Preparation method of kidney organoid with increased proportion of proximal tubules in vitro

Abstract

The invention discloses a preparation method of a kidney-like organ with an increased in-vitro proximal tubule proportion, and belongs to the field of cytology. The method comprises the following steps: inducing kidney organs by using human embryonic stem cells WIBR3, culturing the WIBR3 cells until the WIBR3 cells can be passaged, and digesting the WIBR3 cells by using Accutase digestive juice; after the digestion is completed, stopping the digestion by using a DMEM-F12 culture medium, collecting a cell suspension, centrifuging, discarding supernate, collecting cell precipitates, and counting; resuspending the collected cell precipitate by using a stem cell culture medium, paving the cell precipitate in a pore plate, adding an apoptosis inhibitor, and uniformly mixing; and carrying out suspension culture on the obtained system by using a kidney organ culture medium for promoting the differentiation of the proximal tubules to obtain the kidney organs with the proportion of the proximal tubules increased. The preparation method is simple, the culture efficiency is high, and the prepared kidney organ with the increased proximal tubule proportion can effectively simulate the kidney organ with the increased proximal tubule proportion in vivo and can also be used for chemical substance kidney toxicity detection.

Description

Technical Field

[0001] The present invention belongs to the field of cytology, and particularly relates to a method for preparing kidney organoids with an increased proportion of proximal tubules in vitro. Background Art

[0002] Organoids are three-dimensional structures composed of stem cells or cells derived from human tissues through self-assembly. They can simulate near-physiological cell composition and behavior, have structures and functions similar to native organs, and many organoids can be cultured for a long time while maintaining the stability of the genome and epigenome. Compared with animal experiments, human-derived organoids are derived from human cells, have genomes and epigenetic modifications close to those of normal humans, are more conducive to studying a series of research processes that are difficult to accurately model in animal experiments, and are more convenient for extensive high-throughput screening and real-time imaging due to their experimental convenience; compared with cell experiments, organoids can exhibit cell composition and behavior close to human physiology and can study the processes of tissue and organ formation during embryonic development. Existing organoid research is mainly applied to the mechanism research of infectious diseases and antiviral drug testing, the research of monogenic genetic diseases, the precise treatment of tumors and other fields. Although these organoids provide a key common technical basis for more precise screening of environmental pollutants and chemicals that affect human health, there is currently no good reason in the field of environmental toxicology for this powerful tool.

[0003] Although kidney organoids derived from human embryonic stem cells or induced pluripotent stem cells are now used in kidney disease models, previous induction protocols often have problems such as a low proportion of proximal tubules and low induction efficiency. The renal proximal tubule is a highly specialized part of the nephron, performing most of the reabsorption and secretion of the kidney, which is very crucial and important for the kidney structure.

[0004] Despite some existing methods for inducing kidney organoids, it is a huge difficulty to quickly evaluate kidney organoids with enhanced proximal tubules. Previous methods can only detect the molecular markers of proximal tubules by flow cytometry and immunofluorescence, with a long evaluation time and cumbersome methods. There is a need for a direct and rapid method to evaluate the proximal tubule formation efficiency of kidney organoids. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing kidney organoids with an increased proportion of proximal tubules in vitro, which has a simple preparation method, high culture efficiency, and the prepared kidney organoids with an increased proportion of proximal tubules can effectively simulate kidney organoids with an increased proportion of proximal tubules in vivo and can also be used for detecting the renal toxicity of chemical substances.

[0006] The technical solution adopted by the present invention to achieve the above purpose is:

[0007] A method for preparing kidney organoids with an increased proportion of proximal tubules in vitro, comprising the following steps:

[0008] (1) Induce kidney organoids using human embryonic stem cells WIBR3. When the WIBR3 cells are cultured to the passageable stage, digest them with Accutase digestion solution.

[0009] (2) After digestion is completed, use DMEM-F12 medium to terminate digestion, collect the cell suspension and centrifuge it, discard the supernatant, collect the cell pellet and count it.

[0010] (3) Resuspend the collected cell pellet with stem cell medium and plate it in a well plate, then add an apoptosis inhibitor and mix well.

[0011] (4) Use kidney organoid medium to perform suspension culture on the system obtained in step (3) to obtain kidney organoids with an increased proportion of proximal tubules.

[0012] Further, in step (1), the WIBR3 cells are pre-cultured in a six-well plate coated with Matrigel (a basement membrane matrix extracted from mouse EHS sarcoma). The culture conditions are as follows: The medium used is stem cell medium mTeSR1, and it is cultured in a 37°C incubator for 4 days.

[0013] Further, in step (1), the digestion conditions are as follows: Add 500 μL of Accutase digestion solution to each well of a six-well plate and digest it in a 37°C incubator for 3 - 10 minutes.

[0014] Further, when using DMEM-F12 to terminate digestion in step (2), add a volume that is 3 - 4 times the volume of the Accutase digestion solution.

[0015] Further, the centrifugation conditions in step (2) are as follows: Centrifuge at 1000 - 1200 rpm for 3 - 5 minutes.

[0016] Further, after resuspending with stem cell medium mTeSR1 in step (3), plate it in a six-well plate at a cell density of 1×10 5 - 5×10 5 cells per well.

[0017] Further, the apoptosis inhibitor selected in step (3) is Y-27632 (a ROCK inhibitor), and the concentration is 2 - 10 μM.

[0018] Further, the steps of using kidney organoid medium to perform suspension culture in step (4) include:

[0019] Replace the medium after culturing for 1 - 2 days and perform suspension culture using kidney organoid medium S1;

[0020] Replace the medium after culturing for 2 - 4 days and perform suspension culture using kidney organoid medium S2;

[0021] Replace the culture medium after 2 - 4 days of culture, and culture in suspension using renal organoid medium S3;

[0022] Replace the culture medium after 4 - 6 days of culture, and culture in suspension using renal organoid medium S4;

[0023] Replace the culture medium after 4 - 6 days of culture, and culture in suspension using renal organoid medium S5;

[0024] Continue to culture for 2 - 3 days to obtain renal organoids with an increased proportion of proximal tubules.

[0025] Furthermore, the renal organoid media S1 - S5 in step (4) include basal medium Advanced RPMI 1640 or DMEM / F12, 100x L - Glutamax (L - glutamine), 0.5 - 1 nM Dorsomorphin, 1 - 2 μM CHIR99021 (6 - [[2 - [[4 - (2,4 - dichlorophenyl)-5-(5 - methyl - 1H - imidazol - 2 - yl)-2 - pyrimidinyl]amino]ethyl]amino]-3 - pyridinecarbonitrile), and FGF9 (fibroblast growth factor 9), where renal organoid medium S3 uses 25 - 100 ng / mL of FGF9, renal organoid media S2 and S4 use 200 - 500 ng / mL of FGF9, and renal organoid media S1 and S5 use 25 - 500 ng / mL of FGF9.

[0026] Furthermore, the following small molecule substances are also added to the renal organoid medium S3 in step (4):

[0027] Triiodothyronine, 2.5 - 25 pg / mL;

[0028] Hydrocortisone, 100 - 500 nM;

[0029] TGF - α (transforming growth factor α), 10 - 50 ng / mL;

[0030] CHIR99021, 0.5 - 5 μM;

[0031] DAPT (a γ - secretase inhibitor), 2.5 - 10 μM;

[0032] BMP7 (bone morphogenetic protein 7), 10 - 50 ng / mL;

[0033] LIF (leukemia inhibitory factor), 5 - 50 ng / mL;

[0034] ITS - X (insulin - transferrin - selenium), 5 - 10 ng / mL.

[0035] Further, when performing suspension culture using the renal organoid medium S3 in step (4), an apoptosis inhibitor Y-27632 is added to prevent apoptosis of the organoid cells.

[0036] Further, during the suspension culture in step (4), the corresponding fresh renal organoid medium is replaced every day, and 2-4 mL of the renal organoid medium is added to each well of a six-well plate and cultured in an incubator at 37°C.

[0037] The beneficial effects achieved by the present invention are as follows:

[0038] The present invention changes the culture medium components of metanephric mesenchyme and primitive renal tubules, and at the same time proposes a method for quickly identifying whether renal organoids are cultured mature. The method for preparing renal organoids with an increased proximal tubule ratio proposed by the present invention is simple to operate, has a wide application range, high culture efficiency, and the prepared renal organoids with an increased proximal tubule ratio can effectively simulate renal organoids with an increased proximal tubule ratio in vivo, and can also be used for detecting the renal toxicity of chemical substances. Description of the Drawings

[0039] Figure 1 It is a morphological diagram of mature renal organoids prepared in the embodiment.

[0040] Figure 2 It is a comparison diagram of the staining results of renal proximal tubules evaluated by immunofluorescence method for the renal organoids prepared by the method of the present invention and the existing method.

[0041] Figure 3 It is a diagram of the results of the identification of the renal tubule structure and the evaluation results of proximal tubule-specific endocytosis prepared by the method of the present invention.

[0042] Figure 4 It is a diagram of the results of the evaluation of the toxicity of organic pollutant E to the prepared renal organoids with an increased proximal tubule ratio.

[0043] Figure 5 It is Figure 4 The histogram of the statistical results of the fluorescence intensity in

[0044] Figure 6 It is a diagram of the results of the evaluation of the toxicity of water samples to the prepared renal organoids with an increased proximal tubule ratio.

[0045] Figure 7 It is Figure 6 The histogram of the statistical results of the fluorescence intensity in Detailed Embodiments

[0046] To make the technical features and advantages or technical effects in the above technical solutions of the present invention more obvious and understandable, the following is a detailed description in combination with embodiments and drawings.

[0047] Embodiment:

[0048] In the embodiments of the present invention, the renal organoid culture media S1 to S5 refer to the culture media for culturing renal organoids, including Advanced RPMI 1640 basal medium or DMEM / F12 basal medium, 100x L-Glutamax, 0.5 - 1 nM Dorsomorphin, 1 - 2 μM CHIR99021, 50 - 100 ng / mL FGF9; 2.5 pg / mL Triiodothyronine, 0.1 μM Hydrocortisone, 5 ng / mL ITS-X, 5 ng / mL LIF, 10 ng / mL TGF-α, 2.5 μM DAPT, 10 ng / mL BMP-7, 10 μM Y-27632;

[0049] The preparation steps of renal organoids with an increased proportion of proximal tubules derived from embryonic stem cells WIBR3 include:

[0050] (1) Highly efficient suspension culture of renal organoid precursors:

[0051] ① Induce renal organoids using human embryonic stem cells WIBR3. Culture WIBR3 cells in a six-well plate pre-coated with Matrigel. The culture medium is the stem cell culture medium mTeSR1. Culture in a 37°C incubator for 4 days until subculture is possible. When digesting, add 500 μL of Accutase digestion solution to each well of a six-well plate and digest in a 37°C incubator for 3 minutes;

[0052] ② Add DMEM / F12 with a volume three times that of the digestion solution to terminate digestion. Collect the cell suspension and centrifuge at 1000 rpm for 3 minutes. Discard the supernatant, collect the cell pellet and count;

[0053] ③ Resuspend with mTeSR1 and seed at a cell density of 1×10 5 / well in an ultra-low attachment six-well plate. Add 10 μM apoptosis inhibitor Y-27632, mix gently, and perform the following suspension culture.

[0054] (2) Induction of renal organoids with an increased proportion of proximal tubules: Culture in a 37°C incubator according to the following protocol. Observe the state of renal organoids every day and replace with the corresponding fresh specific renal organoid culture medium for induction culture. After 1 day of suspension culture, replace the medium and culture in the renal organoid specific medium S1 in suspension; after 4 days of culture, culture in the renal organoid specific medium S2 in suspension; after 2 days of culture, culture in the renal organoid specific medium S3 in suspension; after 4 days of culture, culture in the renal organoid specific medium S4 in suspension; after 4 days of culture, culture in the renal organoid specific medium S5 in suspension, and continue to culture for 2 days to obtain mature renal organoids with an increased proportion of proximal tubules, such asFigure 1 as shown

[0055] Immunofluorescence staining experiment of kidney organoids:

[0056] Identify the kidney organoids with an increased proportion of proximal tubules prepared in the above embodiments, and perform immunofluorescence staining experiments on the biomarkers of the glomerulus (PODXL), renal proximal tubules (LTL), and distal tubules (CDH1), which are important components of the kidney organoids. The specific steps are as follows:

[0057] 1) Fixation: Discard the culture medium of the induced mature kidney organoids, and fix them with 4% paraformaldehyde at 37°C for 60 min;

[0058] 2) Permeabilization: Discard 4% paraformaldehyde, and permeabilize the kidney organoids with 0.25% Triton X-100 at 37°C for 60 min;

[0059] 3) Blocking: Discard 0.3% BSA, and block the kidney organoids with 3% BSA at 37°C for 60 min;

[0060] 4) Primary antibody incubation: Discard 0.25% Triton X-100, add the primary antibody diluted 1:50 in 3% BSA to the kidney organoids, and incubate at 37°C for 60 min, 50 μL per well;

[0061] 5) Secondary antibody incubation: Wash 3 times with PBS, 1 min each time. Add the secondary antibody diluted 1:50 in 3% BSA and 10 μg / mL DAPI, and incubate at 37°C for 120 min, 50 μL per well. Wash 3 times with PBS, 1 min each time;

[0062] 6) Observe the fluorescence of renal proximal tubules and distal tubules in the kidney organoids using a laser confocal microscope.

[0063] Figure 2 The figure shows a comparison chart of the staining results of renal proximal tubules prepared by the method of the present invention and the existing method evaluated by immunofluorescence. In the figure, LTL represents the proximal tubule, CDH1 represents the distal tubule, DAPI represents the cell nucleus, and Merge represents the image after merging different fluorescence layers. It can be seen from this figure that the fluorescence intensities of the molecular markers of the proximal tubule and the distal tubule obtained by the preparation method of the present invention are significantly higher than those of the existing method before improvement.

[0064] Figure 3The figure shows the results of the identification of the renal tubular structure prepared by the method of the present invention and the evaluation results of proximal tubular specific endocytosis. Among them, DAPI characterizes the cell nucleus, and Merge represents the image after merging different fluorescence layers. The figure shows the immunofluorescence results of important components in the kidney organoids, namely glomerulus (PODXL), renal proximal tubule (LTL), and distal tubule (CDH1). Dextran555 can be specifically endocytosed by the proximal tubules of the kidney organoids, indicating that the improved kidney organoids have endocytic function.

[0065] Toxicity assessment of organic pollutant E in kidney organoids:

[0066] Perform toxicity assessment of organic pollutant E on the kidney organoids with increased proportion of proximal tubules prepared in Example 1:

[0067] ① Prepare a specific kidney organoid induction medium, and prepare a kidney organoid induction medium containing organic pollutant E;

[0068] ② Use the above-mentioned constructed kidney organoids with increased proportion of proximal tubules, and set up a blank control group and an experimental group: the medium in the blank control group is replaced with fresh kidney organoid induction medium; the medium in the experimental group is replaced with a kidney organoid induction medium containing organic pollutant E;

[0069] ③ Continue the culture, collect the cells after culturing for a certain period of time for toxicological assessment, and the results are as Figures 4 - 5 shown.

[0070] Figure 4 The figure shows the results of the toxicity assessment of organic pollutant E on the prepared kidney organoids with increased proportion of proximal tubules. It is the result of immunofluorescence staining of the main components of the kidney organoids, namely glomerulus (PODXL), renal proximal tubule (LTL), distal tubule (CDH1), and collecting duct (GATA3), after the kidney organoids are exposed to 100 or 1000 nM of organic pollutant E.

[0071] Figure 5 As shown Figure 4 in the results of the fluorescence intensity statistics, it can be seen that the relative fluorescence intensities of LTL and CDH1 decrease, indicating that organic pollutant E may damage the renal tubules of the kidney organoids.

[0072] Toxicity assessment of water samples in kidney organoids:

[0073] Perform the following water sample toxicity assessment on the kidney organoids with increased proportion of proximal tubules prepared in the example:

[0074] ① Prepare a specific kidney organoid induction medium, and prepare a kidney organoid induction medium with the water sample to be tested;

[0075] ② Using the above - constructed renal organoids with an increased proximal tubule ratio, a blank control group and an experimental group were set up: In the blank control group, the culture medium was replaced with fresh renal organoid induction medium; in the experimental group, the culture medium was replaced with renal organoid induction medium prepared with the water sample to be tested.

[0076] ③ Continue the culture, collect the cells after culturing for a certain period of time for toxicological evaluation, and the results are as Figures 6 - 7 shown.

[0077] Figure 6 Shown is the result of the water sample toxicity evaluation of the prepared renal organoids with an increased proximal tubule ratio. Among them, Ultrapure water (ultrapure water) is the control group, and L - I and H - I are the water samples of the experimental group. DAPI represents the cell nucleus, and Merge represents the image after combining different fluorescence layers. The immunofluorescence results of the important components in the renal organoids, namely glomerulus (PODXL), renal proximal tubule (LTL), and distal tubule (CDH1), are shown in the figure.

[0078] Figure 7 Shown is Figure 6 the result of the fluorescence intensity statistics in []. It can be seen that the decrease in the relative fluorescence intensity of LTL, CDH1, and PODOL indicates that some water samples may cause damage to the renal organoids.

[0079] Although the present invention has been disclosed above in embodiments, it is not intended to limit the present invention. Appropriate modifications or equivalent replacements made by those of ordinary skill in the art to the technical solutions of the present invention shall be covered within the protection scope of the present invention. The protection scope of the present invention shall be subject to that defined by the claims.

Claims

1. A method for preparing a kidney organoid with an increased proximal tubule ratio in vitro, characterized in that: The following steps are involved: (1) Human embryonic stem cells WIBR3 were used to induce kidney organoids. WIBR3 cells were cultured until they were ready for passage and then digested with Accutase digestion solution; (2) After digestion is completed, use DMEM-F12 medium to terminate digestion, collect the cell suspension and centrifuge, discard the supernatant, collect the cell pellet and count; (3) Resuspend the collected cell pellet in stem cell culture medium and spread it on a well plate, add apoptosis inhibitor and mix well; (4) The system obtained in step (3) is subjected to suspension culture using kidney organoid culture medium to obtain kidney organoids with an increased proportion of proximal tubules.

2. The preparation method according to claim 1, characterized in that In step (1), WIBR3 cells were cultured in a six-well plate covered with Matrigel gel in advance, and the culture conditions were as follows: the culture medium was the stem cell culture medium mTeSR1, and the cells were cultured in a 37° C. incubator for 4 days.

3. The preparation method according to claim 1, characterized in that: The digestion conditions in step (1) are as follows: 500 μL of Accutase digestion solution is added to a well of a six-well plate, and digestion is carried out in a 37° C. incubator for 3-10 minutes.

4. The preparation method according to claim 1, characterized in that: When DMEM-F12 is used to terminate digestion in step (2), the volume added is 3-4 times the volume of the Accutase digestion solution; the centrifugation conditions are: centrifugation at 1000-1200 rpm for 3-5 minutes.

5. The preparation method according to claim 1, characterized in that: In step (3), the cells were resuspended in stem cell culture medium mTeSR1 and the suspension was adjusted to 1×10 5 -5×10 5 The cells were plated in a 6-well plate at a density of 100 μM / well. The apoptosis inhibitor Y-27632 was selected at a concentration of 2-10 μM.

6. The preparation method according to claim 1, characterized in that: The step of using kidney organoid culture medium for suspension culture in step (4) comprises: After 1-2 days of culture, the medium was replaced and the kidney organoid medium S1 was used for suspension culture; After 2-4 days of culture, the medium was replaced and kidney organoid medium S2 was used for suspension culture; After 2-4 days of culture, the medium was replaced and the kidney organoid medium S3 was used for suspension culture; After 4-6 days of culture, the medium was replaced and kidney organoid medium S4 was used for suspension culture; After 4-6 days of culture, the medium was replaced and the kidney organoid medium S5 was used for suspension culture; The culture was continued for 2-3 days to obtain kidney organoids with an increased proportion of proximal tubules.

7. The preparation method according to claim 6, characterized in that: In step (4), the kidney organoid culture media S1-S5 all include the basal culture medium Advanced RPMI 1640 or DMEM / F12, 100x L-glutamine, 0.5-1nM bismorphin, 1-2μM CHIR99021, and FGF9; wherein the kidney organoid culture medium S3 uses 25-100ng / mL of FGF9, the kidney organoid culture media S2 and S4 use 200-500ng / mL of FGF9, and the kidney organoid culture media S1 and S5 use 25-500ng / mL of FGF9.

8. The preparation method according to claim 7, characterized in that: In step (4), the following small molecules are also added to the kidney organoid culture medium S3: triiodothyronine, 2.5-25 pg / mL; Hydrocortisone, 100-500 nM; TGF-α, 10-50 ng / mL; CHIR99021, 0.5-5 μM; DAPT, 2.5-10 μM; BMP7, 10-50 ng / mL; LIF, 5-50 ng / mL; ITS-X, 5-10 ng / mL.

9. The method according to claim 6 or 7, characterized in that When using kidney organoid medium S3 for suspension culture in step (4), apoptosis inhibitor Y-27632 is added to prevent organoid cell apoptosis.

10. The method according to claim 1, characterized in that During the suspension culture in step (4), the corresponding fresh kidney organoid culture medium is replaced every day, and 2-4 mL of kidney organoid culture medium is added to each well of the six-well plate and cultured in a 37°C incubator.

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