Method for rapid construction of kidney organoids and use thereof

Abstract

Provided are a method for rapid construction of kidney organoids, and the use thereof. The method comprises: (1) directly inoculating human induced pluripotent stem cells, or aggregating said cells into spheroids and then inoculating same, and culturing the human induced pluripotent stem cells by using a culture medium containing an inhibition component, the inhibition component containing a GSK-3 inhibitor; (2) by means of using an additive-free basic culture medium or a culture medium containing FGF-9, culturing the cells obtained in step (1); (3) by means of using a culture medium containing a GSK-3 inhibitor and FGF-9, culturing the cells obtained in step (2); (4) by means of using a culture medium containing FGF-9, culturing the cells obtained in step (3); and (5) by means of using the additive-free basic culture medium, culturing the cells obtained in step (4). Compared with existing reported differentiation schemes, the present method can shorten the construction time of kidney organoids to 6-8 days, and can be directly applied to in-vitro toxicological tests, thereby achieving an extremely high practical value.

Description

A method for rapidly constructing kidney organoids and its application Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for rapidly constructing kidney organoids and an application thereof. Background Art

[0002] Kidney disease is a global health threat, often placing heavy physical, psychological, and economic burdens on patients. Due to the complex structure of the kidney, the development of in vitro kidney disease research models has been slow, hindering the study of kidney disease-related pathological mechanisms and drugs.

[0003] Several methods for inducing nephron progenitor cells from human induced pluripotent stem cells and human embryonic stem cells have been reported (e.g., CN110662832A). However, due to the use of embryoid bodies (EBs), the differentiation induction efficiency is low and it is unclear whether each stage of the process is accurately reproduced.

[0004] In addition, in the existing technology, kidney organoids based on stem cell differentiation have gradually become an important model for kidney physiology and disease research. Since scientists first reported the method of establishing kidney organoids in 2014, they have now been successfully applied in the research of glomerular and tubular diseases. For example, CN115466728A discloses a high-throughput human kidney organoid differentiation method based on human induced pluripotent stem cells, using ROCK pathway inhibitors, GSK-3 inhibitors, FGF-9 and Heparin to induce differentiation and obtain mature kidney organoids. However, the current differentiation methods still have many problems and defects. The main problems include three aspects: (1) The differentiation operation is complex, inefficient, and has a long cycle. The complete differentiation cycle is about 25 days; (2) The differentiation quality of kidney organoids is poor. The existing differentiation methods have problems such as incomplete differentiation and low differentiation efficiency; (3) The differentiation cost is too high. Most of the inducers used in the existing differentiation methods are expensive growth factors, which leads to high production costs and is difficult to achieve popularization in a real sense. These problems combined have greatly limited the large-scale production of key tissues or organs such as stem cell-derived endoderm.

[0005] In summary, there is an urgent need for a method that is efficient, low-cost, and has a short cycle time and can construct organoids that can effectively simulate the kidney. Summary of the Invention

[0006] In response to the deficiencies of existing technologies and actual needs, the present invention provides a method for constructing kidney organoids and its application, in order to solve the problems of existing kidney organoids such as long differentiation cycle, low differentiation efficiency, high differentiation cost, and simple kidney structure.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for rapidly constructing a human kidney organoid, the method comprising the following steps:

[0009] (1) culturing human induced pluripotent stem cells by direct seeding or by aggregating them into spheres and then seeding them in a culture medium containing an inhibitory component; the inhibitory component contains a GSK-3 inhibitor;

[0010] (2) culturing the cells obtained in step (1) using a basal medium without additives or a medium containing FGF-9;

[0011] (3) culturing the cells obtained in step (2) with a culture medium containing a GSK-3 inhibitor and FGF-9;

[0012] (4) culturing the cells obtained in step (3) with a culture medium containing FGF-9;

[0013] (5) The cells obtained in step (4) are cultured in a non-additive basal medium to obtain mature kidney organoids.

[0014] The present invention designs a method for constructing human kidney organoids based on human induced pluripotent stem cells, including controlling the induction factors, induction sequence, and induction time. This method uses a small number of factors, has a simple differentiation process, and a short differentiation cycle. Mature organoids with the main structural and functional cells of the kidney can be quickly obtained. Furthermore, the method can be directly applied to in vitro toxicological experiments, demonstrating extremely high practical value. Furthermore, two initial differentiation states are proposed. As shown in Figure 1, induced pluripotent stem cells (iPSCs) can initially be differentiated and cultured in a 2D manner in a 6-well plate. In the third stage of differentiation, after digestion into single cells, they spontaneously aggregate into kidney organoids. iPSCs can initially aggregate into spheres in a 96-well plate at 1,000 to 5,000 cells / well, and then begin to differentiate into kidney organoids in a 3D format. Differentiation from 2D cells (direct seeding) allows for uniformity and large-scale production, while differentiation from 3D cell spheres allows for observation of the entire developmental state of the kidney organoid.

[0015] It is understood that in the present invention, the culture medium containing inhibitory components refers to a culture medium known in the art (such as a complete pluripotent stem cell culture medium) to which inhibitory components are added. For example, the culture medium containing GSK-3 inhibitors and FGF-9 refers to a basal culture medium (such as Advanced RPMI 1640) to which GSK-3 inhibitors and FGF-9 are added. The non-supplemented basal culture medium refers to a culture medium known in the art (such as an adherent culture medium or a cell differentiation medium, etc.) without the addition of inhibitors or growth factors.

[0016] In the present invention, 1-10 μM CHIR99021 can be added on the 5th to 10th day of differentiation to adjust the ratio of the distal and proximal tubules in the kidney organoids, making the structural composition of the organoids more suitable for specific disease models or toxicology experiments; on the 10th to 20th day of differentiation, the organoids can be transferred to the upper layer of the Transwell for gas-liquid interface culture. This method can more clearly observe the renal tubular structure and facilitate the observation of toxicology test results; the kidney organoids can also be cultured in suspension in a 96-well plate. This method can more intuitively observe the size of the kidney organoids and facilitate the establishment of development-related disease models.

[0017] Two long-term culture conditions for kidney organoids can be used in the present invention. One is 3D suspension culture, which is convenient for observing the development of the entire kidney, and the other is gas-liquid interface culture, which is convenient for observing changes in renal tubular structure. Different culture conditions can be selected according to different disease models and toxicological testing requirements.

[0018] Preferably, the GSK-3 inhibitor comprises any one of CHIR99021, LY2090314, CHIR98014, BIO-acetoxime, AZD2858 or SAR502250, or a combination of at least two thereof.

[0019] Preferably, the inhibitory component in step (1) further contains an ALK5 inhibitor.

[0020] Preferably, the ALK5 inhibitor includes any one of SB431542, RepSox, R-268712 or SB525334, or a combination of at least two of them.

[0021] Preferably, the inhibitory component in step (1) contains any one of a combination of CHIR99021 and SB431542, a combination of CHIR99021 and RepSox, a combination of LY2090314 and R-268712, or a combination of SB525334 and SAR502250.

[0022] In the present invention, differentiation process factors are designed. For example, the inhibitory component in step (1) is a combination of CHIR99021 and SB431542, which can further shorten the differentiation cycle of kidney organoids.

[0023] Preferably, the concentration of the GSK-3 inhibitor in the culture medium containing the inhibitory component in step (1) is 1 to 30 µM, including but not limited to 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 22, 25, 28 or 29 µM.

[0024] Preferably, the concentration of the ALK5 inhibitor in the culture medium containing the inhibitory component in step (1) is 1 to 10 µM, including but not limited to 2, 3, 4, 5, 6, 7, 8 or 9 µM.

[0025] Preferably, the concentration of FGF-9 in the FGF-9-containing culture medium in step (2) is 10~200ng / mL.

[0026] Preferably, the concentration of the GSK-3 inhibitor in the culture medium containing the GSK-3 inhibitor and FGF-9 in step (3) is 1-30 µM (for example, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 22, 25, 28 or 29 µM, etc.), and the concentration of FGF-9 is 10-200 ng / mL, including but not limited to 11 ng / mL, 12 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, 50 ng / mL, 80 ng / mL, 100 ng / mL, 150 ng / mL, 160 ng / mL, 180 ng / mL, 185 ng / mL, 190 ng / mL, 195 ng / mL, 196 ng / mL, 198 ng / mL or 199 ng / mL, etc.

[0027] Preferably, the concentration of FGF-9 in the culture medium containing FGF-9 in step (4) is 10-200 ng / mL, including but not limited to 11 ng / mL, 12 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, 50 ng / mL, 80 ng / mL, 100 ng / mL, 150 ng / mL, 160 ng / mL, 180 ng / mL, 185 ng / mL, 190 ng / mL, 195 ng / mL, 196 ng / mL, 198 ng / mL or 199 ng / mL, etc.

[0028] Preferably, the culturing time in step (1) is 1 to 4 days, including but not limited to 1, 2, 3 or 4 days.

[0029] Preferably, the culturing time in step (2) is 1 to 3 days, such as 2 days.

[0030] Preferably, the culturing time in step (3) is 1 to 2 days.

[0031] Preferably, the culturing time in step (4) is 1 to 6 days, including but not limited to 2, 3, 4 or 5 days.

[0032] Preferably, the culturing time in step (5) is 1 to 10 days, including but not limited to 2, 3, 4, 5, 6, 7, 8 or 9 days.

[0033] As a preferred technical solution, the method for constructing human kidney organoids comprises the following steps:

[0034] (1) Human induced pluripotent stem cells were directly seeded or aggregated into spheres and then seeded in a culture medium containing a GSK-3 inhibitor and an ALK5 inhibitor for 1 to 4 days;

[0035] (2) culturing the cells obtained in step (1) with a non-additive basal medium or a medium containing FGF-9 for 1 to 3 days;

[0036] (3) culturing the cells obtained in step (2) with a culture medium containing a GSK-3 inhibitor and FGF-9 for 1 to 2 days;

[0037] (4) culturing the cells obtained in step (3) with a medium containing FGF-9 for 1 to 6 days;

[0038] (5) The cells obtained in step (4) are cultured in a non-additive basal medium for 1 to 10 days to obtain mature kidney organoids.

[0039] For example, in one embodiment of the present invention, human induced pluripotent stem cells are cultured in a medium containing 10 µM CHIR99021 and 1 µM SB431542 for 2 days, cultured in a factor-free medium or a medium containing 50 ng / mL FGF9 for 2 days, cultured in a medium containing 3 µM CHIR99021 and 50 ng / mL FGF9 for 1-2 days, and cultured in a medium containing 50 ng / mL FGF9 for 2 days to obtain successfully differentiated kidney organoids.

[0040] In a second aspect, the present invention provides the application of the method for rapidly constructing human kidney organoids described in the first aspect in renal toxicology testing.

[0041] In a third aspect, the present invention provides a method for toxicological testing using human kidney organoids, the method comprising the following steps:

[0042] (1) Transferring the kidney organoids constructed by the method for rapidly constructing human kidney organoids described in the first aspect to the upper chamber of a Transwell and adding culture medium to the lower chamber;

[0043] (2) Cultivate kidney organoids at the air-liquid interface until clear tubular structures are visible;

[0044] (3) Add the test agent to the culture medium in the lower chamber;

[0045] (4) Observe the toxicity of the test agent by changes in renal tubular morphology and quantify the degree of damage by staining frozen sections.

[0046] Preferably, the test agent includes cisplatin and the like.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The present invention designs a method for rapidly and efficiently inducing the differentiation of kidney organoids using human induced pluripotent stem cells. This method can solve the problems of long differentiation cycle, low differentiation efficiency, high differentiation cost, and different disease models or toxicology experiments targeting different kidney structures of kidney organoids. It is simple and easy to implement, and the construction effect is stable. By optimizing the differentiation process, the differentiation cycle of kidney organoids is further shortened (6 to 8 days), and mature organoids with the main structures and functional cells of the kidney are quickly obtained. Moreover, it can be directly applied to in vitro toxicology experiments, with extremely high practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is a flowchart of kidney organoid differentiation, showing two routes: full 3D differentiation and 2D-to-3D differentiation;

[0050] Figure 2 shows bright field images of days 0, 4, 9, and 22 during the full 3D differentiation of kidney organoids constructed in Scheme 3. The scale bar is 200 µm.

[0051] Figure 3 shows bright field images of kidney organoids constructed in Scheme 3 during 2D-to-3D differentiation on days 3, 6, 11, 16, and 25. The scale bar is 200 μm.

[0052] Figure 4 shows the immunofluorescence staining results of kidney organoids constructed in Scheme 3. The scale bar is 400 µm. LTL marks proximal tubular cells, and Nephrin marks podocytes.

[0053] Figure 5 shows the bright field images of the full 3D differentiation process of kidney organoids constructed in Scheme 5. The scale bar is 200 µm.

[0054] Figure 6 shows the bright field images of the 2D-to-3D differentiation process of kidney organoids constructed in Scheme 5. The scale bar is 200µm.

[0055] Figure 7 shows the immunofluorescence staining results of kidney organoids constructed in Scheme 5. The scale bar is 200 µm. LTL marks proximal tubular cells, and Nephrin marks podocytes.

[0056] Figure 8 shows the results of kidney organoid differentiation time optimization, the scale bar is 200µm;

[0057] Figure 9 is a bright field image of the results of toxicology testing using kidney organoids. The scale bar is 200µm.

[0058] Figure 10 shows the results of kidney organoid damage detection, scale bar 50µm. DETAILED DESCRIPTION

[0059] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0060] Example 1

[0061] In this embodiment, the construction of human kidney organoids includes the following steps: (1) culturing human induced pluripotent stem cells in a culture medium containing inhibitory components by direct seeding or by aggregating them into spheres and then seeding them; the inhibitory components include GSK-3 inhibitors, etc.; (2) culturing the cells obtained in step (1) in a non-additive basal medium or a culture medium containing FGF-9; (3) culturing the cells obtained in step (2) in a culture medium containing GSK-3 inhibitors and FGF-9; (4) culturing the cells obtained in step (3) in a culture medium containing FGF-9; (5) culturing the cells obtained in step (4) in a non-additive basal medium to obtain mature kidney organoids. Multiple groups of schemes are designed, and the culture temperature, cell collection method and other routine operations in each scheme are the same. The only difference is the type, sequence, dosage and induction time of the factors added in each step of the differentiation process, as shown in Table 1. Day 0 refers to the expansion and culture of human induced pluripotent stem cells using mTeSR medium, and the start of differentiation induction (step 1) is recorded as the first day (Day 1), and so on in chronological order. In Table 1, for example, 10µM CHIR99021 refers to culture medium containing 10µM CHIR99021, 3µM CHIR99021 + 50ng / mL FGF9 refers to culture medium containing 3µM CHIR99021 and 50ng / mL FGF9, and Basal medium refers to culture medium alone. For example, the specific experimental procedures for Protocol 5 are as follows; the remaining protocols can be followed accordingly.

[0062] 1. The 2D to 3D differentiation method includes the following steps:

[0063] 1. Day 0: Cell plating

[0064] (1) When hiPSCs grow to 90% confluency, remove the culture medium and wash once with DPBS;

[0065] (2) Add 1 mL of Accutase and digest in an incubator for 3 minutes;

[0066] (3) Resuspend in 1 mL of mTeSR plus medium and pipette into single cells;

[0067] (4) Take 20 µL of cell suspension, stain with trypan blue, and count;

[0068] (5) 24-well plate 2cm 2 Surface area, seeding density is 15,000 cells / cm 2 , requiring 2.4×10 5 Cell volume: take the corresponding volume of cells and centrifuge at 300 × g for 5 min;

[0069] (6) Remove the supernatant, add 2 mL of mTeSR plus medium, 10 µM Y27632, and blow evenly;

[0070] (7) Add to one well of a Matrigel-treated 6-well plate and culture overnight.

[0071] 2. Day 1-Day 3: Induction of differentiation line

[0072] (1) It was observed that after one day of culture, most of the cells adhered to the wall and could begin to differentiate;

[0073] (2) Aspirate the culture medium and add 2 mL of differentiation medium: basal medium + 10 µM CHIR99021 + 1 µM SB431542 to start differentiation;

[0074] (3) Change the fluid every day.

[0075] 3. Day 3-Day 5: Induce differentiation of renal mesoderm cells

[0076] (1) Aspirate the culture medium and add 2 mL of differentiation medium (basal medium without factors) to each well and continue differentiation.

[0077] (2) Change the fluid every day.

[0078] 4. Day 5-Day 6: Induce differentiation of nephron progenitor cells

[0079] Aspirate the medium and add 2 mL of differentiation medium: basal medium + 3 µM CHIR99021 + 50 ng / mL FGF9, and continue differentiation.

[0080] 5. Day 6-Day 9: Induce differentiation of kidney organoids

[0081] (1) Aspirate the culture medium and add 2 mL of differentiation medium: basal medium + 50 ng / mL FGF9, and continue differentiation;

[0082] (2) Change the fluid every day.

[0083] Day 9 and beyond: Kidney organoid maturation

[0084] (1) Aspirate the culture medium and add 2 mL of differentiation medium: basal medium to mature the organoids.

[0085] (2) Change the fluid every day.

[0086] Second, the full 3D culture method includes the following steps:

[0087] Day 0: Cell aggregation

[0088] (1) When hiPSCs grow to 90% confluency, remove the culture medium and wash once with DPBS;

[0089] (2) Add 1 mL of Accutase and digest in an incubator for 3 minutes;

[0090] (3) Resuspend in 1 mL of mTeSR plus and pipette into single cells;

[0091] (4) Take 20 µL of cell suspension, stain with trypan blue, and count;

[0092] (5) 2000 cells per well of a 96-well plate, 4×10 5 The cell number was calculated, and the corresponding volume of cells was taken and centrifuged at 300 × g for 5 min;

[0093] (6) Remove the supernatant, add 20 mL of mTeSR plus medium, 10 µM Y27632, and blow evenly;

[0094] (7) Add 100 µL to each well of an ultra-low adsorption U-shaped 96-well plate and place the plate on a shaker at 70 rpm for overnight incubation.

[0095] 2. Day 1-Day 3: Start inducing differentiation of the original line

[0096] (1) Carefully aspirate the culture medium in the 96-well plate, taking care not to remove the cell spheres;

[0097] (2) Add 100 µL differentiation medium to each well: basal medium + 10 µM CHIR99021 + 1 µM SB431542; start differentiation.

[0098] 3. Day 3-Day 5: Induce differentiation of renal mesoderm cells

[0099] Carefully aspirate the culture medium in the 96-well plate and add 200 µL of differentiation medium: basal medium to each well to continue differentiation.

[0100] 4. Day 5-Day 6: Induce differentiation of nephron progenitor cells

[0101] Carefully aspirate the culture medium in the 96-well plate and add 200 µL differentiation medium (basal medium + 3 µM CHIR99021 + 50 ng / mL FGF9) to each well to continue differentiation.

[0102] 5. Day 6-Day 9: Induce differentiation of kidney organoids

[0103] Carefully aspirate the culture medium in the 96-well plate and add 200 µL of differentiation medium (basal medium + 50 ng / mL FGF9) to each well to continue differentiation.

[0104] Day 9 and beyond: Kidney organoid maturation

[0105] (1) Spherical cell aggregates were observed in 96-well plates to form distinct kidney organoids;

[0106] (2) Carefully aspirate the culture medium in the 96-well plate and add 200 µL of differentiation medium: basal medium to each well to allow the organoids to mature.

[0107] Table 1

[0108] Protocol Day0 - Day1 Day1 - Day3 Day3 - Day4 Day4 - Day5 Day5 - Day6 Day6 - Day7 Day7 - Day8 After Day8 1mTeSR 10µM CHIR99021 10µM CHIR99021 Basal medium Basal medium 3µM CHIR99021 + 50ng / mL FGF9 3µM CHIR99021 + 50ng / mL FGF9 50ng / mL FGF9 2mTeSR 30µM CHIR99021 Basal medium Basal medium 3µM CHIR99021 + 50ng / mL FGF9 3µM CHIR99021 + 50ng / mL FGF9 50ng / mL FGF9 50ng / mL FGF9 3mTeSR 10µM CHIR99021 Basal medium Basal medium 3µM CHIR99021 + 50ng / mL FGF9 50ng / mL FGF9 50ng / mL FGF9 50ng / mL FGF9 4mTeSR 10µM CHIR99021 Basal medium Basal medium 3µM CHIR99021 + 100ng / mL FGF9 100ng / mL FGF9 100ng / mL FGF9 100ng / mL FGF9 5mTeSR 10µM CHIR99021 + 1µM SB431542 Basal medium Basal medium 3µM CHIR99021 + 50ng / mL FGF9 50ng / mL FGF9 50ng / mL FGF9 50ng / mL FGF9 6mTeSR 10µM CHIR99021 + 1µM SB431542 50ng / mL FGF9 50ng / mL FGF9 3µM CHIR99021 + 50ng / mL FGF9 3µM CHIR99021 + 50ng / mL FGF9 50ng / mL FGF9 50ng / mL FGF9 7mTeSR 10µM CHIR99021 + 1µM SB431542 100ng / mL FGF9 100ng / mL FGF9 3µM CHIR99021 + 100ng / mL FGF9 3µM CHIR99021 + 100ng / mL FGF9 100ng / mL FGF9 100ng / mL FGF9 8mTeSR 10µM CHIR99021 + 10µM SB431542 10ng / mL ActivinA 10ng / mL ActivinA 3µMCHIR99021+50ng / mL FGF93µM CHIR99021+50ng / mL FGF950ng / mL FGF950ng / mL FGF99mTeSR10µM CHIR980141+1µM SB431542Basal mediumBasal medium3µM CHIR99021+50ng / mL FGF950ng / mL FGF950ng / mL FGF950ng / mL FGF910mTeSR10µM CHIR99021+1µM RepSoxBasal mediumBasal medium3µM CHIR99021+50ng / mL FGF950ng / mL FGF950ng / mL FGF950ng / mL FGF9

[0109] Example 2

[0110] The human kidney organoids constructed by each group in Example 1 were analyzed.

[0111] For schemes 1-4, the culture differentiation process and results are similar. Taking scheme 3 as an example, during the full 3D differentiation process, the bright field images on days 0, 4, 9 and 22 are shown in FIG2 , and during the 2D-to-3D differentiation process, the bright field images on days 3, 6, 11, 16 and 25 are shown in FIG3 ; during the 2D-to-3D differentiation process of kidney organoids, the bright field images on days 3, 6, 11, 16 and 25 are shown in FIG3 , and the immunofluorescence staining results of kidney organoids are shown in FIG4 , LTL marks proximal tubular cells, and Nephrin marks podocytes; Taking Example 5 as an example, during the full 3D differentiation process, the bright field images on days 1, 4 and 6 are shown in FIG4 . The bright field images on the 2nd, 6th, 9th and 14th days are shown in FIG5 ; during the 2D to 3D differentiation process of the kidney organoids, the bright field images on the 2nd, 4th, 6th, 9th and 18th days are shown in FIG6 ; the immunofluorescence staining results of the kidney organoids are shown in FIG7 , LTL marks the proximal tubular cells, and Nephrin marks the podocytes; it can be seen that the present invention can effectively construct kidney organoids, obtain mature organoids with the main structures and functional cells of the kidney, and can be constructed based on two initial differentiation states. Differentiation starting from 2D cells can achieve uniformity and large-scale production, and differentiation starting from 3D cell spheres can observe the developmental state of the entire kidney organoid.

[0112] In addition, comparing the differentiation processes of each scheme, the results of Schemes 5-7 are shown in Figure 8. Schemes 5-7 can successfully complete differentiation in 6-8 days, and can grow and mature on the 12th day to generate kidney organoids with obvious structures, while the differentiation cycles of Schemes 1-4 and 8-10 are still relatively long. For example, comparing Scheme 5 with Schemes 3, 9, and 10, it is shown that the present invention utilizes a specific combination of GSK-3 inhibition and ALK5 inhibitors to significantly shorten the differentiation cycle of kidney organoids and quickly obtain mature organoids with the main structures and functional cells of the kidney.

[0113] Example 3

[0114] In this example, the kidney organoids constructed according to Scheme 5 in Example 1 were used to conduct toxicological testing experiments.

[0115] The specific experimental process includes:

[0116] (1) Transfer the kidney organoids to the upper chamber of the Transwell and add factor-free basal medium to the lower chamber until it just covers the bottom of the chamber;

[0117] (2) Cultivate kidney organoids at the air-liquid interface until clear tubular structures are visible;

[0118] (3) Add 200 μM cisplatin to the factor-free basal medium below and treat for 3 days as the experimental group; treat with an equal amount of PBS as the control group (CTRL);

[0119] (4) By observing the morphology of renal tubules under a bright field microscope, it can be clearly observed that the renal tubules in the experimental group gradually lost their obvious tubular structure and tended to be disordered. The speed of change in the renal tubular structure indicates the toxicity of the tested drug;

[0120] (5) Kidney organoids were collected 3 days after cisplatin treatment and frozen sections were made. The degree of damage was quantified by immunofluorescence staining of KIM-1.

[0121] The results of renal tubular morphological changes are shown in Figure 9 , which shows that the renal organoid structure is lost after treatment in the experimental group; the results of quantitative damage degree are shown in Figure 10 , which show that the renal organoids treated with cisplatin were frozen sectioned and immunofluorescence stained, with KIM-1 marking renal injury factors and Nephrin marking podocytes, showing that renal damage was significantly increased after cisplatin treatment.

[0122] In summary, the present invention provides a method for rapidly and efficiently inducing the differentiation of kidney organoids using human induced pluripotent stem cells. This method can solve the problems of long differentiation cycle, low differentiation efficiency, high differentiation cost, and different disease models or toxicology experiments targeting different kidney structures of kidney organoids. The method is simple and easy to implement, and the construction effect is stable. By optimizing the differentiation process, the differentiation cycle of kidney organoids is further shortened, and mature organoids with the main structures and functional cells of the kidney can be quickly obtained in 6 to 8 days. Moreover, the method can be directly applied to in vitro toxicology experiments and has extremely high practical value.

[0123] The applicant states that the present invention is intended to illustrate the detailed methods of the present invention through the above-described embodiments, but the present invention is not limited to the above-described detailed methods, that is, it does not mean that the present invention must rely on the above-described detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. A method for rapidly constructing human kidney organoids, characterized in that: The method comprises the following steps: (1) culturing human induced pluripotent stem cells by direct seeding or by aggregating them into spheres and then seeding them in a culture medium containing an inhibitory component; the inhibitory component contains a GSK-3 inhibitor; (2) culturing the cells obtained in step (1) using a basal medium without additives or a medium containing FGF-9; (3) culturing the cells obtained in step (2) with a culture medium containing a GSK-3 inhibitor and FGF-9; (4) culturing the cells obtained in step (3) with a culture medium containing FGF-9; (5) The cells obtained in step (4) are cultured in a non-additive basal medium to obtain mature kidney organoids.

2. The method for constructing a human kidney organoid according to claim 1, wherein: The GSK-3 inhibitor includes any one or a combination of at least two of CHIR99021, LY2090314, CHIR98014, BIO-acetoxime, AZD2858 or SAR502250; Preferably, the inhibitory component in step (1) further contains an ALK5 inhibitor; Preferably, the ALK5 inhibitor includes any one of SB431542, RepSox, R-268712 or SB525334, or a combination of at least two of them.

3. The method for constructing human kidney organoids according to claim 2, wherein: The inhibitory component in step (1) contains any one of a combination of CHIR99021 and SB431542, a combination of CHIR99021 and RepSox, a combination of LY2090314 and R-268712, or a combination of SB525334 and SAR502250.

4. The method for constructing a human kidney organoid according to claim 2 or 3, wherein: The concentration of the GSK-3 inhibitor in the culture medium containing the inhibitory component in step (1) is 1-30 μM; Preferably, the concentration of the ALK5 inhibitor in the culture medium containing the inhibitory component in step (1) is 0.1-10 µM.

5. The method for constructing a human kidney organoid according to any one of claims 1 to 4, characterized in that: The concentration of FGF-9 in the culture medium containing FGF-9 in step (2) is 10-200 ng / mL.

6. The method for constructing a human kidney organoid according to any one of claims 1 to 5, wherein: In step (3), the concentration of the GSK-3 inhibitor in the culture medium containing the GSK-3 inhibitor and FGF-9 is 1-30 μM, and the concentration of FGF-9 is 10-200 ng / mL; Preferably, the concentration of FGF-9 in the culture medium containing FGF-9 in step (4) is 10-200 ng / mL.

7. The method for constructing a human kidney organoid according to any one of claims 1 to 6, wherein: The culturing time in step (1) is 1 to 4 days; Preferably, the culturing time in step (2) is 1 to 3 days; Preferably, the culturing time in step (3) is 1 to 2 days; Preferably, the culturing time in step (4) is 1 to 6 days; Preferably, the culturing time in step (5) is 1 to 10 days.

8. The method for constructing a human kidney organoid according to any one of claims 1 to 7, wherein: The method comprises the following steps: (1) Human induced pluripotent stem cells were directly seeded or aggregated into spheres and then seeded in a culture medium containing a GSK-3 inhibitor and an ALK5 inhibitor for 1 to 4 days; (2) culturing the cells obtained in step (1) with a non-additive basal medium or a medium containing FGF-9 for 1 to 3 days; (3) culturing the cells obtained in step (2) with a culture medium containing a GSK-3 inhibitor and FGF-9 for 1 to 2 days; (4) culturing the cells obtained in step (3) with a medium containing FGF-9 for 1 to 6 days; (5) The cells obtained in step (4) are cultured in a non-additive basal medium for 1 to 10 days to obtain mature kidney organoids.

9. Use of the method for constructing human kidney organoids according to any one of claims 1 to 8 in renal toxicology testing.

10. A method for toxicological testing using human kidney organoids, characterized in that: The method comprises the following steps: (1) Transferring the kidney organoid constructed by the method for constructing a human kidney organoid according to any one of claims 1 to 8 into the upper chamber of a Transwell and adding culture medium into the lower chamber; (2) Cultivate kidney organoids at the air-liquid interface until clear tubular structures are visible; (3) Add the test agent to the culture medium in the lower chamber; (4) Observe the toxicity of the test agent by changes in renal tubular morphology and quantify the degree of damage by staining frozen sections.

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