Cat kidney organoid and culture medium and culture method thereof

Abstract

The invention discloses a cat kidney organoid as well as a culture medium and a culture method thereof, and relates to the technical field of organoid culture. The cat kidney organoid is successfully constructed on the basis of the kidney adult stem cells, and the deficiency of a cat kidney in-vitro model is made up; by adopting an amplification and differentiation culture medium segmented culture mode, comprehensive differentiation of various cell types of the kidney can be promoted, including simultaneous differentiation of near-end renal tubular cells, far-end renal tubular cells, glomerular cells and the like; according to the method, the conditioned culture medium containing Wnt-3A, R-spondin 3 and Noggin is generated by using the L-WRN cell line, and the conditioned culture medium containing Wnt-3A is generated by using the L Wnt-3A cell line, so that the culture method is lower in cost and higher in consistency, and the success rate and the growth speed of cat kidney organoid culture can be increased.

Description

Technical Field

[0001] The invention belongs to the technical field of organoid culture, and relates to cat kidney organoids and a culture medium and a culture method thereof. Background Art

[0002] The kidney is an important organ in the animal body, which can easily lead to serious lesions and death. Chronic kidney disease (CKD) is a common disease in cats and one of the main chronic diseases that cause death in cats. It is estimated that the incidence of CKD in cats is 1.6% to 20%. Although cats of all ages may suffer from chronic kidney disease, the incidence increases with age. The incidence of cats over 15 years old can be as high as 80%. Therefore, chronic kidney disease is considered to be the main cause of death in elderly cats. However, there is a serious shortage of kidney donors for cats, and there is an urgent need to develop new in vitro kidney models to solve the current dilemma.

[0003] Organoids are tissue analogs with a certain spatial structure formed by three-dimensional culture of stem cells or organ progenitor cells in vitro. Organoids can differentiate and self-organize to form some specific functions and structures of the corresponding organs of the main body. Because organoids can simulate organ development and formation and maintain genomic stability during long-term in vitro expansion, they have become an in vitro model that has attracted much attention in recent years.

[0004] At present, the construction of kidney organoids is mainly concentrated in human and mouse sources, and most of them are induced and differentiated by pluripotent stem cells, which have the advantage of being homologous to humans or mice. For example, the CN118240746A patent discloses a culture method for rapidly constructing human kidney organoids, using human induced pluripotent stem cells to be directly inoculated or aggregated into balls for inoculation, and then gradually adding specific growth factors for culture, thereby shortening the kidney organoid culture cycle. The CN116064373A patent discloses a mouse kidney organoid culture medium and a culture method, by adding Wnt pathway activator CHIR99021 and aromatic retinoic acid TTNPB (Arotinoid Acid) to the kidney organoid culture medium, activating the Wnt pathway and retinoic acid (RA) pathway, promoting the differentiation of various cell types of the kidney and forming tubular-like structures, thereby improving the success rate of culture and the growth rate of mouse kidney organoids.

[0005] Currently, there is no method for culturing cat kidney organoids based on renal adult stem cells to obtain multiple renal cell types. Summary of the invention

[0006] To solve the above technical problems, the present invention provides a culture medium and a culture method for cat kidney organoids, and obtains a cat kidney organoid that can be stably propagated, which contains proximal tubular cells, distal tubular cells and glomerular cells, and truly simulates the multiple cell types and spatial organizational structure of the cat kidney.

[0007] The technical solution adopted by the present invention to achieve the technical purpose is:

[0008] The first aspect of the present invention provides a culture medium for cat kidney organoids, comprising: an expansion medium and a differentiation medium, wherein:

[0009] The expansion medium is a basal medium supplemented with the following components at final concentrations: 2% B27, 50 ng / mL EGF, 1.25 mM N-Acetylcysteine, 5 μM A83-01, 100 ng / mL FGF-10, 20 μg / mL gentamicin, 50 ng / mL amphotericin B, 10 μM Y-27632, 1×ITS, and a conditioned medium containing Wnt-3A, R-spondin 3, and Noggin;

[0010] The differentiation medium is a basal medium supplemented with the following components at final concentrations: a conditioned medium containing Wnt-3A (produced by L Wnt-3A cells, containing Wnt-3A), 5 μM A83-01, 3 μM aromatic retinoic acid (TTNPB), 10 μM fludrocortisone acetate, and 10 μM forskolin;

[0011] The basic culture medium is Advanced DMEM / F12 supplemented with the following components at final concentrations: 1% GlutaMAX, 1% Penicillin-Streptomycin and 10 mM HEPES.

[0012] Preferably, the preparation method of the conditioned medium is as follows:

[0013] i) Change 5*10 7 L-WRN cells or L Wnt-3A cells were seeded in 15-cm culture dishes and added with 25–30 mL of culture medium (DMEM / F12 + 10% FBS + 1% Penicillin-Streptomycin);

[0014] ii) After culturing for 60-72 hours, the first batch of conditioned medium was collected, centrifuged at 2000 g / 10 min, and the supernatant was supplemented with fresh medium, and the supernatant was stored at 4°C;

[0015] iii) 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 new medium was added;

[0016] iv) 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.

[0017] A second aspect of the present invention provides a method for culturing cat kidney organoids, which is achieved by the above-mentioned culture medium and comprises the following steps:

[0018] (1) Obtaining cat embryo kidney tissue, extracting primary cells, and preparing kidney adult stem cells;

[0019] (2) dispersing the kidney adult stem cells obtained in step (1) in a mixture of expansion medium and matrix gel, and incubating until the mixture solidifies;

[0020] (3) adding expansion medium and culturing for 7 to 14 days;

[0021] (4) replacing the expansion medium with a differentiation medium, and continuing culturing for 11 to 14 days to obtain cat kidney organoids; the cat kidney organoids contain proximal tubular cells, distal tubular cells, and glomerular cells.

[0022] Preferably, the specific method for preparing kidney adult stem cells in step (1) is as follows:

[0023] 1-1) Obtain cat embryo kidney tissue, remove other unnecessary tissues and cut into pieces;

[0024] 1-2) Add collagenase IV and place on a shaker for digestion. Check under a microscope to see if there are ductal structures. If not, continue digestion.

[0025] 1-3) After digestion is completed, centrifuge, add washing medium (high-glucose DMEM supplemented with 1% fetal bovine serum and 1% penicillin / streptomycin) to resuspend and terminate digestion, centrifuge to remove the supernatant, and the remaining precipitate is a mixture containing adult stem cells and renal tubular fragments.

[0026] Preferably, the step (2) comprises the following steps:

[0027] 2-1) Dispersing adult stem cells in a mixture of expansion medium and matrix gel, and then dropping the mixture into a cell culture plate;

[0028] 2-2) Place the culture plate upside down in a 37°C, 5% CO2 cell culture incubator and incubate for 20 to 30 minutes until the matrix gel solidifies.

[0029] Preferably, the amplification medium in step (3) is incubated in a 37° C. water bath before being added; the amplification medium in step (3) is replaced every 2 to 3 days.

[0030] Preferably, in step (3), the cells are cultured in the expansion medium for at least 2 to 4 days after subculturing.

[0031] Preferably, the differentiation medium in step (4) is incubated in a 37° C. water bath before being added; the differentiation medium in step (4) is replaced every 2 to 3 days.

[0032] A third aspect of the present invention provides the use of the above culture medium or culture method in culturing cat kidney renal organoids.

[0033] The fourth aspect of the present invention provides a cat kidney organoid that can be stably propagated for a long time, which is achieved by the above-mentioned culture medium or culture method. The cat kidney organoid comprises proximal tubular cells, distal tubular cells and glomerular cells, which truly simulates the multiple cell types and spatial tissue structure of the cat kidney.

[0034] The beneficial effects of the present invention are

[0035] (I) Successfully constructed cat kidney organoids based on renal adult stem cells, which can be stably propagated for a long time, making up for the lack of an in vitro model of cat kidney;

[0036] (ii) The use of a segmented culture mode using expansion and differentiation medium can promote the comprehensive differentiation of multiple cell types in the kidney, including the simultaneous differentiation of proximal tubular cells, distal tubular cells and glomerular cells, truly simulating the multiple cell types and spatial organizational structure of the cat kidney;

[0037] (III) Since the added factors such as Noggin, Wnt-3A and R-spondin 3 currently available on the market are expensive and the quality is inconsistent between different batches, the present invention uses the L-WRN cell line to produce a conditioned medium containing Wnt-3A, R-spondin 3 and Noggin, and uses the LWnt-3A cell line to produce a conditioned medium of Wnt-3A. This culture method has lower cost and higher consistency, and can improve the success rate and growth rate of cat kidney organoid culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a bright field image of the kidney organoid of the present invention (scale bar 100 μm), Figure 1 A is the organoid cultured in expansion medium, Figure 1 B is the organoid after culture in differentiation medium;

[0039] Figure 2 The results of the identification of the proximal tubule marker LTL of the kidney organoids and the distal tubule cell marker CALB1 before (upper) and after (lower) differentiation of the kidney organoids of the present invention; scale bar 100 μm;

[0040] Figure 3The diagram shows the identification results of the glomerular cell marker WT1 in the kidney organoids of the present invention before differentiation (upper) and after differentiation (lower); scale bar: 100 μm. DETAILED DESCRIPTION

[0041] The present invention is described in detail below in conjunction with specific implementation methods. The following specific embodiments are helpful for those skilled in the art to further understand the present invention, but do not limit the present invention in any form.

[0042] Example

[0043] 1. Reagents and Materials

[0044] Advanced DMEM / F12 was purchased from Gibco, catalog number 12634010

[0045] HEPES buffer was purchased from Prosai, catalog number PB180325

[0046] GlutaMAX supplement can directly replace L-glutamine in cell culture medium. It was purchased from Pronocell with the catalog number PB180419.

[0047] Penicillin-Streptomycin was purchased from Pronose with the catalog number PB180120. Gentomycin was purchased from Pronose with the catalog number PB180124.

[0048] Amphotericin B was purchased from Purnosai with the catalog number PB180127.

[0049] N-Acetylcysteine ​​was purchased from Aladdin with the product number A105422.

[0050] B27 supplement is B27 serum-free supplement, purchased from Yuanpei Biotechnology, catalog number S440J7

[0051] A83-01 is an inhibitor of TGF-β type I receptor ALK5, ALK4, and ALK7, which can promote the reprogramming of mouse fibroblasts into iPSCs. It was purchased from Aladdin with the catalog number A274862.

[0052] EGF is epidermal growth factor, purchased from Nearshore Protein, catalog number C029

[0053] FGF-10, recombinant human fibroblast growth factor-10, was purchased from Nearshore Protein with the catalog number CR11

[0054] Y-27632, a small molecule inhibitor of the Rho-associated protein kinase p160ROCK, was purchased from Merck with the catalog number SCM075

[0055] 100×ITS, insulin-transferrin-selenium, a universal cell supplement, purchased from Yuanpei, catalog number S450J7

[0056] Aromatic retinoic acid (TTNPB), a potent RAR agonist, was purchased from Maclean with catalog number R872599

[0057] Fludrocortisone acetate, a mineralocorticoid, was purchased from Aladdin with the catalog number F420036

[0058] Forskolin is a potent adenylate cyclase activator, purchased from Aladdin, catalog number F127328

[0059] 2. Preparation of culture medium

[0060] 1. Conditioned medium:

[0061] i) Change 5*10 7 L-WRN cells (BioWind, CRL-3276) and L Wnt-3A (BioWind, CRL-2647) cells were seeded in 15-cm culture dishes and added with 25–30 mL of culture medium (DMEM / F12 + 10% FBS + 1% Penicillin-Streptomycin);

[0062] ii) After 60-72 hours of culture, the first batch of conditioned medium was collected and centrifuged at 2000 g / 10 min to obtain the supernatant.

[0063] Supplement with fresh culture medium (DMEM / F12+10% FBS+1% Penicillin-Streptomycin),

[0064] The supernatant was stored at 4°C;

[0065] iii) After continuing to culture for 36 to 48 hours, the second batch of conditioned medium was collected, the supernatant was taken by centrifugation at 2000 g / 10 min, and new medium (DMEM / F12+10% FBS+1% Penicillin-Streptomycin) was added;

[0066] iv) 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] Among them, the conditioned medium used in the expansion medium is produced by L-WRN cells and contains Wnt-3A, R-spondin3, and Noggin; the conditioned medium used in the differentiation medium is produced by L Wnt-3a and only contains Wnt-3A.

[0068] 2. Expansion medium:

[0069] Advanced DMEM / F12, 1% GlutaMAX, 1% Penicillin-Streptomycin, 10mM HEPES, 2% B27, 50ng / mL EGF, 1.25mM N-Acetylcysteine, 5μM A83-01, 100ng / mL FGF-10, 20μg / mL gentamicin, 50ng / mL amphotericin B, 10μM Y-27632, 1×ITS, 40% conditioned medium containing Wnt-3A, R-spondin3 and Noggin

[0070] N-Acetylcysteine, A83-01, and FGF-10 reagents are all powders, which can be dissolved according to the instructions. Taking the preparation of 50mL of expansion medium as an example, the specific formula is as follows:

[0071] Components volume GlutaMAX 500μL Penicillin-Streptomycin 500μL HEPES, 1M 500μL B27，50× 1mL EGF, 1 mg dissolved in 5 mL sterile water, 200 μg / mL 12.5μL N-Acetylcysteine, dissolved in DMSO, 100 mM 625μL A83-01, dissolved in DMSO, 10 mM 25μL FGF-10, 500 μg dissolved in 5 mL sterile water, 100 μg / mL 50μL Gentamycin, 10 mg / mL 100μL Amphotericin B, 250 μg / mL 10μL Y-27632, 10mM 50μL ITS, 100× 500μL Conditioned medium (Wnt-3A, R-spondin 3, Noggin) 20mL

[0072] Finally, all components were adjusted to 50 mL with Advanced DMEM / F12.

[0073] 3. Differentiation medium:

[0074] Advanced DMEM / F12, 1% GlutaMAX, 1% Penicillin-Streptomycin, 10 mM HEPES, 40% conditioned medium containing Wnt-3A, 5 μM A83-01, 3 μM aromatic retinoic acid (TTNPB), 10 μM fludrocortisone acetate, 10 μM forskolin

[0075] Aromatic retinoic acid (TTNPB), fludrocortisone acetate, and forskolin reagents are all powders and can be dissolved according to the instructions.

[0076] Taking the preparation of 50 ml differentiation medium as an example, the specific formula is as follows:

[0077] Components volume GlutaMAX 500μL Penicillin-Streptomycin 500μL HEPES, 1M 500μL Conditioned medium (Wnt-3A) 20mL Aromatic retinoic acid (TTNPB), dissolved in DMSO, 10 mM 15μL Fludrocortisone acetate, dissolved in DMSO, 10 mM 50μL Forskolin, dissolved in DMSO, 10 mM 50μL

[0078] Finally, all components were adjusted to 50 mL with Advanced DMEM / F12.

[0079] 3. Cultivation of Feline Kidney Organoids

[0080] 1. Preparation of kidney organoids derived from adult kidney stem cells

[0081] (1) Tissue collection: Place the cat embryo kidney tissue obtained by dissection into a centrifuge tube containing 45 mL of ice-cold basal culture medium.

[0082] (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.

[0083] (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).

[0084] (4) Tissue digestion: Transfer tissue fragments to a 15 mL centrifuge tube, add an appropriate amount of preheated collagenase IV 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.

[0085] (5) After digestion is complete, add 10 mL of washing medium (high-glucose DMEM supplemented with 1% fetal bovine serum and 1% penicillin / streptomycin, where the glucose concentration in the high-glucose DMEM medium is 4500

[0086] mg / L) and centrifuged at 200 g for 5 min at 4°C to terminate the digestion and wash the cells.

[0087] (6) Resuspend the pellet in an appropriate amount of washing medium (high-glucose DMEM supplemented with 1% fetal bovine serum and 1% penicillin / streptomycin), 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 or 50 duct structures), 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.

[0088] (7) Aspirate the supernatant and resuspend the cells to be prepared into organoids in a mixture of expansion medium and matrix gel (volume ratio 1:3).

[0089] (8) Drop the mixture of cells and matrix gel on the bottom of a preheated 24-well cell culture plate, with each drop being approximately 30 to 35 μL.

[0090] (9) Place the culture plate upside down in a 37°C, 5% CO2 cell culture incubator and incubate for 20 to 30 minutes.

[0091] (10) Prepare the required amount of organoid expansion medium and preheat the organoid expansion medium in a 37°C water bath.

[0092] (11) After the matrix gel solidifies, turn the culture plate over and carefully add 300-500 μL of the above expansion medium into each well along the well wall. Place the culture plate in a cell culture incubator.

[0093] (12) Replace the amplification medium every 2 to 3 days. Carefully aspirate the amplification medium from each well and then add fresh pre-warmed amplification medium.

[0094] 2. Inducing differentiation

[0095] The kidney organoids were cultured in the expansion medium for 7 to 10 days. After subculturing, they were cultured in the expansion medium for at least 2 to 4 days. The expansion medium was then replaced with the differentiation medium. The differentiation medium was replaced every 2 to 3 days and the culture was continued for 11 to 14 days. The morphology of the organoids after culture was observed. Figure 1 .

[0096] 3. Organoid identification

[0097] The cultured organoids were collected, fixed, dehydrated, and paraffin sections were prepared. Cell markers were identified by immunofluorescence, including the proximal tubule marker LTL, the distal tubule marker CALB1, and the glomerular cell marker WT1. The identification results are shown in Figures 2-3 . Figure 2 This is the result of identification of the proximal tubule marker LTL and the distal tubule marker CALB1 in renal organoids before and after differentiation by immunofluorescence staining. Blue represents CALB1 and green represents LTL. Green-positive cells were detected in the organoids, indicating that the proximal tubule marker LTL was expressed in renal organoids before and after differentiation. At the same time, the upper picture is the organoid before differentiation, in which no blue-positive cells were detected, and the lower picture is the organoid after differentiation, in which blue-positive cells were detected, indicating that the distal tubule marker CALB1 was not expressed in renal organoids before differentiation, but CALB1 was expressed after differentiation.

[0098] Figure 3 This is the result of identifying the glomerular marker WT1 in renal organoids before and after differentiation by immunofluorescence staining. Blue represents DAPI and green represents WT1. 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 the renal organoid did not express the glomerular marker WT1 before differentiation, but expressed WT1 after differentiation.

[0099] In summary, through our culture system, we successfully cultured cat kidney organoids containing proximal tubules, distal tubules, and glomeruli.

[0100] 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. Culture medium for feline kidney organoids, including: Expansion medium and differentiation medium, wherein: The expansion medium is a basal medium supplemented with the following components at final concentrations: 2% B27, 50 ng / mL EGF, 1.25 mM N-Acetylcysteine, 5 μM A83-01, 100 ng / mL FGF-10, 20 μg / mL gentamicin, 50 ng / mL amphotericin B, 10 μM Y-27632, 1×ITS, and a conditioned medium containing Wnt-3A, R-spondin 3, and Noggin; The differentiation medium is a basal medium supplemented with the following components at final concentrations: a conditioned medium containing Wnt-3A, 5 μM A83-01, 3 μM aromatic retinoic acid, 10 μM fludrocortisone acetate, and 10 μM forskolin; The basic culture medium is Advanced DMEM / F12 supplemented with the following components at final concentrations: 1% GlutaMAX, 1% Penicillin-Streptomycin and 10 mM HEPES.

2. The culture medium according to claim 1, characterized in that The preparation method of the conditioned medium is as follows: i) Change 5*10 7 L-WRN cells or L Wnt-3A cells were seeded in 15 cm culture dishes and added with 25-30 mL of culture medium, which was DMEM / F12 supplemented with 10% FBS and 1% Penicillin-Streptomycin; ii) After culturing for 60-72 hours, the first batch of conditioned medium was collected, centrifuged at 2000 g / 10 min, and the supernatant was supplemented with fresh medium, and the supernatant was stored at 4°C; iii) 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 new medium was added; iv) 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.

3. A method for culturing cat kidney organoids, achieved by the culture medium of claim 1 or 2, comprising the following steps: (1) Obtaining cat embryo kidney tissue, extracting primary cells, and preparing kidney adult stem cells; (2) dispersing the kidney 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 to 14 days; (4) replacing the expansion medium with a differentiation medium, and continuing culturing for 11 to 14 days to obtain cat kidney organoids; the cat kidney organoids contain proximal tubular cells, distal tubular cells, and glomerular cells.

4. The culture method according to claim 3, characterized in that The specific method for preparing kidney adult stem cells in step (1) is as follows: 1-1) Obtain cat embryo kidney tissue, remove other unnecessary tissues and cut into pieces; 1-2) Add collagenase IV and place on a shaker for digestion. Check under a microscope to see if there are ductal structures. If not, continue digestion. 1-3) After digestion is completed, centrifuge, add washing medium to resuspend to terminate digestion, centrifuge to remove supernatant, and the remaining precipitate is a mixture containing adult stem cells and renal tubular fragments. The washing medium is high-glucose DMEM supplemented with 1% fetal bovine serum and 1% penicillin / streptomycin.

5. The culture method according to claim 3, characterized in that: The step (2) comprises the following steps: 2-1) Dispersing adult stem cells in a mixture of expansion medium and matrix gel, and then dropping the mixture into a cell culture plate; 2-2) Place the culture plate upside down in a 37°C, 5% CO2 cell culture incubator and incubate for 20 to 30 minutes until the matrix gel solidifies.

6. The culture method according to claim 3, characterized in that: The amplification medium in step (3) is incubated in a 37° C. water bath before being added; the amplification medium in step (3) is replaced every 2 to 3 days.

7. The culture method according to claim 3, characterized in that: In the step (3), the cells are cultured in the expansion medium for at least 2 to 4 days after subculturing.

8. The culture method according to claim 3, characterized in that: The differentiation medium in step (4) is incubated in a 37° C. water bath before being added; the differentiation medium in step (4) is replaced every 2 to 3 days.

9. Use of the culture medium of claim 1 or the culture method of claim 3 in culturing cat kidney renal organoids.

10. A cat kidney organoid that can be stably propagated for a long time, which is achieved by the culture medium described in claim 1 or the culture method described in claim 3, wherein the cat kidney organoid comprises proximal tubular cells, distal tubular cells and glomerular cells.

Images