Construction method and application of kidney organ model

Abstract

The invention relates to the technical field of organoid culture, in particular to a construction method and application of a kidney organoid model. Comprising the following steps: S1, carrying out digestion and flow sorting on renal cortex tissues to obtain renal stem cells; s2, carrying out plane culture on the kidney stem cells to obtain kidney epithelial progenitor cells; s3, performing three-dimensional culture on the renal epithelial progenitor cells to obtain the kidney organ model. According to the method, an adult renal cortex tissue is taken as a starting cell source, a kidney organ model with mature near-end and far-end renal tubule phenotypes is efficiently constructed through an optimized plane amplification and three-dimensional induction culture system, and the formation of kidney organs can be efficiently induced in a short time; the finally obtained kidney organ model can be used for simulating acute kidney injury in vitro, and an efficient tool is provided for kidney disease mechanism research, drug toxicity screening and personalized treatment model construction.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, and in particular to a method for constructing and applying a kidney organoid model. Background Technology

[0002] As vital metabolic and excretory organs, the kidneys are closely associated with various kidney diseases, such as acute kidney injury, chronic renal failure, and tubulointerstitial disease, when their function is impaired or fails. Currently, in vitro models based on primary kidney cells are key tools for studying kidney development mechanisms, disease pathology, and drug screening. However, their application is severely limited by issues such as the scarcity of donor tissue, insufficient cell purity, and difficulties in maintaining in vitro function.

[0003] Traditional methods rely heavily on embryonic kidney tissue or induced pluripotent stem cells (iPSCs) to construct kidney organoids. However, embryonic kidney tissue faces ethical controversies and limited availability, while iPSC-induced kidney organoids suffer from long culture cycles (typically 4-6 weeks), low structural maturity, and significant batch-to-batch variability, making it difficult to realistically simulate the physiological functions of adult kidneys. Existing kidney organoid models can mostly only simulate a single structure of the proximal or distal renal tubule, failing to simultaneously reproduce the functional phenotype of a complete nephron, resulting in insufficient accuracy in complex disease model studies such as acute kidney injury.

[0004] Chinese invention patent application CN118076727A provides a method for rapid construction of kidney organoids and nephritis models, as well as their applications and evaluation systems. It obtains kidney organoids by enzymatically digesting kidney cells to obtain single cells, and then differentiating and culturing them in a cell culture incubator. However, it does not solve the aforementioned technical problems.

[0005] In summary, establishing a kidney organoid culture system that is widely available in cells, easy to operate, and can simultaneously reproduce the phenotypes of mature proximal and distal renal tubules is a key problem that urgently needs to be solved in this field. Summary of the Invention

[0006] To address the aforementioned technical problems, the first aspect of this invention provides a method for constructing a kidney organoid model, comprising:

[0007] S1. Renal cortical tissue was digested and flow cytometry-sorted to obtain renal stem cells;

[0008] S2. The renal stem cells are subjected to planar culture to obtain renal epithelial progenitor cells;

[0009] S3. The renal epithelial progenitor cells are cultured in three dimensions to obtain a renal organoid model.

[0010] In one implementation, step S1 includes:

[0011] S11. Digest the renal cortex tissue to obtain digestive juice;

[0012] S12. After the digestive fluid is filtered, the filtrate is centrifuged, resuspended in FACS buffer, filtered to obtain a single-cell suspension, the cells are labeled with CD24 antibody, and renal stem cells are obtained by flow cytometry sorting.

[0013] In one implementation, step S1 includes:

[0014] S11. Use sterile PBS (phosphate buffer) to clean and disinfect the renal cortical tissue discarded during the operation. Cut the tissue into small pieces (1mm×1mm) and soak them in collagenase. Digest the tissue at 37°C. When the digestion solution becomes obviously turbid and large pieces of tissue disappear, add 10ml of LDM medium to stop the digestion and obtain the digestion solution.

[0015] S12. Filter the digestion solution using a cell filter and collect the filtrate; centrifuge the filtrate and collect the precipitate, resuspend it in FACS buffer, filter it using a cell filter to obtain a single-cell suspension, label the cells with CD24 antibody, and obtain renal stem cells by flow cytometry.

[0016] In one embodiment, the collagenase is Liberase. TM TM research grade solution.

[0017] In one embodiment, the cell filter has a size of 50-200 μm; it is further optionally 70 μm.

[0018] In one embodiment, the centrifugal force in step S1 is 300-500g, and the centrifugation time is 2-10min.

[0019] In one embodiment, the step of labeling cells with CD24 antibody in S12 specifically includes: adding CD24 antibody to the single-cell suspension and incubating on ice for 30-50 min, washing twice with FACS buffer, and resuspending in DMEM / F12 medium.

[0020] In one embodiment, the FACS buffer is composed of PBS buffer and FBS (fetal bovine serum), wherein the mass ratio of PBS buffer to FBS is 49:1.

[0021] In one embodiment, the amount of CD24 antibody added is based on 10 6 Calculate the amount of antibody added to each cell by adding 10 μL.

[0022] In one embodiment, step S2 includes: seeding the renal stem cells into a planar amplification culture medium for amplification culture, amplifying the cells until the cell confluence is >95%, then passaged the cells, and digesting them to obtain renal epithelial progenitor cells.

[0023] In one embodiment, step S2 includes: seeding the renal stem cells into a planar expansion culture medium and placing it at 37°C with 5% CO2. 2 Culture in a cell culture incubator, changing the culture medium every 2-3 days. When the cell confluence is >95%, passage the cells, discard the culture medium in the flask, add trypsin digestion solution and digest for 3-5 minutes. Observe under a microscope when the cells appear round, transparent and in suspension. Add an equal volume of planar amplification culture medium, centrifuge, collect the precipitate, resuspend, and obtain renal epithelial progenitor cells.

[0024] In one embodiment, the seeding density of the renal stem cells is 10-1. 3 -10 5 pcs / cm 2 ; Preferably 10 4 pcs / cm 2 .

[0025] In one embodiment, the planar amplification medium comprises: a basal medium and a growth regulator; the basal medium is Advanced DMEM / F12 medium.

[0026] In one embodiment, the growth regulator comprises, based on the volume of Advanced DMEM / F12 culture medium:

[0027] 0.5-2% N2 nutritional supplement; 1-4% B27 nutritional supplement; 1-5% penicillin-streptomycin solution; 20-50 ng / mL epithelial growth factor; 1-50 μM ROCK kinase inhibitor; 70-100 ng / mL Wnt signaling pathway activator; 1-5 μM MGF-β signaling inhibitor; 0.3-0.5 μg / mL hydrocortisone; 0.5 × 10 -10 -1.5×10 -10 M choleraemycin, 4.5-5.5 ng / mL insulin; 1.3 × 10 -4 -2.3×10 -4 M adenine; 4.5-5.5 μg / mL transferrin, 1.5 × 10 -9 -2.5×10 -9 M triiodothyronine.

[0028] In one embodiment, the Wnt signaling pathway activator includes recombinant human Wnt3a V3 protein and recombinant human RSPO1 protein, wherein the weight ratio of recombinant human Wnt3a V3 protein to recombinant human RSPO1 protein is (2-3):1; further optionally, it is 2.5:1.

[0029] In one embodiment, the growth regulator comprises, based on the volume of Advanced DMEM / F12 culture medium:

[0030] 1% N2 nutritional supplement; 2% B27 nutritional supplement; 1% penicillin-streptomycin solution; 20 ng / mL epidermal growth factor (EGF); 10 μM ROCK kinase inhibitor; 50 ng / mL recombinant human Wnt3a V3 protein; 20 ng / mL recombinant human RSPO1 protein; 1 μM M GF-β signaling inhibitor; 0.5 μg / mL hydrocortisone; 1.5 × 10 -10 M choleraemycin, 5.5 ng / mL insulin; 2.3 × 10 -4 M adenine; 5.5 μg / mL transferrin, 2.5 × 10 -9 M triiodothyronine.

[0031] In one embodiment, step S3 includes: digesting the renal epithelial progenitor cells with trypsin digestion solution, centrifuging, resuspending and counting them in BM, centrifuging again, resuspending them in 100% matrix gel at 1E6 cells / 100μL, adding the matrix gel containing cells as droplets to a preheated suspension culture plate, incubating at 37°C to solidify the matrix gel, and then adding organoid culture medium and culturing for 5-10 days to obtain a kidney organoid model.

[0032] In one embodiment, the organoid culture medium comprises: Advanced DMEM / F12 medium and growth regulators; the growth regulators include: 0.5-2% N2 nutrient supplement, 1-4% B27 nutrient supplement, 5-15 μM γ-secretase inhibitor, 10-20 ng / mL tumor suppressor-M, 5-15 μM fludrocortisone, 5-15 μM MGF-β receptor inhibitor, and 150-200 ng / mL cell growth factor.

[0033] In one embodiment, the cell growth factors include human epidermal growth factor, hepatocyte growth factor and fibroblast growth factor, wherein the weight ratio of human epidermal growth factor, hepatocyte growth factor and fibroblast growth factor is (1-3):1:(4-6); further optionally, it is 2:1:4.

[0034] In one embodiment, the organoid culture medium comprises: Advanced DMEM / F12 medium and growth regulators; the growth regulators include: 1% N2 nutrient supplement, 1% B27 nutrient supplement, 10 μM γ-secretase inhibitor (DAPT), 20 ng / mL OSM, 10 μM fludrocortisone, 10 μM MGF-β receptor inhibitor, and 50 ng / mL recombinant human epidermal growth factor, 40 ng / mL recombinant human hepatocyte growth factor, and 100 ng / mL recombinant human fibroblast growth factor.

[0035] The second aspect of the present invention provides an application of a method for constructing a kidney organoid model, the method being used to prepare an in vitro model of acute kidney injury.

[0036] Beneficial effects:

[0037] This invention provides a method for constructing a kidney organoid model and its application, which has the following advantages:

[0038] (1) This invention uses adult renal cortical tissue as the starting cell source and efficiently constructs a renal organoid model with mature proximal and distal renal tubular phenotypes through an optimized planar amplification and three-dimensional induction culture system.

[0039] (2) The present invention optimizes the planar amplification medium. By selecting Advanced DMEM / F12 medium as the basic medium and adding specific growth regulators, such as epithelial growth factor, hydrocortisone, triiodothyronine, etc., the cell amplification efficiency is significantly improved, and the growth rate can be kept stable during the passage process.

[0040] (3) The three-dimensional induction system of this invention uses a specific organoid culture medium. Through the synergistic effect of factors such as DAPT and OSM, it promotes the rapid maturation of organoids. The resulting kidney organoid model can be used to simulate acute kidney injury in vitro, providing an efficient tool for the study of kidney disease mechanisms, drug toxicity screening and the construction of personalized treatment models.

[0041] (4) The culture process of the present invention is simple and the differentiation cycle is short. It can efficiently induce the formation of kidney organoids in a short time, providing a feasible approach for long-term in vitro culture of kidney organoids and has broad application prospects. Attached Figure Description

[0042] Figure 1 The results of expression detection of cell surface markers obtained after planar amplification culture in step S2 of Example; among them, CD13 is a marker of mature renal tubular epithelial cells, and CD24 is a marker of renal tubular epithelial precursor cells;

[0043] Figure 2The results of expression detection of cell markers in kidney organoids are shown in the example.

[0044] Figure 3 The transmission electron microscopy results of cells and kidney organoids obtained after planar amplification culture in step S2 of the embodiment;

[0045] Figure 4 H&E stained section of kidney organoid as shown in the example;

[0046] Figure 5 Immunofluorescence staining image of kidney organoids in the embodiment;

[0047] Figure 6 The image shows the qPCR results of the kidney organoid hypoxia injury model as an example.

[0048] Figure 7 Figure showing the qPCR results optimized for kidney organoid inducing factors. Detailed Implementation

[0049] The following is some information about the raw materials involved in this invention.

[0050] Table 1

[0051]

[0052]

[0053] Note: Unless otherwise specified, the solvents of the solutions involved in this invention are all water; the reagent concentrations are all mass concentrations; the room temperature is 25°C; and the raw materials, consumables and equipment used are all commercially available.

[0054] Example

[0055] The first aspect of this embodiment provides a method for constructing a kidney organoid, comprising:

[0056] S1. Renal cortical tissue is digested and sorted by flow cytometry to obtain renal stem cells; specific steps include:

[0057] S11. Use sterile PBS (phosphate buffer) to clean and disinfect the renal cortical tissue discarded during the operation. Cut the tissue into small pieces (1mm×1mm) and soak them in collagenase. Digest the tissue at 37°C. When the digestion solution becomes obviously turbid and large pieces of tissue disappear, add 10ml of LDM medium to stop the digestion and obtain the digestion solution.

[0058] S12. Filter the digestion solution using a 70 μm cell filter and collect the filtrate; after centrifuging the filtrate for 5 min with 300 g, remove the centrifuge tube without shaking it, spray the tube with alcohol and wipe it dry, then place it in a biosafety cabinet, resuspend it with 2 mL of FACS buffer, and filter it using a 70 μm cell filter to obtain a single-cell suspension.

[0059] Take 20 μL of single-cell suspension and add 20 μL of 0.4% trypan blue staining solution, mix well and count. Every 10... 6 After adding 10 μL of CD24 antibody to each cell, incubate on ice for 30 min, add FACS buffer to make up to 15 mL, and centrifuge at 200 g for 5 min; discard the supernatant, resuspend the cells in 1 mL of FACS buffer, add FACS buffer to make up to 15 mL, and centrifuge at 200 g for 5 min; discard the supernatant, resuspend the cells in 1 mL of LDM / F12 medium, and separate the renal stem cells by flow cytometry.

[0060] S2. Perform planar culture of the renal stem cells to obtain renal epithelial progenitor cells; the specific steps include:

[0061] The renal stem cells were used at 10 4 pcs / cm 2 Inoculate the culture medium at the specified inoculation density into the planar amplification medium and incubate at 37°C with 5% CO2. 2 Culture in a cell culture incubator, changing the culture medium every 2-3 days. When the cell confluence is >95%, passage the cells, discard the culture medium in the flask, add trypsin digestion solution and digest for 3 minutes. Observe under a microscope when the cells appear round, transparent and in suspension. Add an equal volume of planar amplification culture medium, centrifuge, collect the precipitate, resuspend, and obtain renal epithelial progenitor cells.

[0062] The planar amplification medium comprises: a basal medium and growth regulators; the basal medium is Advanced DMEM / F12 medium. The growth regulators include:

[0063] 1% N2 nutritional supplement;

[0064] 2% B27 nutritional supplement;

[0065] 1% penicillin-streptomycin solution;

[0066] 20 ng / mL epidermal growth factor (EGF);

[0067] 10μM ROCK kinase inhibitor;

[0068] 50 ng / mL recombinant human Wnt3a V3 protein;

[0069] 20 ng / mL recombinant human RSPO1 protein;

[0070] 1μMTGF-β signaling inhibitor;

[0071] 0.5 μg / mL hydrocortisone;

[0072] 1.5×10 -10 M choleraemycin;

[0073] 5.5 ng / mL insulin;

[0074] 2.3×10 -4 M-adenine;

[0075] 5.5 μg / mL transferrin;

[0076] 2.5×10 -9 M triiodothyronine.

[0077] S3. The renal epithelial progenitor cells are cultured in three dimensions to obtain a renal organoid model; the specific steps include:

[0078] The renal epithelial progenitor cells were digested with trypsin digestion solution, centrifuged, resuspended in BM for counting, and centrifuged again. They were then resuspended in 100% matrix gel at a ratio of 1E6 cells / 100μL. The matrix gel containing cells was added dropwise (100μL per drop) to a preheated suspension culture plate and incubated at 37°C to allow the matrix gel to solidify. Subsequently, organoid culture medium was added and cultured for 7 days to obtain a kidney organoid model.

[0079] The organoid culture medium comprises: Advanced DMEM / F12 medium and growth regulators; the growth regulators include:

[0080] 1% N2 nutritional supplement;

[0081] 1% Vitamin B27 supplement;

[0082] 10 μM γ-secretase inhibitor (DAPT);

[0083] 20 ng / mL of OSM (suppressant tumor-M);

[0084] 10 μM Fludrocortisone;

[0085] 10 μMTGF-β receptor inhibitor;

[0086] 50 ng / mL recombinant human epidermal growth factor;

[0087] 40 ng / mL recombinant human hepatocyte growth factor;

[0088] 100 ng / mL recombinant human fibroblast growth factor.

[0089] The second aspect of this embodiment provides an application of a method for constructing a kidney organoid model, which is used to prepare an in vitro model of acute kidney injury.

[0090] Performance testing

[0091] 1. Cell marker assay

[0092] 1) Flow cytometry analysis was used to analyze cell surface markers (CD13 and CD24) obtained after planar amplification culture in step S2 of Example 1 to verify whether the cells possess renal tubular epithelial characteristics and precursor cell potential, providing a qualified cell source for subsequent three-dimensional organoid culture. The detection results are shown below. Figure 1 Among them, CD13 is a marker of mature renal tubular epithelial cells, indicating that the cells have a functional epithelial phenotype; CD24 is a marker of renal tubular epithelial precursor cells, suggesting that the cells retain the ability to proliferate and differentiate.

[0093] like Figure 1 As shown, the planar cultured cells highly expressed CD13 and CD24, demonstrating the successful enrichment of a population of renal tubular epithelial cells with both mature and precursor characteristics.

[0094] 2) The qPCR method was used to detect the cell markers of the kidney organoids obtained in the examples, with primary kidney tissue and plane culture as controls. The detection results are shown in [Figure 1]. Figure 2 As can be seen from the figure, the markers of the proximal kidney organoids (HNF4A, LRP2), loop of Henle (SLC12A1), and distal renal tubules (AQP3) obtained after three-dimensional culture in the embodiments of this application are upregulated compared with those obtained by planar culture, and are close to the level of primary kidney tissue.

[0095] 2. Transmission electron microscopy results

[0096] Transmission electron microscopy was performed on the cells (EM-2D) obtained after planar amplification culture in step S2 of Example, and on the resulting kidney organoids (EM-3D-Day7). The results are shown in [Figure number missing]. Figure 3 and Figure 4 .like Figure 3 As shown, cells form cystic structures after three-dimensional culture. Figure 4 As shown, H&E staining revealed that the obtained kidney organoids had a sac-like, hollow, three-dimensional structure.

[0097] 3. Immunofluorescence detection results

[0098] The kidney organoids obtained in the examples were stained with DAPI (4',6-diamidinyl-2-phenylindole), and then immunofluorescence was performed to detect the kidney organoid cell markers HNF4A, LRP2, and NKCC2. The detection results are shown in [Figure 1]. Figure 5 .

[0099] like Figure 5 As shown, immunofluorescence staining technology was used to verify the protein expression of HNF4A, LRP2 and NKCC2 markers.

[0100] 4. Determination of specific injury markers in proximal and distal nephrons after hypoxic injury to renal organoids

[0101] The kidney organoids obtained in the examples were cultured in reduced factor medium (Advanced DMEM / F12 with N-2 nutrient supplement (1X) and B-27 nutrient supplement (1X)) (DIFF.n), followed by hypoxia (1% oxygen) culture in reduced factor medium for 48 h (DIFF.h), and then hypoxia (1% oxygen) culture in reduced factor medium for 48 h followed by reoxygenation for 5 days (DIFF.h / n). The qPCR results are shown in […]. Figure 6 The figure shows that the expression of specific damage markers in the proximal (HAVCR1) and distal (LCN2) nephrons is upregulated. These results demonstrate that the kidney organoids constructed in this invention can effectively simulate the biological characteristics of kidney cells, providing an effective in vitro model for studying kidney development, disease mechanisms, and drug screening.

[0102] 5. An investigation into changes in organoid maturity after increases or decreases in DAPT, OSM, and Fludrocortisone factors.

[0103] This application investigates the effect of altering the combination of inducing factors in organoid culture medium on organoid maturation. The expression of the proximal marker HNF4A was detected using qPCR, and the results are shown below. Figure 7 Where D stands for DAPT, O for OSM, and F for Fludrocortisone. The figure shows that the combination of DAPT, OSM, and Fludrocortisone (DOF) results in the highest expression, representing the optimal combination of inducing factors.

Claims

1. A method for constructing a kidney organoid model, characterized in that, include: S1. Renal cortical tissue was digested and flow cytometry-sorted to obtain renal stem cells; S2. The renal stem cells are subjected to planar culture to obtain renal epithelial progenitor cells; S3. The renal epithelial progenitor cells are cultured in three dimensions to obtain a renal organoid model.

2. The method for constructing a kidney organoid model according to claim 1, characterized in that, Step S1 includes: S11. Digest the renal cortex tissue to obtain digestive juice; S12. After the digestive fluid is filtered, the filtrate is centrifuged, resuspended in FACS buffer, filtered to obtain a single-cell suspension, the cells are labeled with CD24 antibody, and renal stem cells are obtained by flow cytometry sorting.

3. The method for constructing a kidney organoid model according to claim 2, characterized in that, The specific steps of labeling cells with CD24 antibody in S12 include: adding CD24 antibody to the single cell suspension and incubating on ice for 30-50 min, washing twice with FACS buffer, and then resuspending in DMEM / F12 medium.

4. The method for constructing a kidney organoid model according to claim 1, characterized in that, The S2 step includes: seeding the renal stem cells into a planar amplification culture medium for amplification culture, amplifying culture until the cell confluence is >95%, performing passage culture, and digesting to obtain renal epithelial progenitor cells.

5. The method for constructing a kidney organoid model according to claim 4, characterized in that, The planar amplification medium comprises: basal medium and growth regulators; the basal medium is Advanced DMEM / F12 medium.

6. The method for constructing a kidney organoid model according to claim 5, characterized in that, Based on the volume of Advanced DMEM / F12 medium, the growth regulator comprises: 0.5-2% N2 nutritional supplement; 1-4% B27 nutritional supplement; 1-5% penicillin-streptomycin solution; 20-50 ng / mL epithelial growth factor; 1-50 μM ROCK kinase inhibitor; 70-100 ng / mL Wnt signaling pathway activator; 1-5 μM MGF-β signaling inhibitor; 0.3-0.5 μg / mL hydrocortisone; 0.5 × 10 -10 -1.5×10 -10 M choleraemycin, 4.5-5.5 ng / mL insulin; 1.3 × 10 -4 -2.3×10 -4 M adenine; 4.5-5.5 μg / mL transferrin, 1.5 × 10 -9 -2.5×10 -9 M triiodothyronine.

7. The method for constructing a kidney organoid model according to claim 1, characterized in that, The S3 step includes: digesting the renal epithelial progenitor cells with trypsin digestion solution, centrifuging, resuspending and counting them in BM, centrifuging again, and resuspending them in 100% matrix gel at 1E6 cells / 100μL. The matrix gel containing the cells is dropped onto a preheated suspension culture plate and incubated at 37°C to solidify the matrix gel. Then, organoid culture medium is added and cultured for 1-5 days to obtain a kidney organoid model.

8. The method for constructing a kidney organoid model according to claim 7, characterized in that, The organoid culture medium comprises: Advanced DMEM / F12 medium and growth regulators; the growth regulators include: 0.5-2% N2 nutrient supplement, 1-4% B27 nutrient supplement, 5-15 μM γ-secretase inhibitor, 10-20 ng / mL tumor suppressor-M, 5-15 μM fludrocortisone, 5-15 μM MGF-β receptor inhibitor and 150-200 ng / mL cell growth factor.

9. The method for constructing a kidney organoid model according to claim 8, characterized in that, The cell growth factors include human epidermal growth factor, hepatocyte growth factor and fibroblast growth factor, and the weight ratio of human epidermal growth factor, hepatocyte growth factor and fibroblast growth factor is (1-3):1:(4-6).

10. An application of a method for constructing a kidney organoid model according to any one of claims 1-8, characterized in that, The construction method described above is used to prepare an in vitro model of acute kidney injury.

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