drop the cell mixture in a circular droplet shape onto a 6-well plate for cell suspension culture, with the diameter of the droplet being about 4 mm, and the appropriate distance between the droplets; place the cell plate upright in a 37°C, 5% CO2 incubator for 5 min, then place it upside down in a 37°C, 5% CO2 incubator for 40 min, add 2 ml of RGF medium to each well, and place it in a 37°C, 5% CO2 incubator for culture; change the medium every 2 - 4 days.

[0064] To verify whether the products obtained by the above culture method can simulate real polycystic kidney organoids, in this example, sequencing comparison and morphological comparison were performed on the above culture products. Among them, the sequencing comparison results are as Figure 2 , Figure 6 shown. ADPKD is mainly caused by PKD1 gene mutation. By sequencing the DNA of polycystic kidney organoids, the mutations in the organoids are identified. Figure 2 The sequencing identification diagram of the culture products shows that there are heterozygous point mutations in exon 1 of the PKD1 gene and exon 18 of the PKD1 gene in two polycystic kidney patients respectively. Figure 6 In the comparison of the sequencing of normal kidney tissue, polycystic kidney tissue, and polycystic kidney organoids, through RNA sequencing, it shows that polycystic kidney organoids can simulate polycystic kidney tissue to a certain extent, but are different from normal kidney tissue.

[0065] Under the differential interference contrast microscope, it can be seen that the polycystic kidney organoids cultured in this example have cyst formation ( Figure 3), the typical morphology of the cultured product has a high similarity to polycystic kidney tissue. Figure 4 ). According to the transmission electron microscopy images of the cultured product Figure 5 ), compared with normal kidney UB organoids, PKD organoids showed mitochondrial structural disorders and increased autophagy. At the same time, through the Upset diagram Figure 6 ), a comparison of human PKD tissues, mouse PKD tissues, adult-derived PKD organoids of the present invention, IPS-derived PKD organoids, etc. in the GEO database shows that the adult-derived PKD organoids of the present invention are the most similar to human PKD tissues and can be used as a reliable model for the pathogenesis of human PKD.

[0066] Example 2

[0067] In this example, it was designed to use the organoid tissue obtained by culturing in Example 1 above for drug screening. In order to obtain a drug model with better growth conditions, in this example, the active ingredients promoting the culture of PKD organoids were screened. Since cAMP agonists can promote the single-cell growth of PKD organoids, three cAMP agonists, Bucladesine, Forskolin, and 8-Br-cAMP, were screened in this example to test the culture effect.

[0068] First, after digesting the polycystic kidney organoids obtained by culturing in Example 1 above into single cells, different concentrations of the above agonists were added to the cell culture environment. Among the cAMP agonists, the effects of Forskolin and other compounds on the single-cell growth of PKD organoids at each concentration are as Figure 1 shown. Consistent with cAMP-dependent cyst formation, the addition of cAMP agonists to the RGF culture medium components promoted the single-cell growth of PKD organoids, and 10 μM Forskolin was the most effective.

[0069] Based on the above research conclusions, in this example, another PKD disease model culture medium and culture method are provided, which can be used as a drug screening strategy based on PKD organoids Figure 7 ), and the specific steps are as follows: After digesting the PKD organoids obtained by culturing in Example 1 into single cells, they were re-inoculated into 96-well plates and cultured with RGF medium + 10 μM Forskolin for 7 days to fully promote vesicle expansion and simulate the progression state of PKD disease, and then the drug screening strategy was implemented. By summarizing the relevant pathways involved in the pathogenesis of PKD and the small molecule drugs reported in the literature, a small-scale small molecule compound library containing 20 small molecules was formulated and applied to PKD organoids for drug screening Figure 8 ).

[0070] Using the above drug screening method, this embodiment provides the application of GSK2193874 as a polycystic kidney disease inhibitor. The inhibitory effect of GSK2193874 on PKD organoids is as Figure 9 shown. It can be seen that with the increase in the concentration of GSK2193874 and the prolongation of the culture time, the vesicles of PKD organoids have significantly shrunk. On the one hand, it confirms the feasibility of applying PKD organoids to the drug screening strategy, and also confirms the inhibitory activity of GSK2193874 on polycystic kidney disease.

[0071] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for in vitro culture of polycystic kidney 3D organoids, characterized in that, It includes the following steps: separating the renal pathological cyst wall tissue of patients with autosomal dominant polycystic kidney disease into single cells, resuspending with Matrigel, aggregating the single cells into clusters for culture, and culturing with RGF medium to obtain polycystic kidney 3D organoids; the components of the RGF medium are as follows: 90 - 110 units / ml Penicillin - 90 - 110 μg / ml Streptomycin Solution; HEPES buffer, 9 - 11 mM; GlutaMAX, 1x; B27, 1x; Primocin, 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; Retinoic Acid, 1 - 3 μM; GDNF, 90 - 110 ng / ml; FGF20, 90 - 110 ng / ml; each component is diluted in Advanced DMEM / F12.

2. The method for in vitro culture of polycystic kidney 3D organoids according to claim 1, characterized in that, The separation method of the single cells is as follows: cleaning and cutting the cyst wall tissue into pieces, adding a DMEM / F12 mixture containing hyaluronidase and collagenase IV into an incubator for digestion for a period of time, and adding a DMEM / F12 mixture to terminate digestion; adding a red blood cell lysate to the cells after terminating digestion, lysing red blood cells in a water bath and adding a DMEM / F12 mixture to terminate red blood cell lysis to obtain the single cells.

3. The method for in vitro culture of polycystic kidney 3D organoids according to claim 1, characterized in that, After the single cells are resuspended with Matrigel, they are inoculated in a culture plate as a gel droplet, the diameter of the inoculated gel droplet is 3 - 5 mm, and the inoculated culture plate is cultured upright and then inverted for a period of time to make the inoculated cells closer to a sphere; adding the above RGF medium to the culture plate after the inverted culture is completed, culturing in a CO2 incubator, and changing the medium every 2 - 4 days.

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