Culture medium and culture method for mouse kidney organoid

Abstract

The invention relates to a culture method for mouse kidney organs, which comprises the following steps: (1) extracting primary cells from mouse kidney tissues, and preparing adult stem cells; (2) dispersing the adult stem cells in a mixed solution of an amplification culture medium and matrigel, and incubating until the mixed solution is solidified; (3) adding an amplification culture medium, culturing for 7-10 days, and continuously culturing in the amplification culture medium for at least 2-4 days after passage; and (4) replacing the amplification culture medium with a differential culture medium, and continuously culturing for 11-14 days to obtain the mouse kidney organoid. According to the method, the amplification culture medium and the differentiation culture medium are adopted for culturing the kidney organoid in two stages, comprehensive differentiation of multiple cell types of the kidney is promoted, the culture period of the mouse kidney organoid can be shortened, and the differentiation success rate is increased.

Description

Technical Field

[0001] The present invention relates to the field of organoid culture, and in particular to a culture medium and a culture method for mouse kidney organoids. Background Art

[0002] Organoids are "mini organs" with three-dimensional spatial structures that are induced by self-assembly of adult stem cells, embryonic stem cells or induced pluripotent stem cells under 3D culture conditions in vitro. They have complex structures similar to real organs and multiple cell types, and can simulate the physiological and pathological states of the source tissue or organ to the greatest extent.

[0003] At present, most of the kidney organoids are induced and differentiated by pluripotent stem cells, including nephron organoids and ureter / collecting duct organoids. For example, the CN117070440A patent discloses a method for efficiently constructing human kidney organoids, which is based on hPSCs induction of kidney organoids, induced by pluripotent embryonic stem cells (ES) and their artificial induced pluripotent stem cells (iPSC). This method is time-consuming and laborious, has high technical requirements, has certain uncontrollability in the differentiation direction, poor genomic stability, and cannot be further proliferated. Disadvantages; at the same time, iPSC-derived organoids are similar to the early stages of human kidney development, contain non-kidney cell types, and are not ideal systems for simulating adult diseases or studying potential regenerative therapies. The CN105378062A patent discloses organoids and their uses including separation and cultivation of SRC+ kidney cell group sources. The method requires first sorting out the SRC+ kidney cell group by flow or magnetic beads, and then culturing it into organoids. The preparation process is relatively cumbersome and lacks glomerular cells. Patent CN116218762A discloses a culture medium for renal tubular organoids and a method for culturing renal tubular organoids, wherein the method prepares renal tubular organoids lacking glomerular cells.

[0004] Currently, there is no method for culturing mouse kidney organoids based on primary cells derived from kidney tissue that contains multiple kidney tissue cell types (including proximal tubules, Medullary loops, distal tubules and glomeruli). Summary of the invention

[0005] To solve the above technical problems, the present invention includes the following aspects:

[0006] A first aspect of the present invention provides a method for culturing mouse kidney organoids, the method comprising the following steps:

[0007] (1) Extracting primary cells from mouse kidney tissue to prepare adult stem cells;

[0008] (2) dispersing the adult stem cells obtained in step (1) in a mixture of expansion medium and matrix gel, and incubating until the mixture solidifies;

[0009] (3) adding expansion medium and culturing for 7-10 days, and continuing to culture in the expansion medium for at least 2-4 days after subculturing;

[0010] (4) replacing the expansion medium with a differentiation medium and continuing the culture for 11-14 days to obtain mouse kidney organoids;

[0011] The mouse kidney organoids contain proximal tubular cells, medullary loop cells, distal tubular cells and glomerular cells.

[0012] Preferably, the mouse kidney tissue is C57BL / 6 mouse kidney tissue.

[0013] Preferably, the specific method for preparing adult stem cells in step (1) is as follows:

[0014] (i) Tissue collection: mice were sacrificed, dissected, and the isolated kidneys were placed in basal culture medium;

[0015] (ii) trimming tissue: removing fat and other tissues;

[0016] (iii) Digestion of tissue: Add collagenase IV and digest on a shaker;

[0017] (iv) Microscopic examination: Pipette up and down 10-20 times to promote tissue fragmentation and examine microscopically for the presence of ductal structures. If not, continue digestion;

[0018] (v) Termination of digestion: Add washing medium to resuspend and terminate digestion, filter with a 70 μm cell sieve, centrifuge and remove the supernatant. The remaining precipitate is the primary cells containing adult stem cells.

[0019] Preferably, the basal culture medium in step (i) comprises Advanced DMEM / F12, HEPES, GlutaMAX, a penicillin-streptomycin mixture and Y-27632.

[0020] More preferably, the basic culture medium in step (i) comprises Advanced DMEM / F12, 0.5-2% HEPES, 0.5-2% GlutaMAX, 0.5-2% penicillin-streptomycin mixture and 5-20 μM Y-27632. The above component content percentages are all volume percentages.

[0021] Further preferably, the basal culture medium in step (i) comprises Advanced DMEM / F12, 1% HEPES, 1% GlutaMAX, 1% penicillin-streptomycin mixture and 10 μM Y-27632. The above component content percentages are all volume percentages.

[0022] Preferably, the washing medium added in step (v) is a high-glucose DMEM medium containing 1% fetal bovine serum and 1% penicillin / streptomycin. The above component content percentages are all volume percentages.

[0023] Preferably, the expansion medium in steps (2) and (3) comprises Advanced DMEM / F12, HEPES, GlutaMAX, a penicillin-streptomycin mixture, gentamicin, amphotericin B, a conditioned medium I containing Wnt-3A, R-spondin 3 and Noggin, N-acetylcysteine, B27 supplement, A83-01, EGF, FGF-10 and Y-27632.

[0024] More preferably, the expansion medium in steps (2) and (3) comprises Advanced DMEM / F12, 0.5-2% HEPES, 0.5-2% GlutaMAX, 0.5-2% penicillin-streptomycin mixture, 10-50 μg / ml gentamicin, 20-100 μg / ml amphotericin B, 30-50% conditioned medium I containing Wnt-3A, R-spondin 3 and Noggin, 0.5-2 mM N-acetylcysteine, 0.5-3% B27 supplement, 1-10 μM A83-01, 20-100 ng / ml EGF, 50-200 ng / ml FGF-10 and 5-20 μM Y-27632. The above component content percentages are all volume percentages.

[0025] Further preferably, the expansion medium in steps (2) and (3) comprises Advanced DMEM / F12, 1% HEPES, 1% GlutaMAX, 1% penicillin-streptomycin mixture, 20 μg / ml gentamicin, 50 μg / ml amphotericin B, 40% conditioned medium I containing Wnt-3A, R-spondin 3 and Noggin, 1.25 mM N-acetylcysteine, 1.5% B27 supplement, 5 μM A83-01, 50 ng / ml EGF, 100 ng / ml FGF-10 and 10 μM Y-27632. The above component content percentages are all volume percentages.

[0026] Further preferably, the expansion medium in steps (2) and (3) is composed of Advanced DMEM / F12, 1% HEPES, 1% GlutaMAX, 1% penicillin-streptomycin mixture, 20 μg / ml gentamicin, 50 μg / ml amphotericin B, 40% conditioned medium I containing Wnt-3A, R-spondin 3 and Noggin, 1.25 mM N-acetylcysteine, 1.5% B27 supplement, 5 μM A83-01, 50 ng / ml EGF, 100 ng / ml FGF-10 and 10 μM Y-27632. The above component content percentages are all volume percentages.

[0027] Preferably, the preparation method of the conditioned medium I containing Wnt-3A, R-spondin 3 and Noggin is as follows:

[0028] (a) 5*10 7 L-WRN cells were seeded in a 15 cm culture dish and 25-30 ml of culture medium was added;

[0029] (b) After 60-72 h of culture, the first batch of conditioned medium was collected, centrifuged at 2000 g / 10 min, the supernatant was supplemented with fresh medium, and the supernatant was stored at 4°C;

[0030] (c) After continuing to culture for 36 to 48 hours, the second batch of conditioned medium was collected, centrifuged at 2000 g / 10 min, and the supernatant was taken and supplemented with new medium;

[0031] (d) After continuing to culture for 36 to 48 hours, the third batch of conditioned medium was collected and centrifuged at 2000 g / 10 min to obtain the supernatant. All conditioned medium were combined, mixed, aliquoted, and stored at -80°C.

[0032] Preferably, the culture medium in step (a) comprises a mixture of DMEM / F12, 10% FBS and 1% penicillin-streptomycin.

[0033] Preferably, the matrix gel in step (2) is Matrigel.

[0034] Preferably, in step (2), the volume ratio of the amplification medium to the matrix gel is 1:1.

[0035] Preferably, the step (2) comprises the following steps:

[0036] (i) dispersing adult stem cells in a mixture of expansion medium and matrix gel;

[0037] (ii) dropping a mixture of adult stem cells, expansion medium and matrix gel into a cell culture plate;

[0038] (iii) Incubate the culture plate in a cell culture incubator until the mixture solidifies.

[0039] Preferably, in step (iii), the culture plate is placed upside down in a cell culture incubator at 37° C. and 5% CO 2 and incubated for 20-30 minutes until the mixed solution solidifies.

[0040] Preferably, the expansion medium in step (3) is incubated in a 37° C. water bath before being added.

[0041] Preferably, the expansion medium in step (3) is replaced every 2-3 days.

[0042] Preferably, the differentiation medium in step (4) contains aromatic retinoic acid, fludrocortisone acetate and forskolin.

[0043] More preferably, the differentiation medium in step (4) comprises A83-01, conditioned medium II containing Wnt-3A, aromatic retinoic acid, fludrocortisone acetate and forskolin.

[0044] Further preferably, the differentiation medium in step (4) comprises Advanced DMEM / F12, HEPES, GlutaMAX, a penicillin-streptomycin mixture, a conditioned medium II containing Wnt-3A, A83-01, aromatic retinoic acid, fludrocortisone acetate and forskolin.

[0045] Preferably, the differentiation medium in step (4) contains 1-5 μM aromatic retinoic acid, 5-20 μM fludrocortisone acetate and 5-20 μM forskolin.

[0046] More preferably, the differentiation medium in step (4) contains 3 μM aromatic retinoic acid, 10 μM fludrocortisone acetate and 10 μM forskolin.

[0047] Preferably, the differentiation medium in step (4) comprises 1-10 μM A83-01, 30-50% of conditioned medium II containing Wnt-3A, 1-5 μM aromatic retinoic acid, 5-20 μM fludrocortisone acetate and 5-20 μM forskolin. The above component content percentages are all volume percentages.

[0048] More preferably, the differentiation medium in step (4) comprises 5 μM A83-01, 40% conditioned medium II containing Wnt-3A, 3 μM aromatic retinoic acid, 10 μM fludrocortisone acetate and 10 μM forskolin. The above component content percentages are all volume percentages.

[0049] Preferably, the differentiation medium in step (4) comprises Advanced DMEM / F12, 0.5-2% HEPES, 0.5-2% GlutaMAX, 0.5-2% penicillin-streptomycin mixture, 1-10 μM A83-01, 30-50% conditioned medium II containing Wnt-3A, 1-5 μM aromatic retinoic acid, 5-20 μM fludrocortisone acetate and 5-20 μM forskolin. The above component content percentages are all volume percentages.

[0050] More preferably, the differentiation medium in step (4) comprises Advanced DMEM / F12, 1% HEPES, 1% GlutaMAX, 1% penicillin-streptomycin mixture, 5 μM A83-01, 40% conditioned medium II containing Wnt-3A, 3 μM aromatic retinoic acid, 10 μM fludrocortisone acetate and 10 μM forskolin. The above component content percentages are all volume percentages.

[0051] Preferably, the preparation method of the conditioned medium II containing Wnt-3A is as follows:

[0052] (a) 5*10 7 LWnt-3A cells were seeded in a 15 cm culture dish and 25-30 ml of culture medium was added;

[0053] (b) After 60-72 h of culture, the first batch of conditioned medium was collected, centrifuged at 2000 g / 10 min, the supernatant was supplemented with fresh medium, and the supernatant was stored at 4°C;

[0054] (c) After continuing to culture for 36 to 48 hours, the second batch of conditioned medium was collected, centrifuged at 2000 g / 10 min, and the supernatant was taken and supplemented with new medium;

[0055] (d) After continuing to culture for 36 to 48 hours, the third batch of conditioned medium was collected and centrifuged at 2000 g / 10 min to obtain the supernatant. All conditioned medium were combined, mixed, aliquoted, and stored at -80°C.

[0056] Preferably, the culture medium in step (a) comprises a mixture of DMEM / F12, 10% FBS and 1% penicillin-streptomycin.

[0057] Preferably, the differentiation medium in step (4) is replaced every 2-3 days.

[0058] A second aspect of the present invention provides a culture medium composition for culturing mouse kidney organoids, the culture medium composition comprising an expansion medium and a differentiation medium, the expansion medium comprising Advanced DMEM / F12, HEPES, GlutaMAX, a penicillin-streptomycin mixture, gentamicin, amphotericin B, a conditioned medium I comprising Wnt-3A, R-spondin 3 and Noggin, N-acetylcysteine, B27 supplement, A83-01, EGF, FGF-10 and Y-27632, and the differentiation medium comprising Advanced DMEM / F12, HEPES, GlutaMAX, a penicillin-streptomycin mixture, a conditioned medium II comprising Wnt-3A, A83-01, aromatic retinoic acid, fludrocortisone acetate and forskolin.

[0059] Preferably, the expansion medium comprises Advanced DMEM / F12, 0.5-2% HEPES, 0.5-2% GlutaMAX, 0.5-2% penicillin-streptomycin mixture, 10-50 μg / ml gentamicin, 20-100 μg / ml amphotericin B, 30-50% conditioned medium I comprising Wnt-3A, R-spondin 3 and Noggin, 0.5-2 mM N-acetylcysteine, 0.5-3% B27 supplement, 1-10 μM A83-01, 20-100 ng / ml EGF, 50-200 ng / ml FGF-10 and 5-20 μM Y-27632; the differentiation medium comprises Advanced DMEM / F12, 0.5-2% HEPES, 0.5-2% GlutaMAX, 0.5-2% penicillin-streptomycin mixture, 1-10 μM A83-01, 30-50% of conditioned medium II containing Wnt-3A, 1-5 μM aromatic retinoic acid, 5-20 μM fludrocortisone acetate and 5-20 μM forskolin. The above component content percentages are all volume percentages.

[0060] More preferably, the expansion medium comprises Advanced DMEM / F12, 1% HEPES, 1% GlutaMAX, 1% penicillin-streptomycin mixture, 20 μg / ml gentamicin, 50 μg / ml amphotericin B, 40% conditioned medium I comprising Wnt-3A, R-spondin 3 and Noggin, 1.25 mM N-acetylcysteine, 1.5% B27 supplement, 5 μM A83-01, 50 ng / ml EGF, 100 ng / ml FGF-10 and 10 μM Y-27632; the differentiation medium comprises Advanced DMEM / F12, 1% HEPES, 1% GlutaMAX, 1% penicillin-streptomycin mixture, 5 μM A83-01, 40% conditioned medium II comprising Wnt-3A, 3 μM aromatic retinoic acid, 10 μM fludrocortisone acetate and 10 μM forskolin. The above component content percentages are all volume percentages.

[0061] Further preferably, the expansion medium consists of Advanced DMEM / F12, 1% HEPES, 1% GlutaMAX, 1% penicillin-streptomycin mixture, 20 μg / ml gentamicin, 50 μg / ml amphotericin B, 40% conditioned medium I containing Wnt-3A, R-spondin 3 and Noggin, 1.25 mM N-acetylcysteine, 1.5% B27 supplement, 5 μM A83-01, 50 ng / ml EGF, 100 ng / ml FGF-10 and 10 μM Y-27632; the differentiation medium consists of Advanced DMEM / F12, 1% HEPES, 1% GlutaMAX, 1% penicillin-streptomycin mixture, 5 μM A83-01, 40% conditioned medium II containing Wnt-3A, 3 μM aromatic retinoic acid, 10 μM fludrocortisone acetate and 10 μM forskolin. The above component content percentages are all volume percentages.

[0062] Preferably, the preparation method of the conditioned medium I containing Wnt-3A, R-spondin 3 and Noggin is as follows:

[0063] (a) 5*10 7 L-WRN cells were seeded in a 15 cm culture dish and 25-30 ml of culture medium was added;

[0064] (b) After 60-72 h of culture, the first batch of conditioned medium was collected, centrifuged at 2000 g / 10 min, the supernatant was supplemented with fresh medium, and the supernatant was stored at 4°C;

[0065] (c) After continuing to culture for 36 to 48 hours, the second batch of conditioned medium was collected, centrifuged at 2000 g / 10 min, and the supernatant was taken and supplemented with new medium;

[0066] (d) After continuing to culture for 36 to 48 hours, the third batch of conditioned medium was collected and centrifuged at 2000 g / 10 min to obtain the supernatant. All conditioned medium were combined, mixed, aliquoted, and stored at -80°C.

[0067] Preferably, the culture medium in step (a) comprises a mixture of DMEM / F12, 10% FBS and 1% penicillin-streptomycin.

[0068] Preferably, the preparation method of the conditioned medium II containing Wnt-3A is as follows:

[0069] (a) 5*10 7 LWnt-3A cells were seeded in a 15 cm culture dish and 25-30 ml of culture medium was added;

[0070] (b) After 60-72 h of culture, the first batch of conditioned medium was collected, centrifuged at 2000 g / 10 min, the supernatant was supplemented with fresh medium, and the supernatant was stored at 4°C;

[0071] (c) After continuing to culture for 36 to 48 hours, the second batch of conditioned medium was collected, centrifuged at 2000 g / 10 min, and the supernatant was taken and supplemented with new medium;

[0072] (d) After continuing to culture for 36 to 48 hours, the third batch of conditioned medium was collected and centrifuged at 2000 g / 10 min to obtain the supernatant. All conditioned medium were combined, mixed, aliquoted, and stored at -80°C.

[0073] Preferably, the culture medium in step (a) comprises a mixture of DMEM / F12, 10% FBS and 1% penicillin-streptomycin.

[0074] The third aspect of the present invention provides use of the above-mentioned culture medium composition in culturing mouse kidney organoids, wherein the mouse kidney organoids comprise proximal tubular cells, medullary loop cells, distal tubular cells and glomerular cells.

[0075] A fourth aspect of the present invention provides the use of the above-mentioned differentiation medium in promoting differentiation of mouse kidney organoid cells, wherein the mouse kidney organoid cells simultaneously differentiate into one or more of proximal tubular cells, medullary loop cells, distal tubular cells and glomerular cells.

[0076] Preferably, the mouse kidney organoid cells are simultaneously differentiated into proximal tubular cells, medullary loop cells, distal tubular cells and glomerular cells.

[0077] Preferably, the mouse kidney organoid cells are differentiated into distal tubular cells and glomerular cells simultaneously.

[0078] The technical effects produced by the present invention are:

[0079] 1. Compared with the iPSC-derived kidney organoids used in most current technologies, the kidney organoids prepared using adult stem cells in the present invention have the following advantages: (1) The stem cell source is easier to obtain; (2) The prepared kidney organoids are more mature, closer to fully developed kidney tissue, and more suitable for the study of adult diseases; (3) The prepared kidney organoids have a more single cell type, consisting entirely of kidney cells without containing other cells.

[0080] 2. The present invention creatively uses expansion medium and differentiation medium to culture kidney organoids in two stages. The inventors unexpectedly discovered that the differentiation medium selected by the present invention can promote the comprehensive differentiation of multiple cell types in the kidney, including the simultaneous differentiation of proximal tubular cells, medullary loop cells, distal tubular cells and glomerular cells.

[0081] 3. Additive factors such as Noggin, Wnt-3A and R-spondin 3 are expensive, and the quality of different batches is inconsistent. The present invention uses L-WRN cell line to produce conditioned medium containing Wnt-3A, R-spondin 3 and Noggin, and uses LWnt-3A cell line to produce conditioned medium of Wnt-3A. This culture method has lower cost and higher consistency, and can improve the success rate and growth rate of mouse kidney organoid culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 is a bright field image of a kidney organoid of the present invention, wherein Figure 1 A is the kidney organoid cultured in expansion medium. Figure 1 B is kidney organoid cultured in differentiation medium;

[0083] Figure 2 is the HE staining result of the kidney organoid of the present invention, wherein Figure 2 A is the kidney organoid cultured in expansion medium. Figure 2 B is kidney organoid cultured in differentiation medium;

[0084] Figure 3 It is the identification result of the proximal tubule marker LTL and the medullary loop cell marker Nkcc2 of the kidney organoid of the present invention;

[0085] Figure 4 It is the identification result of the distal tubule cell marker Calb1 before (upper) and after (lower) differentiation of the kidney organoid of the present invention;

[0086] Figure 5These are the results of identification of the glomerular cell marker WT1 in the kidney organoids of the present invention before differentiation (upper) and after differentiation (lower). DETAILED DESCRIPTION

[0087] Experimental Example 1: Cultivation of Mouse Kidney Organoids

[0088] 1. Preparation of kidney organoids derived from adult stem cells

[0089] (1) Tissue collection: The kidney tissue of 12-week-old C57BL / 6 mice obtained by dissection was placed in a centrifuge tube containing 45 ml of ice-cold basal culture medium (components see Table 1).

[0090] Table 1 Basic culture medium components, suppliers and working concentrations

[0091] Reagents brand Working concentration (%, v / v) AdvancedDMEM / F12 Gibco - HEPES Punosa 1％ GlutaMAX Punosa 1％ Penicillin-Streptomycin mixture Punosa 1％ Y-27632 Merck 10μM

[0092] (2) Tissue trimming: Assess whether the tissue block contains other unwanted tissues and remove as much other tissue as possible using surgical scissors or a scalpel and forceps.

[0093] (3) In a biosafety cabinet, transfer the tissue to a 6-cm cell culture dish, cut a piece of tissue along the largest longitudinal section with sterile surgical scissors, fix it with fixative, dehydrate the tissue, and prepare paraffin sections. Transfer the remaining tissue pieces to a sterile 1.5-mL centrifuge tube and use surgical scissors to cut the tissue into as small pieces as possible (no more than 4 minutes).

[0094] (4) Tissue digestion: Transfer tissue fragments to a 15 mL centrifuge tube, add an appropriate amount of preheated collagenase IV (C8160, Solebol) digestion solution, wrap the centrifuge tube with plastic wrap and place it on a shaker for digestion. Examine the tube under a microscope every 15 minutes to see if there is a ductal structure. If not, continue digestion.

[0095] (5) When digestion is complete, add 10 ml of washing medium (high-glucose DMEM containing 1% fetal bovine serum and 1% penicillin / streptomycin), filter through a 70 μm cell sieve, and centrifuge at 200 g for 5 min at 4°C. The remaining precipitate is the primary cells containing adult stem cells.

[0096] (6) Resuspend the pellet in an appropriate amount of tissue preservation solution, count the cells, transfer the cell suspension containing the required number of cells to a new centrifuge tube (1000 cells per well of a 24-well plate), and centrifuge at 200 g for 5 min at 4°C. If the cell pellet is red, it may be contaminated with red blood cells and can be treated with red blood cell lysis buffer.

[0097] (7) Aspirate the supernatant and resuspend the cells to be prepared into organoids in a mixture of expansion medium (components see Table 2) and Matrigel (Matrigel, Corning, 356234) in a volume ratio of 1:1.

[0098] Table 2 Expansion medium components, suppliers and working concentrations

[0099]

[0100] The preparation method of the conditioned medium I containing Wnt-3A, R-spondin 3 and Noggin is as follows: (a) 5*10 7 L-WRN cells (BioWind, CRL-3276) were inoculated in a 15 cm culture dish and 25-30 ml of culture medium (DMEM / F12+10% FBS+1% penicillin-streptomycin mixture) was added; (b) after culturing for 60-72 h, the first batch of conditioned medium was collected, the supernatant was centrifuged at 2000 g / 10 min, fresh medium was added and the supernatant was stored at 4°C; (c) after continuing to culture for 36-48 h, the second batch of conditioned medium was collected, the supernatant was centrifuged at 2000 g / 10 min, and new medium was added; (d) after continuing to culture for 36-48 h, the third batch of conditioned medium was collected, the supernatant was centrifuged at 2000 g / 10 min, all conditioned medium were combined, mixed, aliquoted and stored at -80°C.

[0101] (8) Drop the mixture of cells, expansion medium and matrix gel on the bottom of a preheated 24-well cell culture plate, with each drop being approximately 35 μL.

[0102] (9) Place the culture plate upside down in a 37°C, 5% CO2 cell culture incubator and incubate for 20-30 min until the mixture solidifies.

[0103] (10) Prepare the required amount of organoid expansion medium (see Table 2 for components) and place the organoid expansion medium in a 37°C water bath.

[0104] (11) After the matrix gel solidifies, turn the culture plate over and carefully add 500 μl of the above expansion medium into each well along the well wall. Place the culture plate in a cell culture incubator.

[0105] (12) Replace the expansion medium every 2-3 days. Carefully aspirate the expansion medium from each well and then add fresh pre-warmed expansion medium.

[0106] 2. Inducing differentiation

[0107] The kidney organoids were cultured in the expansion medium for 7-10 days. After subculturing, they were cultured in the expansion medium for at least 2-4 days. The expansion medium was then replaced with the differentiation medium (components are shown in Table 3). The differentiation medium was replaced every 2-3 days and the culture was continued for 11-14 days. The morphology of the organoids after culture was shown in Table 3. Figure 1 .

[0108] Table 3 Differentiation medium components, suppliers and working concentrations

[0109] Reagents brand Working concentration (%, v / v) AdvancedDMEM / F12 Gibco - HEPES Punosa 1％ GlutaMAX Punosa 1％ Penicillin-Streptomycin mixture Punosa 1％ Wnt-3A Conditioned Medium II 40％ A83-01 Aladdin 5μM Aromatic retinoic acid (TTNPB) Maclean 3μM Fludrocortisone acetate Aladdin 10μM Forskolin Aladdin 10μM

[0110] The preparation method of the conditioned medium II containing Wnt-3A is as follows: (a) 5*10 7 LWnt-3A cells (BioWind, CRL-2647) were inoculated in a 15 cm culture dish and 25-30 ml of culture medium (DMEM / F12+10% FBS+1% penicillin-streptomycin mixture) was added; (b) after culturing for 60-72 h, the first batch of conditioned medium was collected, the supernatant was obtained by centrifugation at 2000 g / 10 min, fresh medium was added and the supernatant was stored at 4°C; (c) after continuing to culture for 36-48 h, the second batch of conditioned medium was collected, the supernatant was obtained by centrifugation at 2000 g / 10 min, and new medium was added; (d) after continuing to culture for 36-48 h, the third batch of conditioned medium was collected, the supernatant was obtained by centrifugation at 2000 g / 10 min, all conditioned medium were combined, mixed, aliquoted and stored at -80°C.

[0111] 3. Organoid identification

[0112] The cultured organoids were collected, fixed, dehydrated, and paraffin sections were prepared. Histomorphology was identified by hematoxylin-eosin staining, and cell markers were identified by immunofluorescence, including the proximal tubule marker LTL, the loop cell marker Nkcc2, the distal tubule marker Calb1, and the glomerular cell marker. The identification results are shown in Figure 2-5 .

[0113] Figure 2 It is the result of histomorphological identification of renal organoids before and after differentiation by hematoxylin-eosin staining. The cell morphology of renal organoids is consistent with that of kidney tissue. Figure 3 This is the identification result of the proximal tubule marker LTL and the medullary loop cell marker Nkcc2 in the pre-differentiated kidney organoids by immunofluorescence staining. In the upper figure, blue represents DAPI and green represents LTL. Green-positive cells were detected in the organoids, indicating that the differentiated kidney organoids express the proximal tubule marker LTL. In the lower figure, blue represents DAPI and red represents Nkcc2. Red-positive cells were detected in the organoids, indicating that the differentiated kidney organoids express the medullary loop cell marker Nkcc2.

[0114] Figure 4 This is the result of identification of distal tubule marker Calb1 in kidney organoids before and after differentiation by immunofluorescence staining. Blue represents DAPI and green represents Calb1. The upper picture is the organoid before differentiation, and no green-positive cells were detected. The lower picture is the organoid after differentiation, and green-positive cells were detected, indicating that kidney organoids do not express distal tubule marker Calb1 before differentiation, but express Calb1 after differentiation. The statistical results of the number of distal tubules before and after differentiation are shown in Table 4.

[0115] Figure 5 This is the result of identifying the glomerular marker WT1 in kidney organoids before and after differentiation by immunofluorescence staining. Blue represents DAPI and red represents WT1. The upper picture is the organoid before differentiation, and no red-positive cells were detected. The lower picture is the organoid after differentiation, and red-positive cells were detected, indicating that the kidney organoid did not express the glomerular marker WT1 before differentiation, but expressed WT1 after differentiation. The statistical results of the number of glomerular cells before and after differentiation are shown in Table 5.

[0116] Table 4 Statistical results of the number and differentiation rate of distal tubules in mouse kidney organoids before and after differentiation

[0117] method Number of organoids containing distal tubule cells Distal tubule differentiation rate Before differentiation culture 46 9.1％ After differentiation culture 512 88.6％

[0118] Table 5 Statistical results of glomerular number and differentiation rate of mouse kidney organoids before and after differentiation

[0119] method Number of organoids containing glomerular cells Glomerular differentiation rate Before differentiation culture 4 1.2％ After differentiation culture 316 58.9％

[0120] In summary, the kidney organoid culture method of the present invention can successfully culture mouse kidney organoids containing proximal tubules, medullary loops, distal tubules and glomeruli.

[0121] Although specific embodiments of the present invention have been described, it will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the scope or spirit of the present invention. Therefore, the present invention is intended to cover all such changes and modifications that fall within the scope of the appended claims and their equivalents.

Claims

1. A method for culturing mouse kidney organoids, characterized in that: The culture method comprises the following steps: (1) Extracting primary cells from mouse kidney tissue to prepare adult stem cells; (2) dispersing the adult stem cells obtained in step (1) in a mixture of expansion medium and matrix gel, and incubating until the mixture solidifies; (3) adding expansion medium and culturing for 7-10 days, and continuing to culture in the expansion medium for at least 2-4 days after subculturing; (4) replacing the expansion medium with a differentiation medium, and continuing culturing for 11-14 days to obtain mouse kidney organoids; the mouse kidney organoids contain proximal tubular cells, medullary loop cells, distal tubular cells, and glomerular cells.

2. The culture method according to claim 1, characterized in that The expansion medium in steps (2) and (3) comprises Advanced DMEM / F12, HEPES, GlutaMAX, a penicillin-streptomycin mixture, gentamicin, amphotericin B, a conditioned medium I containing Wnt-3A, R-spondin 3 and Noggin, N-acetylcysteine, B27 supplement, A83-01, EGF, FGF-10 and Y-27632.

3. The culture method according to claim 2, characterized in that The preparation method of the conditioned medium I containing Wnt-3A, R-spondin 3 and Noggin is as follows: (a) 5*10 7 L-WRN cells were seeded in a 15 cm culture dish and 25-30 ml of culture medium was added; (b) After 60-72 h of culture, the first batch of conditioned medium was collected, centrifuged at 2000 g / 10 min, the supernatant was supplemented with fresh medium, and the supernatant was stored at 4°C; (c) After continuing to culture for 36 to 48 hours, the second batch of conditioned medium was collected, centrifuged at 2000 g / 10 min, and the supernatant was taken and supplemented with new medium; (d) After continuing to culture for 36 to 48 hours, the third batch of conditioned medium was collected and centrifuged at 2000 g / 10 min to obtain the supernatant. All conditioned medium were combined, mixed, aliquoted, and stored at -80°C.

4. The culture method according to claim 1, characterized in that The differentiation medium in step (4) contains aromatic retinoic acid, fludrocortisone acetate and forskolin.

5. The culture method according to claim 4, characterized in that The differentiation medium in step (4) comprises A83-01, conditioned medium II containing Wnt-3A, aromatic retinoic acid, fludrocortisone acetate and forskolin.

6. The culture method according to claim 5, characterized in that: The differentiation medium in step (4) comprises Advanced DMEM / F12, HEPES, GlutaMAX, a penicillin-streptomycin mixture, a conditioned medium II containing Wnt-3A, A83-01, aromatic retinoic acid, fludrocortisone acetate and forskolin.

7. The culture method according to claim 5 or 6, characterized in that: The preparation method of the conditioned medium II containing Wnt-3A is as follows: (a) 5*10 7 LWnt-3A cells were seeded in a 15 cm culture dish and 25-30 ml of culture medium was added; (b) After 60-72 h of culture, the first batch of conditioned medium was collected, centrifuged at 2000 g / 10 min, the supernatant was supplemented with fresh medium, and the supernatant was stored at 4°C; (c) After continuing to culture for 36 to 48 hours, the second batch of conditioned medium was collected, centrifuged at 2000 g / 10 min, and the supernatant was taken and supplemented with new medium; (d) After continuing to culture for 36 to 48 hours, the third batch of conditioned medium was collected and centrifuged at 2000 g / 10 min to obtain the supernatant. All conditioned medium were combined, mixed, aliquoted, and stored at -80°C.

8. A culture medium composition for culturing mouse kidney organoids, characterized in that: The culture medium composition comprises an expansion medium and a differentiation medium, wherein the expansion medium comprises Advanced DMEM / F12, HEPES, GlutaMAX, a mixture of penicillin and streptomycin, gentamicin, amphotericin B, a conditioned medium I comprising Wnt-3A, R-spondin 3 and Noggin, N-acetylcysteine, B27 supplement, A83-01, EGF, FGF-10 and Y-27632, and the differentiation medium comprises Advanced DMEM / F12, HEPES, GlutaMAX, a mixture of penicillin and streptomycin, a conditioned medium II comprising Wnt-3A, A83-01, aromatic retinoic acid, fludrocortisone acetate and forskolin.

9. Use of the culture medium composition according to claim 8 in culturing mouse kidney organoids, characterized in that: The mouse kidney organoids contain proximal tubular cells, medullary loop cells, distal tubular cells and glomerular cells.

10. Use of the differentiation medium of claim 8 in promoting differentiation of mouse kidney organoid cells, characterized in that: The mouse kidney organoid cells are simultaneously differentiated into one or more of proximal tubular cells, medullary loop cells, distal tubular cells and glomerular cells.

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