Method for rapidly preparing hiPSC-sourced kidney organoid

Abstract

The invention relates to the technical field of biology, in particular to a method for rapidly preparing a hiPSC-sourced kidney organ. The method comprises the following steps: gradually carrying out induced differentiation, digestion and centrifugation on stem cells by adopting a middle mesoderm induction culture medium and other culture mediums to obtain a cell mixture, incubating, and carrying out induced culture by adopting a kidney precursor induction culture medium and the like to obtain the kidney organ. The culture medium, the cell factors and the like used in the method are conventional reagents, the production cycle of the kidney precursor organ-like globules can be stably controlled within 18 days, and the globules can be continuously differentiated into kidney organ-like mature bodies after resuscitation; and an important cell population related to kidney tissues and having appearance morphological characteristics highly consistent with those of a mature body which is directly differentiated from hIPSC into a kidney organoid without cryopreservation. According to the kidney organ product provided by the invention, the production of kidney organs can be increased through the steps of cryopreservation and resuscitation, the difference between batches is reduced, and the standardization and large-scale production process can be realized.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a method for rapidly preparing hiPSC-derived kidney organoids. Background Technology

[0002] Currently, the core idea for the rapid preparation of hiPSC-derived kidney organoids is to optimize the differentiation process by precisely regulating key signaling pathways to accelerate the induction of kidney progenitor cells and the maturation of organoids. The main methods include: I. Preparation of Kidney Organoids by Sequential Addition of Growth Factors. This involves adding specific growth factors (such as CHIR99021, noggin, and FGF9) in stages to efficiently direct the differentiation of hiPSCs into metanephric mesenchymal (MM) and ureteral bud (UB) progenitor cells, which is the foundation for forming complete nephron structures. However, this method suffers from incomplete maturity and function. Rapidly differentiated organoids often remain in a "semi-mature" stage, lacking the complete transport functions of key nephrons such as the proximal tubules. For example, the renal tubular cells in traditional organoids cannot effectively mimic the active reabsorption process of substances such as glucose and sodium ions. The lack of vascularized structures in organoids leads to functional limitations of internal cells due to insufficient nutrient and oxygen supply.

[0003] II. Microfluidic Bioprinting Technology. Microfluidic bioprinters can precisely mix MM and UB progenitor cells and print them into filamentous structures with a core-shell structure. This structure better mimics the in vivo microenvironment, promoting organoid formation and exhibiting mature nephron structures within two weeks. However, there are technical challenges in reproducibility and scalability. While microfluidic bioprinting technology can improve structural consistency, the equipment is expensive and the operation is complex, making large-scale production difficult. Sequential addition of growth factors is sensitive to culture conditions, resulting in significant batch-to-batch variations and affecting experimental reproducibility. Since kidney organoids cannot be directly cryopreserved during and after differentiation from hiPSCs, the entire production process is difficult to repeat and scale up.

[0004] III. Optimizing the Culture System. Employing specific culture medium formulations and conditions (such as using ultra-low adsorption plates and horizontal shakers) can significantly improve organoid formation efficiency and uniformity. For example, existing protocols have shortened the entire differentiation process to approximately 23 days. However, this presents technical hurdles and high costs. Optimizing the culture system (such as using specific inhibitors or 3D culture) requires highly specialized experimental skills, and the culture medium is expensive. While rapid maturation methods (such as 48-hour signal modulation) can accelerate differentiation, they may introduce non-physiological stress, affecting the long-term stability of organoids. While significant progress has been made in the rapid preparation of hiPSC-derived kidney organoids (e.g., sequential addition of growth factors, microfluidic bioprinting, and optimized culture systems), limitations remain in functional validation. Currently, the industry lacks a standardized evaluation system for kidney organoid products, making direct comparison of organoid functional data from different laboratories difficult. Although organoid nephrotoxicity testing (e.g., cisplatin, gentamicin) is superior to animal models, in vivo experiments are still needed to verify their clinical relevance. Summary of the Invention

[0005] To address the technical problems existing in the prior art, the present invention aims to provide a method for rapidly preparing hiPSC-derived kidney organoids. The main technical problem solved by this invention is how to efficiently induce differentiation of human IPSCs in vitro into kidney organoids capable of cryopreservation and thawing, wherein the kidney organoids provided by this method maintain consistent cell viability and maturity before and after cryopreservation and thawing.

[0006] According to a first aspect of the present invention, the present invention provides a method for rapidly preparing hiPSC-derived kidney organoids, comprising the following steps: (1) In a container, take stem cells and resuspend them in mTeSR1 complete medium, seed them into culture well plates for adherent culture, discard the mTeSR1 complete medium, and gradually induce differentiation by using mesodermal induction medium, kidney progenitor induction medium and kidney organoid maturation medium in sequence. Discard the medium, add digestive solution for digestion, pipette the cells, centrifuge to collect the cell pellet, resuspend the cell pellet to obtain a cell suspension, centrifuge the cell suspension to obtain a cell mixture; (2) The cell mixture described in step (1) is mixed with Pulse medium and incubated. The Pulse medium is then discarded, and the mixture is induced and cultured sequentially using renal progenitor induction medium and renal organoid mature body maintenance medium to obtain the renal organoid.

[0007] In some embodiments, the stem cells in step (1) are hiPSC stem cells; the cell density of the stem cells in mTeSR1 complete culture medium is 3 × 10⁻⁶. 4 -1×10 5 per mL.

[0008] In some embodiments, the mTeSR1 complete culture medium contains Y-27632 at a concentration of 6-12 μmol / L.

[0009] In some embodiments, the temperature for adherent culture in step (1) is 36.5 - 37.5 °C, and the culture time is 20-30 h.

[0010] In some embodiments, step (1), which involves discarding the complete mTeSR1 culture medium and sequentially inducing differentiation using mesodermal induction medium, renal progenitor induction medium, and renal organoid maturation medium, includes the following steps: The mTeSR1 complete medium was aspirated, and the mid-section mesodermal induction medium was added to the container for the first induction differentiation treatment. Then the mid-section mesodermal induction medium was aspirated, and the renal progenitor induction medium was added to the container for the second induction differentiation treatment. Finally, the renal progenitor induction medium was aspirated, and the renal organoid maturation medium was added to the container for the third induction differentiation treatment. In some embodiments, the first induced differentiation treatment lasts for 4-6 days, the temperature is 36.5-37.5℃, and the medium is changed daily during the first induced differentiation treatment; the second induced differentiation treatment lasts for 2-4 days, the temperature is 36.5-37.5℃, and the medium is changed daily during the second induced differentiation treatment; the third induced differentiation treatment lasts for 4-6 days, the temperature is 36.5-37.5℃, and the medium is changed daily during the third induced differentiation treatment.

[0011] In some embodiments, the preparation of the mesodermal induction medium in step (1) includes: adding CHIR-99021 to Essential 6 Medium, mixing well, and obtaining the mesodermal induction medium; the concentration of CHIR-99021 in the mesodermal induction medium is 6-12 μmol / L; The preparation of the renal progenitor induction medium includes: adding FGF9 and Heparin to Essential 6 Medium, mixing well, and obtaining the renal progenitor induction medium; in the renal progenitor induction medium, the concentration of FGF9 is 150-250 ng / ml, and the concentration of Heparin is 0.5-2 μg / ml.

[0012] In some embodiments, the preparation of the kidney organoid maturation culture medium in step (1) includes: PenStrep, Triiodothyronine, Hydrocortisone, ITS, Transforming growth factor-α, FGF9, CHIR99021, DAPT, BMP7, LIF, and Y27632 were added to DMEM / F12 medium and mixed well to obtain the kidney organoid maturation medium. In the kidney organoid maturation culture medium, the volume percentage concentration of PenStrep was 0.5%-2%, the concentration of Triiodothyronine was 1-3 pg / mL, the concentration of Hydrocortisone was 0.05-0.2 μmol / L, the concentration of ITS was 3-8 ng / mL, the concentration of Transforming Growth Factor-α was 6-12 ng / mL, the concentration of FGF9 was 35-65 ng / mL, the concentration of CHIR99021 was 0.8-1.6 μmol / L, the concentration of DAPT was 1.5-3 μmol / L, the concentration of BMP7 was 7.5-12.5 ng / mL, the concentration of LIF was 3-6 ng / mL, and the concentration of Y27632 was 6-12 μmol / L.

[0013] In some embodiments, step (1) of adding digestive fluid for digestion includes: Wash with DPBS buffer, then add digestion solution for digestion, and then aspirate the digestion solution. The digestion solution is a mixture of cell dissociation reagent and Y27632; in the digestion solution, the concentration of Y-27 is 6-12 μmol / L, and the concentration of cell dissociation reagent is 0.5X-1.5X; the digestion temperature is 36.5-37.5℃, and the digestion time is 3-5 min. X represents the concentration multiple.

[0014] In some embodiments, the blowing of cells in step (1) includes the following steps: Add Essential 6 Medium to the container, pipette the cells to obtain a mixture, centrifuge the mixture at 1000-1500 rpm for 3-6 min, remove the supernatant, and obtain the cell pellet; the Essential 6 Medium contains 8%-12% FBS (fetal bovine serum) by volume.

[0015] In some embodiments, step (1) of resuspending the cell pellet includes the following steps: The cell pellet was mixed with Essential 6 Medium to obtain a cell suspension, thus completing the resuspending of the cell pellet. The Essential 6 Medium contained 8%-12% FBS by volume.

[0016] In some embodiments, the centrifugation of the cell suspension in step (1) includes the following steps: The cell suspension was counted, and the cell density in the suspension was adjusted to 2 × 10⁻⁶. 5 - 2.5 × 10 5 Cells / mL: Place the cell suspension in a container, then centrifuge at 300-400 ×g for 3-5 min, then centrifuge at 70-100 ×g for 30-60 s, and finally repeat centrifugation at 70-100 ×g for 30-60 s to obtain a cell mixture.

[0017] In some embodiments, the preparation of the Pulse medium in step (2) includes the following steps: adding CHIR-99021 to Essential 6 Medium and mixing well to obtain the Pulse medium; the concentration of CHIR-99021 in the Pulse medium is 6-12 μmol / L; the incubation temperature in step (2) is 36.5-37.5℃ and the incubation time is 40-80 min.

[0018] In some embodiments, step (2) involves sequentially using renal progenitor induction culture medium and renal organoid maintenance culture medium for induction culture, including the following steps: Add renal progenitor induction medium to the container for the fourth induction differentiation treatment, then remove the renal progenitor induction medium and add renal organoid mature body maintenance medium to the container for the fifth induction differentiation treatment. In some embodiments, the temperature of the fourth induced differentiation treatment is 36.5-37.5℃, the duration of the fourth induced differentiation treatment is 3-5.5 days, and the medium is changed every 2 days during the fourth induced differentiation treatment; the temperature of the fifth induced differentiation treatment is 36.5-37.5℃, the duration of the fifth induced differentiation treatment is 11-13 days, and the medium is changed every 2 days during the fifth induced differentiation treatment.

[0019] In some embodiments, the preparation of the renal progenitor induction culture medium in step (2) includes: The preparation of the renal progenitor induction medium includes: adding FGF9 and Heparin to Essential 6 Medium, mixing well, and obtaining the renal progenitor induction medium; in the renal progenitor induction medium, the concentration of FGF9 is 150-250 ng / ml, and the concentration of Heparin is 0.5-2 μg / ml.

[0020] In some embodiments, the preparation of the kidney organoid mature body maintenance culture medium in step (2) includes: adding Heparin to Essential 6 Medium and mixing it to obtain the kidney organoid mature body maintenance culture medium; the concentration of Heparin in the kidney organoid mature body maintenance culture medium is 0.5-2 μg / ml.

[0021] According to a second aspect of the present invention, the present invention provides a kidney organoid obtained by the above-described method for rapid preparation of hiPSC-derived kidney organoids.

[0022] According to a third aspect of the invention, the invention provides the use of kidney organoids in screening drugs for the prevention or treatment of kidney diseases.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: Compared to other differentiation schemes, the preparation method provided in this invention uses common reagents (all transparent and publicly available) in the culture medium, cytokines, small molecule inhibitors, and other components used in the differentiation of hiPSC-derived kidney organoids. The production cycle for cryopreservable kidney progenitor organoid glomeruli can be stably controlled within 18 days. Furthermore, after thawing, these glomeruli can continue to differentiate into mature kidney organoids, maintaining a high degree of consistency in appearance and morphological characteristics with those differentiated directly from hIPSCs without cryopreservation, as well as important cell populations related to kidney tissue. For example, regardless of whether the organoid glomeruli have undergone cryopreservation and thawing, continued differentiation into mature kidney organoids ensures the expression of endothelial cell markers (CD31), loop of Henle epithelial cell markers (E-cadherien / NKCC2), glomerular upper podocyte markers (NPHS1 / MafB), renal collecting duct markers (PAX2), and proximal tubular markers (LTL / CUBN). This means that various important renal unit structures tend to be more complete and closer to real human kidney organs. Therefore, the kidney organoid products provided by this invention can increase the production of kidney organoid products in the same batch through cryopreservation and thawing steps, and reduce batch-to-batch variations, further realizing the standardization and large-scale production process. Moreover, compared with kidney organoids produced by microfluidic bioprinting technology, this method has reduced material costs and machine maintenance costs. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 To implement the results of Case 1, the mature kidney organoids were observed and photographed under a 20x microscope in bright field. The differentiation of the single cells after cryopreservation and thawing continued for 17 days through steps (8), (9), (10), and (11). Figure 2 To implement the results of observing and photographing mature kidney organoids obtained by continuing the differentiation of kidney organoid glomeruli in step (11) for 12 days under bright field with a 20x microscope in Case 1; Figure 3 To implement Case 1, the results of observing and photographing mature kidney organoids obtained by directly undergoing differentiation from step (1) to step (11) for 30 days without cryopreservation under a 10x microscope in a bright field are shown.

[0026] Figure 4 The images show the pathological verification results of the mature kidney organoids that were directly differentiated from IPSCs 30 days ago and were not cryopreserved, as described in Case 1. These include HE results observed under a 10x bright field microscope under an upright microscope, immunoenzyme histochemical staining results (CD31, E-Cad, NPHS, PAX2, NKCC2), and fluorescence staining results (MFAB, CUBN, LTL) taken under a 10x microscope.

[0027] Figure 5 The pathological verification results of the kidney organoids obtained by directly differentiating from IPSC for 18 days in Case 1, freezing them in liquid nitrogen for 120 days, and continuing the differentiation of step (11) for a total of 12 days after thawing are shown in the figure (observed under a 10x microscope). Detailed Implementation

[0028] To better understand the technical solution of the present invention, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] The main reagents and consumables used in the following examples are shown in Table 1 below.

[0030] Table 1

[0031] Implementation Case 1 A method for rapidly preparing hiPSC-derived kidney organoids includes the following steps: (1) Preparation and digestion of hiPSC cells HiPSCs were cultured in 6-well plates in a 5% CO2 incubator using mTsSR1 complete medium (Stemcell, 85850) at 37°C and 5% CO2 concentration. Cell confluence reached 80%-90% and the cells were in good condition (smooth clonal morphology and tight cell junctions). The culture medium was aspirated, and the cells were washed with DPBS (Thermo Fisher, 14190367) buffer. The DPBS buffer was then aspirated, and 2 mL of digestion solution (TrypLE Select (1X), Thermo Fisher, 12563011) was added for digestion (37°C, 4 min). The digestion solution was then aspirated. (2) hiPSC terminates digestion Add 3 mL of mTeSR1 complete culture medium (containing Y-27632 at a concentration of 6 μmol / L) to a 6-well plate, pipette the cells 3 times to obtain a mixture, centrifuge at 1000 rpm for 3 min, remove the supernatant to obtain the cell pellet; (3) hiPSC seed plate The cell pellet from step (2) was mixed with mTeSR1 complete culture medium (containing Y-27632 at a concentration of 6 μmol / L) to obtain a cell suspension. Cell counting was performed under a microscope using a disposable hemocytometer (Merck Millipore, MDH-2N1-50PK), and the cell density was adjusted to 1.2 × 10⁻⁶. 5 Cells / mL were transferred to new 6-well plates and cultured in a 5% CO2 incubator at 37°C for 1 day. (4) First induction of differentiation treatment Remove the mTeSR1 complete medium from the 6-well plate in step (3), add 2 mL of mid-section mesoderm induction medium for the first induction differentiation treatment. The first induction differentiation treatment is carried out in a 5% CO2 incubator at 37℃ for 5 days, and the medium needs to be changed every day. The preparation of the mesodermal induction medium includes: CHIR-99021 (Wako, 034-23103) was added to Essential 6 Medium (Thermofisher, A1516401) and mixed well to obtain the mesodermal induction medium; the concentration of CHIR-99021 in the mesodermal induction medium was 6 μmol / L. (5) Second induction of differentiation treatment Remove the mid-section mesodermal induction medium from step (4), wash the 6-well plate once with 3 mL of DMEM / F12 (LifeTechnologies, 11330032) medium, then remove the DMEM / F12 medium, and then add 2 mL of renal progenitor induction medium to the 6-well plate for the second induction differentiation treatment. The second induction differentiation treatment is carried out in a 5% CO2 incubator at 37°C for 3 days, and the medium needs to be changed every day. The preparation of the renal progenitor induction culture medium includes: FGF9 (R&D Systems, 273-F9) and Heparin (Sigma, H3149) were added to Essential 6 Medium and mixed to obtain the renal progenitor induction medium. The concentration of FGF9 in the renal progenitor induction medium was 200 ng / ml and the concentration of Heparin was 1 μg / ml. (6) Third induction of differentiation treatment Remove the renal progenitor induction medium from step (5), add 2 mL of renal organoid maturation medium for the third induction differentiation treatment. The third induction differentiation treatment is carried out in a 5% CO2 incubator at 37℃ for 5 days, and the medium needs to be changed every day. The preparation of the kidney organoid maturation culture medium includes: PenStrep (Life Technologies, 15140122), Triiodothyronine (Sigma, T6397), Hydrocortisone (Sigma, H0888), ITS (Sigma, I1884), Transforming growth factor-α (Life Technologies, PHG0051), FGF9, CHIR99021 (Wako, 034-23103), DAPT (SantaCruz, SC-201315), BMP7 (R&D Systems, 354-BP), LIF (ISOkine, IK0700), and Y27632 (TOCRIS, RDS1254) were added to DMEM / F12 medium and mixed well to obtain the kidney organoid maturation medium. In the kidney organoid maturation culture medium, the volume percentage concentration of PenStrep was 1%, the concentration of Triiodothyronine was 2.5 pg / mL, and the concentration of Hydrocortisone was 0.1 μmol / L. The concentration of ITS was 5 ng / mL, and the concentration of Transforming Growth Factor-α was 10 ng / mL. The concentrations of FGF9 and CHIR99021 were 50 ng / mL, DAPT and BMP7 were 10 ng / mL, LIF and Y27632 were 5 ng / mL and 10 μmol / L, respectively. (7) Cell digestion Remove the kidney organoid maturation culture medium from step (6), add 2 mL of DPBS buffer to each well and wash 3 times, remove the DPBS buffer, then add 2 mL of digestion solution (a mixture of TrypLE Select and Y-27), in which the concentration of TrypLE Select is 1X and the concentration of Y-27 is 6 μmol / L, place in an incubator at 37℃ for 3 min, remove and remove the digestion solution, add 1 mL of Essential 6 Medium (containing 10% FBS by volume), pipette and blow the cells to obtain a mixture, centrifuge at 1000 rpm for 3 min, remove the supernatant and obtain the cell pellet; (8) Preparation of cell mixture Mix the cell pellet obtained in step (7) with Essential 6 Medium (containing 10% FBS by volume), resuspend, and adjust the cell density to 2 × 10⁻⁶ cells / mL. 5 Cells / mL, take 200μL of the cell suspension after adjusting the cell density and transfer it to a 1.5ml low-adsorption EP tube. Place it in a low-temperature centrifuge (4℃) and centrifuge at 300×g for 3min. Then rotate the 1.5ml low-adsorption EP tube 180° around its long axis and return it to the low-temperature centrifuge (4℃). Centrifuge at 70×g for 30s. Then rotate the 1.5ml low-adsorption EP tube 180° around its long axis and return it to the low-temperature centrifuge (4℃). Centrifuge again at 70×g for 30s. Discard the supernatant to obtain the cell mixture. (9) Inoculation of cell mixtures The cell mixture from step (8) was transferred into a 6-well plate, and 1 mL of Pulse medium was added for incubation. The incubation was carried out in a 5% CO2 incubator at 37°C for 1 hour. The preparation of the Pulse medium includes: adding CHIR-99021 to Essential 6 Medium, mixing well, and obtaining the Pulse medium; in the Pulse medium, the concentration of CHIR-99021 is 5 μmol / L; (10) Fourth induction of differentiation treatment Pulse medium was removed, and 1 mL of renal progenitor induction medium was added for the fourth differentiation induction treatment. The fourth differentiation induction treatment was carried out in a 5% CO2 incubator at 37℃ for 5 days, with the medium being changed every 2 days. The renal progenitor induction culture medium in step (10) is prepared in the same way as the renal progenitor induction culture medium in step (5); (11) Fifth induction of differentiation treatment and maintenance of mature bodies The renal progenitor induction medium was aspirated to obtain renal organoid glomeruli. 1 mL of renal organoid mature body maintenance medium was added for the fifth induction differentiation treatment; the fifth induction differentiation treatment included: Kidney organoid glomeruli were pipetted into 6-well plates until they detached from the bottom and became suspended, yielding a suspension. This suspension was then transferred to ultra-low adsorption 6-well plates (CORNING, 3471) for suspension culture at 37°C in a 5% CO2 incubator for 12 days, with the medium changed every 2 days, to obtain mature kidney organoids. Mature kidney organoids were then continuously cultured in maintenance medium for mature kidney organoids, with the medium changed every 2 days.

[0032] The preparation of the maintenance culture medium for mature kidney organoids includes: adding Heparin to Essential 6 Medium and mixing well to obtain the maintenance culture medium for mature kidney organoids; the concentration of Heparin in the maintenance culture medium for mature kidney organoids is 1 μg / ml.

[0033] (12) Cryopreservation of kidney organs Group 1: Aspirate the organoid cryopreservation solution and mix by pipetting. The cell pellet of the kidney organoids digested by TrypLE Selec in step (7) (most of which are now in a single-cell state) is then added to further adjust the cell density in the cell suspension to 1×10⁻⁶. 6 1 unit / ml, transferred to cryovials; Group 2: In addition, take the suspension from step (11) (using a Pasteur pipette to blow the kidney organoids in the 6-well plate until the kidney organoids detach from the bottom of the plate and become suspended), transfer it to a 15ml centrifuge tube, let it stand for 3 minutes, discard the supernatant, use a wide-tipped pipette or a Pasteur pipette to draw up the organoid cryopreservation solution and blow to mix the above kidney organoids, reduce excessive mechanical damage, maintain the integrity of the organoids, control the density of organoids in the cryopreservation solution to 15~20 per ml, transfer it to cryopreservation tubes, and the volume of cell suspension in each cryopreservation tube is 1 ml.

[0034] After transferring the cryovials from the first and second groups to the programmed cooling box (cryopreservation box), immediately place them in a -80℃ freezer for 48 hours; then remove the cryovials from the first and second groups from the cryopreservation box and quickly transfer them to liquid nitrogen for long-term storage (90-180 days).

[0035] Effect verification I. Cryopreservation and thawing experiment Group 1: Cell pellets of kidney organoids digested by TrypLE Selec in step (12) (cell pellets that have been frozen in liquid nitrogen for 120 days) were selected for this purpose, and the specific steps included as follows: Take out the cryopreservation tubes of the first group from the liquid nitrogen tank in step (12) and quickly complete the thawing procedure: place the cryopreservation tubes of the first group in a 37°C water bath for rewarming; after the cryopreservation liquid thaws, unscrew the tube caps and use a pipette tip to transfer the suspended cells and cryopreservation liquid together to a 15 ml centrifuge tube, add 3 times the volume of DF-12 medium (i.e. DMEM / F12 (with HEPES) in Table 1, the same below), continue to pipette tip to blow 5 times, centrifuge at 350 ×g for 5 minutes, aspirate the supernatant, and wash twice with DF-12 medium in the same way. Then, starting from step (8) in the above implementation case one, prepare the cell mixture and repeat the subsequent experiments (steps (8)-(11)) to obtain the mature kidney organoids of the first group.

[0036] The second group: kidney organoid glomeruli that had been frozen in liquid nitrogen for 120 days in step (12) were selected. The specific steps included the following: Remove the cryovials (containing 15-20 kidney organoids / ml) from the second group in step (12) from the liquid nitrogen tank and quickly complete the thawing procedure: place the cryovials in a 37°C water bath for rewarming; after the cryopreservation liquid thaws, unscrew the cap and use a Pasteur pipette to transfer the organoids and cryopreservation liquid together to a 15 ml centrifuge tube, add 3 times the volume of DF-12 medium, continue to pipette and blow 5 times, let stand at room temperature for 3-5 minutes, discard the supernatant, and wash twice with DF-12 medium in the same way. Then add 2 mL of kidney organoid mature body maintenance medium for the fifth induction differentiation treatment (refer to the method described in step (11)), and change the medium every 2 days. Continue culturing for 12 days to obtain mature kidney organoids, which will be used as the second group of mature kidney organoids.

[0037] The third group: The mature kidney organoids obtained by differentiation directly undergoing steps (1) to (11) (a total of 30 days from step (1) to step (11)) without cryopreservation (i.e. the mature kidney organoids obtained after continuous suspension culture for 12 days in step (11)) were selected as the mature kidney organoids of the third group.

[0038] The morphology of mature kidney organoids derived from revived single cells or kidney organoid glomeruli was observed and photographed under bright field using a 20x microscope. Figure 1 , Figure 2 and Figure 3 As shown), where Figure 1 The mature kidney organoids (Group 1) are obtained by differentiating from single cells that have been cryopreserved and then thawed and continue to undergo the above steps (8), (9), (10), and (11) for a total of 17 days. Figure 2 The second group consists of mature kidney organoids obtained by directly subjecting renal organoid glomeruli that have undergone cryopreservation and resuscitation to the fifth induction differentiation treatment (11) (a total of 12 days of suspension culture). The third group consists of mature kidney organoids obtained from the same batch that have undergone differentiation directly from steps (1) to (11) for a total of 30 days without cryopreservation. Figure 3 Compared with the organoids shown in the figure, they showed no significant changes in appearance characteristics such as organoid integrity, gloss and translucency, and cell viability, and all of them showed "mulberry"-like wrinkled structures on the outside of the organoids.

[0039] II. Pathological Verification of Kidney Organoid Maturity In step (11), at least five mature kidney organoids were selected from the suspension for pathological verification of maturity, following the method described in the literature by Xinyu Chen (Human striatal organoids derived from pluripotent stem cells recapitulate striata development and compartments). Specifically, the following steps were included: Mature kidney organoids were prepared into 15 μm thick frozen sections. Following the instructions of the HRP / DAB IHC Detection kit (Abcam, ab236466), immunofluorescence histochemistry or immunoenzyme histochemistry was performed to identify markers for renal endothelial cell markers (CD31), loop of Henle epithelial cell markers (E-cadherien / NKCC2), glomerular superior podocyte markers (NPHS1 / MafB), renal collecting duct markers (PAX2), and proximal tubular markers (LTL / CUBN). The staining results (CD31 / E-cadherien / NPHS / PAX2 / NKCC2) and HE results obtained by immunoenzyme histochemistry were observed under a 10x bright-field microscope. The fluorescence staining results (MFAB / CUBN / LTL) were captured under a 10x microscope using a cell fluorescence imaging microscope system.

[0040] The results are as follows Figure 4 and Figure 5 As shown, the kidney organoids from the same batch, after being thawed and further differentiated into mature kidney organoids after 120 days of cryopreservation, retained various important cell populations that were present in mature kidney organoids that were directly differentiated without cryopreservation.

[0041] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A method for rapidly preparing hiPSC-derived kidney organoids, characterized in that, Includes the following steps: (1) In a container, take stem cells and place them in mTeSR1 complete medium for adherent culture. Discard the mTeSR1 complete medium and induce differentiation stepwise by using mesodermal induction medium, kidney progenitor induction medium and kidney organoid maturation medium in sequence. Discard the medium, add digestive solution for digestion, pipette the cells, centrifuge to collect the cell pellet, resuspend the cell pellet to obtain a cell suspension, centrifuge the cell suspension to obtain a cell mixture; (2) The cell mixture described in step (1) is mixed with Pulse medium and incubated. The Pulse medium is then discarded, and the mixture is induced and cultured sequentially using renal progenitor induction medium and renal organoid mature body maintenance medium to obtain the renal organoid.

2. The method for rapid preparation of hiPSC-derived kidney organoids according to claim 1, characterized in that, The stem cells mentioned in step (1) are hiPSC stem cells; the initial cell density of the stem cells in mTeSR1 complete culture medium is 3×10⁻⁶. 4 - 1×10 5 per mL.

3. The method for rapid preparation of hiPSC-derived kidney organoids according to claim 2, characterized in that, The mTeSR1 complete culture medium contains Y-27632 at a concentration of 6-12 μmol / L.

4. The method for rapid preparation of hiPSC-derived kidney organoids according to claim 1, characterized in that, The temperature for adherent culture in step (1) is 36.5-37.5℃, and the culture time is 20-30 h.

5. The method for rapidly preparing hiPSC-derived kidney organoids according to claim 1, characterized in that, Step (1) involves discarding the complete mTeSR1 culture medium and sequentially inducing differentiation using mid-section mesodermal induction medium, kidney progenitor induction medium, and kidney organoid maturation medium, which includes the following steps: The mTeSR1 complete medium was aspirated, and mesodermal induction medium was added to the container for the first induction differentiation treatment. Then, the mesodermal induction medium was aspirated, and renal progenitor induction medium was added to the container for the second induction differentiation treatment. Finally, the renal progenitor induction medium was aspirated, and renal organoid maturation medium was added to the container for the third induction differentiation treatment.

6. The method for rapid preparation of hiPSC-derived kidney organoids according to claim 5, characterized in that, The first induced differentiation treatment lasts for 4-6 days at a temperature of 36.5-37.5℃, with the medium changed daily during the first induced differentiation treatment; the second induced differentiation treatment lasts for 2-4 days at a temperature of 36.5-37.5℃, with the medium changed daily during the second induced differentiation treatment; the third induced differentiation treatment lasts for 4-6 days at a temperature of 36.5-37.5℃, with the medium changed daily during the third induced differentiation treatment.

7. The method for rapid preparation of hiPSC-derived kidney organoids according to claim 1, characterized in that, The preparation of the mesodermal induction medium in step (1) includes: adding CHIR-99021 to Essential 6 Medium and mixing well to obtain the mesodermal induction medium; the concentration of CHIR-99021 in the mesodermal induction medium is 6-12 μmol / L; The preparation of the renal progenitor induction medium includes: adding FGF9 and Heparin to Essential 6 Medium, mixing well, and obtaining the renal progenitor induction medium; in the renal progenitor induction medium, the concentration of FGF9 is 150-250 ng / ml, and the concentration of Heparin is 0.5-2 μg / ml.

8. The method for rapid preparation of hiPSC-derived kidney organoids according to claim 1, characterized in that, The preparation of the kidney organoid maturation culture medium in step (1) includes: PenStrep, Triiodothyronine, Hydrocortisone, ITS, Transforming growth factor-α, FGF9, CHIR99021, DAPT, BMP7, LIF, and Y27632 were added to DMEM / F12 medium and mixed well to obtain the kidney organoid maturation medium. In the kidney organoid maturation culture medium, the volume percentage concentration of PenStrep was 0.5%-2%, the concentration of Triiodothyronine was 1-3 pg / mL, the concentration of Hydrocortisone was 0.05-0.2 μmol / L, the concentration of ITS was 3-8 ng / mL, the concentration of Transforming Growth Factor-α was 6-12 ng / mL, the concentration of FGF9 was 35-65 ng / mL, the concentration of CHIR99021 was 0.8-1.6 μmol / L, the concentration of DAPT was 1.5-3 μmol / L, the concentration of BMP7 was 7.5-12.5 ng / mL, the concentration of LIF was 3-6 ng / mL, and the concentration of Y27632 was 6-12 μmol / L.

9. The method for rapid preparation of hiPSC-derived kidney organoids according to claim 1, characterized in that, Step (1) involves adding digestive fluid for digestion, including: Wash with DPBS, then add digestive solution for digestion, and then aspirate the digestive solution. The digestion solution is a mixture of cell dissociation reagent and Y-27; in the digestion solution, the concentration of Y-27 is 6-12 μmol / L, and the concentration of cell dissociation reagent is 0.5X-1.5X; the digestion temperature is 36.5-37.5℃, and the digestion time is 3-5 min.

10. The method for rapid preparation of hiPSC-derived kidney organoids according to claim 1, characterized in that, Step (1) of blowing the cells includes the following steps: Add Essential 6 Medium to the container, pipette the cells to obtain a mixture, centrifuge the mixture at 1000-1500 rpm for 3-6 minutes, remove the supernatant, and obtain the cell pellet; the Essential 6 Medium contains 8%-12% FBS by volume.

11. The method for rapid preparation of hiPSC-derived kidney organoids according to claim 1, characterized in that, Step (1) involves resuspending the cell pellet, which includes the following steps: The cell pellet was mixed with Essential 6 Medium to obtain a cell suspension, thus completing the resuspending of the cell pellet. The Essential 6 Medium contained 8-12% FBS by volume.

12. The method for rapid preparation of hiPSC-derived kidney organoids according to claim 1, characterized in that, Step (1) involves centrifuging the cell suspension, which includes the following steps: The cell suspension was counted, and the cell density in the suspension was adjusted to 2 × 10⁻⁶. 5 - 2.5 × 10 5 To obtain a cell mixture, place the cell suspension in a container and centrifuge at 300-400 ×g for 3-5 min, then centrifuge at 70-100 ×g for 30-60 s, and finally repeat the centrifugation at 70-100 ×g for 30-60 s to obtain a cell mixture.

13. The method for rapidly preparing hiPSC-derived kidney organoids according to claim 1, characterized in that; The preparation of the Pulse medium in step (2) includes the following steps: adding CHIR-99021 to Essential 6 Medium and mixing well to obtain the Pulse medium; the concentration of CHIR-99021 in the Pulse medium is 6-12 μmol / L; the incubation temperature in step (2) is 36.5-37.5℃ and the incubation time is 40-80 minutes.

14. The method for rapid preparation of hiPSC-derived kidney organoids according to claim 1, characterized in that, Step (2) involves sequentially inducing culture using renal progenitor induction medium and renal organoid mature body maintenance medium, including the following steps: The renal progenitor induction medium was added to the container for the fourth induction differentiation treatment. Then the renal progenitor induction medium was aspirated, and the renal organoid mature body maintenance medium was added to the container for the fifth induction differentiation treatment.

15. The method for rapid preparation of hiPSC-derived kidney organoids according to claim 14, characterized in that, The fourth induction differentiation treatment is carried out at a temperature of 36.5-37.5℃ for 3-5.5 days, with the medium changed every 2 days during the treatment. The fifth induction differentiation treatment is carried out at a temperature of 36.5-37.5℃ for 11-13 days, with the medium changed every 2 days during the treatment.

16. The method for rapid preparation of hiPSC-derived kidney organoids according to claim 14, characterized in that, The preparation of the renal progenitor induction culture medium includes: The preparation of the renal progenitor induction medium includes: adding FGF9 and Heparin to Essential 6 Medium, mixing well, and obtaining the renal progenitor induction medium; in the renal progenitor induction medium, the concentration of FGF9 is 150-250 ng / ml, and the concentration of Heparin is 0.5-2 μg / ml; The preparation of the maintenance culture medium for mature kidney organoids includes: adding Heparin to Essential 6 Medium and mixing well to obtain the maintenance culture medium for mature kidney organoids; the concentration of Heparin in the maintenance culture medium for mature kidney organoids is 0.5-2 μg / ml.

17. A kidney organoid obtained by the rapid preparation method of hiPSC-derived kidney organoids according to any one of claims 1-16.

18. The use of the kidney organoids of claim 17 in screening drugs for the prevention or treatment of kidney diseases.

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