3D culture method of vascularized human kidney organoid and application of 3D culture method

Abstract

The invention discloses a 3D culture method of vascularized human kidney organoid and application thereof. The 3D culture method of the human kidney organoid comprises the following steps: S1, preparing a pig kidney CdECM gel solution; s2, resuspending the hPSCs by using a first differential culture medium, performing induced differentiation for 36-60 hours, and continuously culturing for 12-36 hours after half amount of the liquid is changed; replacing the culture system with a second differential culture medium, culturing for 3-7 days, and replacing half of the liquid every other day during the culture process to obtain primary human kidney organs; and S3, uniformly mixing the primary human kidney organ obtained in the step S2 and the pig kidney CdECM gel solution obtained in the step S1, standing for 10-15 minutes, transferring into a cell culture plate after gel formation, culturing for 6-8 days, and changing a half amount of the solution every other day in the period, so as to obtain the human kidney organ. According to the method, a 3D growth environment closer to the in-vivo environment is provided for kidney organ differentiation, and kidney organ blood vessel differentiation is promoted.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a 3D culture method for vascularized human kidney organoids and its application. Background Technology

[0002] Since Takasato's research team successfully obtained 3D kidney organoids containing glomerular podocytes and renal tubules from monolayer cultured intermediate mesodermal (IM) suspensions in 2014, the induction of 3D human kidney organoids from human pluripotent stem cells (hPSCs) has rapidly progressed. Currently, hPSCs can differentiate into structurally rich 3D kidney organoids, including major kidney structures such as glomerular podocytes, proximal tubules, distal tubules, and connecting ducts, with some proximal tubules exhibiting reabsorption function. However, due to the lack of vascular networks in in vitro culture, the differentiation maturity of kidney organoids still needs further improvement. Vascularization plays a crucial role in organoid maturation and cell type development, and is an important condition for achieving mature organoid differentiation.

[0003] However, the reported induction methods often have the following problems: 1) Kim JW et al. used porcine kidneys to remove extracellular matrix to improve the vascularization of kidney organoids; the disadvantage of this method is that during the differentiation of kidney organoids, only the bottom layer cells in contact with the dECM-coated matrix gel can directly receive the signaling factors in the matrix gel. It is necessary to add the pro-angiogenic factor VEGF to the culture medium to promote the interaction between the kidney organoids and the bottom layer dECM in order to achieve an increase in the overall vascularization of the kidney organoids. 2) Garreta E et al. used porcine kidneys to remove extracellular matrix and 3D culture hPSCs to differentiate into human kidney organoids; the disadvantage is that in this method, porcine kidney dECM cannot promote the vascularization differentiation of kidney organoids. It is necessary to use human kidney dECM or to co-culture with vascular endothelial organoids to achieve the vascularization of kidney organoids. From the perspective of material sources, using human kidneys for dECM is not conducive to the widespread application of the technology due to issues such as the source of materials and ethics. From the perspective of culture methods, it is necessary to add other components such as fibrinogen, transglutaminase and thrombin, or to add vascular endothelial organoids for co-culture, which is more complicated and not conducive to the stable reproducibility of the technology.

[0004] Therefore, in order to control material costs and sources while achieving good vascularization induction, there is an urgent need to develop a 3D culture method for human kidney organoids based on porcine kidneys without extracellular matrix. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a 3D culture method for vascularized human kidney organoids. This method, without the need to add fibrinogen or other components, utilizes readily available porcine kidneys to encapsulate human kidney organoids in the extracellular matrix for culture. This not only provides a 3D growth environment that is closer to the in vivo environment for kidney organoid differentiation, but also enables a greater degree of reception of signaling factors from the porcine kidney extracellular matrix by the encapsulated kidney organoids, forming a 3D suspension culture and promoting vascular differentiation of kidney organoids.

[0006] The present invention also proposes a vascularized human kidney organoid prepared by the above method.

[0007] This invention also proposes applications of the aforementioned vascularized human kidney organoids.

[0008] According to a first aspect of the present invention, a method for 3D culture of vascularized human kidney organoids is provided, the method comprising the following steps: S1: The cortical portion of the pig kidney was decellularized to obtain a decellularized matrix, which was then freeze-dried and reconstituted to obtain a decellularized pig kidney extracellular matrix gel solution. S2: Human pluripotent stem cells were resuspended in the first differentiation medium and induced to differentiate for 36-60 h. After half of the medium was replaced, the cells were cultured for another 12-36 h to obtain embryoid bodies. Then the culture system was replaced with the second differentiation medium and cultured for 3-7 days, with half of the medium replaced every other day, to obtain primary human kidney organoids. S3: Mix the primary human kidney organoids obtained in step S2 with the porcine kidney decellularized gel solution obtained in step S1, and let it stand at 37°C for 10-15 min until gelation occurs. Then transfer it to a cell culture plate and culture for 6-8 days, changing half of the medium every other day to obtain vascularized human kidney organoids.

[0009] In some embodiments of the present invention, the decellularization process in step S1 includes the following steps: A1: After chopping the cortical part of the pig kidney, rinse it with ddH2O, then extract it with 2%-4% Triton X-100 aqueous solution for 10-15 h, then transfer it to 2%-6% sodium deoxycholate aqueous solution and shake it at room temperature for 20-30 h to decellularize. Finally, rinse it with ddH2O 2-4 times. This is one cycle. Perform two cycles in total and collect the decellularized cortical fragments. A2: Add pepsin to the decellularized cortical fragments obtained in step A1 and digest at room temperature for 6-24 h to obtain a decellularized matrix hydrogel. The mass ratio of pepsin to cortical fragments is 1:(8-12).

[0010] In some embodiments of the present invention, the solvent for reconstitution in step S1 includes 10×PBS buffer.

[0011] In some embodiments of the present invention, step S1 further includes adjusting the pH of the solution to 7-8 after digestion.

[0012] In some embodiments of the present invention, step S1 further includes adjusting the pH of the solution to 7.2-7.6 after digestion.

[0013] In some embodiments of the present invention, the porcine kidney extracellular matrix-free gel solution described in step S1 can gel at 37°C.

[0014] In some embodiments of the present invention, the first differentiation medium in step S2 includes a basal medium and additives. The basal medium includes IMDM medium, F12 medium, and PFHM-II medium. The additives in the first differentiation medium include the following components at final concentrations: 2.2-2.8 mg / mL bovine serum albumin, 0.05-0.2% (v / v) insulin-transferrin-seleno-aminoethanol (ITS-X), 0.002%-0.005% 1-thioglycerol (αMTG), 0.03-0.07 mg / mL ascorbic acid-2-phosphate, 0.5-2% (v / v) Glutamax, 0.5-2% (v / v) lipid concentrate, 1.7-3.3 mg / mL polyvinyl alcohol, 1-10 μM GSK-3 inhibitor, 2-10 μM ROCK inhibitor, and 0.7-1.3 mM β-mercaptoethanol.

[0015] In some embodiments of the present invention, the first differentiation medium in step S2 includes a basal medium and additives. The basal medium includes IMDM medium, F12 medium, and PFHM-II medium. The additives in the first differentiation medium include the following components at final concentrations: 2.4-2.7 mg / mL bovine serum albumin, 0.8-1.2% (v / v) ITS-X, 0.002%-0.004% αMTG, 0.04-0.06 mg / mL ascorbic acid-2-phosphate, 0.8-1.2% (v / v) Glutamax, 0.8-1.2% (v / v) lipid concentrate, 2-3 mg / mL polyvinyl alcohol, 4-6 μM GSK-3 inhibitor, 5-7 μM ROCK inhibitor, and 0.7-1.3 mM β-mercaptoethanol.

[0016] In some embodiments of the present invention, the volume ratio of the IMDM medium, F12 medium and PFHM-II medium is (38~44):(42~48):(4~6).

[0017] In some embodiments of the present invention, the GSK-3 inhibitor includes CHIR99021.

[0018] In some embodiments of the present invention, the ROCK inhibitor includes Y-27632.

[0019] In some embodiments of the present invention, the second differentiation medium in step S2 includes a basal medium and additives, wherein the basal medium includes DMEM medium, and the additives in the second differentiation medium include the following components at final concentrations: 10%-20% KnockOut serum substitute, 0.5-2% (v / v) non-essential amino acid solution, 0.5-2% (v / v) Glutamax, 0.5-2% (v / v) HEPES and 1.7-3.3 mg / mL polyvinyl alcohol.

[0020] In some embodiments of the present invention, the second differentiation medium in step S2 includes a basal medium and additives, wherein the basal medium includes DMEM medium, and the additives in the second differentiation medium include the following components at final concentrations: 13%-17% KnockOut serum substitute, 0.8-1.2% (v / v) non-essential amino acid solution, 0.8-1.2% (v / v) Glutamax, 0.8-1.2% (v / v) HEPES and 2-3 mg / mL polyvinyl alcohol.

[0021] In some embodiments of the present invention, step S2 includes: resuspending human pluripotent stem cells in a first differentiation medium and inducing differentiation for 45-50 h, changing half the medium and continuing to culture for 22-26 h to obtain embryoid bodies; then changing the culture system to a second differentiation medium and culturing for 4-6 days, changing half the medium every other day during this period, to obtain primary human kidney organoids.

[0022] In some embodiments of the present invention, the porcine kidney extracellular matrix-free gel solution described in step S3 is diluted to a concentration of 0.6-1% (w / v) with a third differentiation culture medium before being mixed with the primary human kidney organoid.

[0023] In some embodiments of the present invention, the porcine kidney extracellular matrix-free gel solution described in step S3 is diluted to a concentration of 0.7-0.9% (w / v) with a third differentiation culture medium before being mixed with the primary human kidney organoid.

[0024] In some embodiments of the present invention, the third differentiation medium is a second differentiation medium supplemented with 1%-3% fetal bovine serum.

[0025] In some embodiments of the present invention, 15 μL of diluted porcine kidney extracellular matrix-free gel solution is added for every 5-10 primary human kidney organoids.

[0026] According to a second aspect of the present invention, a 3D culture method for preparing vascularized human kidney organoids as described in the first aspect of the present invention is provided.

[0027] According to a third aspect of the present invention, the application of the vascularized human kidney organoids described in the second aspect of the present invention in disease model construction, drug screening, or toxicity testing is proposed.

[0028] The present invention has at least the following beneficial effects: The 3D culture method for vascularized human kidney organoids provided by this invention employs a 3D encapsulation approach, completely encapsulating the kidney organoids within a porcine kidney CdECM. This culture method more closely resembles the physical support and signal transduction mechanisms required for in vivo development of kidney organoids, achieving superior vascularization effects compared to other ECMs such as Collagen and Matrigel without the need for VEGF supplementation. This 3D encapsulation suspension culture method better replicates the actual microenvironment of the human kidney, providing a simple and feasible method for obtaining vascularized kidney organoids in vitro in the future. Attached Figure Description

[0029] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a schematic diagram of the CdECM fabrication process and the gelation result diagram in Example 1 of the present invention; wherein, the scale bar of the bright field image is 5 mm, and the scale bar of the HE staining image is 100 μm; Figure 2 This is a flowchart illustrating step 3 in Embodiment 1 of the present invention; Figure 3 The figures show the results of screening at different CdECM concentrations and serum concentrations in the experimental examples of this invention. A shows the immunofluorescence staining results of kidney organoids encapsulated with CdECM at different concentrations, with markers being glomerular podocyte PODXL (647 nm, blue), vascular endothelial CD31 (488 nm, green), and vascular endothelial progenitor marker CD146 (568 nm, red), with a scale bar of 100 μm. B shows the immunofluorescence staining results under different serum concentration conditions, with markers being glomerular podocyte PODXL (647 nm, blue), vascular endothelial CD31 (488 nm, green), and vascular endothelial progenitor marker CD146 (568 nm, red), with a scale bar of 100 μm. C shows the quantitative results of the vascular area ratio of kidney organoids and the CD31 (PECAM1) qPCR results (fold change) under different serum concentration conditions. OG in the figure refers to kidney organoids. Figure 4The images show the results of screening at different culture times in the experimental examples of this invention. A shows bright-field images of kidney organoids cultured for 5, 7, and 9 days after differentiation on day 8, encapsulated in CdECM. The red circles in the image at day D8+9 indicate off-target cells resulting from prolonged culture. B shows the immunofluorescence staining results of primary kidney organoids cultured for 3, 5, and 7 days after differentiation on day 8, encapsulated in CdECM. The markers are glomerular podocyte PODXL (647 nm, blue) and vascular endothelial CD31 (488 nm, green). The scale bar is 100 μm. C shows the percentage of vascular area, vascular connectivity density, and average vascular length of the same batch of samples at D8+3, D8+5, and D8+7 in image B, quantified using the Angio Tool. Figure 5 The images show the results of vascularized kidney organoids obtained through in vitro 3D encapsulation culture in the experimental examples of this invention. A is a schematic diagram of the differentiation induction process; B shows the differentiation effects of different matrix gels used for encapsulation compared using bright-field and immunofluorescence methods. On day 15, the kidney organoid markers were glomerular podocyte PODXL (568 nm, red), vascular endothelial CD31 (488 nm, green), and vascular endothelial progenitor marker CD146 (647 nm, blue). The markers for kidney organoids encapsulated in different matrix gels during 3D suspension culture were glomerular podocyte PODXL (647 nm, blue) and vascular endothelial CD31 (488 nm, green). The bright-field scale bar is 200 μm, and the immunofluorescence scale bar is 100 μm. μm; C shows the immunofluorescence staining results based on Figure B, including the percentage of vascular area, vascular connectivity density, and average vascular length quantitatively calculated using AngioTool, as well as the qPCR quantitative results of mature vascular endothelial CD31 (PECAM1) expressed in kidney organoids cultured in different ECMs compared with kidney organoids; D shows the continuous monolayer images and 3D reconstruction images of vascularized kidney organoids obtained after 7 days of culture in CdECM, with a Z-axis difference of 2 μm between each monolayer, a slice size of 16 μm, and a total of 8 slices; Figure 6This is a diagram showing the verification results of vascularized kidney organoids differentiated from CdECM using a mouse model in the experimental examples of this invention. In this diagram, A shows the HE staining results of the transplanted kidney organoids encapsulating CdECM under the kidney capsule of immunodeficient mice at week 11, and the transplantation site was removed one week later. The scale bar is 100 μm. B shows the immunofluorescence staining results of the transplantation site, with markers being glomerular podocyte PODXL (568 nm, red), vascular endothelial CD31 (488 nm, green), and vascular endothelial precursor marker CD146 (647 nm, blue). The scale bar is 20 μm. Detailed Implementation

[0030] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0031] Unless otherwise specified in the following examples and test cases, all instruments and reagents are commercially available and obtained through conventional purchasing channels.

[0032] In the following examples and test cases, the unit of "w / v" is g / mL, that is, 1% (w / v) refers to 10 mg / mL.

[0033] Example 1 This embodiment provides a 3D culture method for vascularized human kidney organoids and successfully induced vascularized human kidney organoids. The specific steps of the method are as follows: 1. Materials and reagents: 1) Cell preparation: Human pluripotent stem cells (hPSCs) were resuscitated and seeded at an initial density of 30% into 6-well cell culture plates coated with Matrigel (BioCoat, 354277). The cells were cultured in mTeSR-plus medium (Stemcell Technologies, 100-0276) until the density reached 60%-80%, and then passaged into 10 cm cell culture dishes pre-coated with Matrigel for later use.

[0034] 2) Preparation of culture medium: ① First Differentiation Medium: The BPEL medium used in the first stage of kidney organoid differentiation includes basal medium and additives. The basal medium consists of 41% (v / v) IMDM (Gibco, 12440053), 45% (v / v) F12 (Gibco, 12440053), and 5% (v / v) protein-free hybridoma cell medium (Gibco, 12040077). The additives account for 9% (v / v) of the first differentiation medium and are components with the following final concentrations: 2.5 mg / mL bovine serum albumin (BSA) (Sigma-Aldrich, 821006), 0.1% (v / v) insulin-transferrin-selenoethanolamine (Gibco, 51500056), 0.0039% (v / v) 1-thioglycerol (Sigma-Aldrich, M6145), 0.05 mg / mL... mg / mL ascorbic acid-2-phosphate (AA2P) (Sigma-Aldrich, A8960), 1% (v / v) Glutamax (Gibco, 35050061), 1% (v / v) lipid concentrate (Gibco, 11905031), 2.5 mg / mL polyvinyl alcohol (Sigma-Aldrich, P8136), 3.3 μM CHIR99021 (Stemcell Technologies, 72054), 8 μM Y-27632 (Stemcell Technologies, 72304), and 100 μM β-mercaptoethanol (Gibco, 21985023).

[0035] ② Second differentiation medium: The Stage II medium used for the second stage of kidney organoid differentiation includes the basal medium DMEM and the following additives at final concentrations: 15% (v / v) KnockOut serum substitute (KOSR) (Gibco, A3181502), 1% (v / v) non-essential amino acid solution (NEAA) (Gibco, 11140050), 1% (v / v) Glutamax, 1% (v / v) HEPES (Gibco, 15630080) and 2.5 mg / mL polyvinyl alcohol (Sigma-Aldrich, P8136).

[0036] ③ Third differentiation medium: The Stage II + 1.5% FBS medium used in the third stage of kidney organoid differentiation includes Stage II medium and 1.5% (v / v) fetal bovine serum FBS (Gibco, 10270-106).

[0037] 3) Preparation of porcine kidney decellularized extracellular matrix (CdECM) gel: Porcine kidney cortex was minced and rinsed with ddH2O (Beyotime, R0022) until white. It was then extracted in 3% Triton X-100 (Sigma-Aldrich, T9284) aqueous solution for 12 h, followed by decellularization in 4% sodium deoxycholate aqueous solution (BBI, A600150-0250) with shaking at room temperature for 24 h. Finally, it was rinsed three times in ddH2O. This cycle constituted one extraction and decellularization, and a total of two cycles were performed to obtain decellularized CdECM fragments. The prepared CdECM samples were retained and stained with hematoxylin and eosin (HE) to determine the degree of decellularization. To prepare a 1% (w / v) CdECM hydrogel, 100 mg of CdECM fragments were placed in 10 mL of 1 mg / mL pepsin (Sigma-Aldrich, P7012) hydrochloric acid solution (0.01 M HCl) and digested at room temperature for 12 h (6-24 h is acceptable). After ultracentrifugation (4℃, 30000 g, 30 min), the supernatant was collected to obtain a homogeneous, semi-transparent digestion solution. The pH of the digestion solution was adjusted to neutral with 1 mol / L NaOH and 1 mol / L HCl solution. Then, 1 mL of 10×PBS solution (Thermo Fisher, AM9624) was added at 4℃ to adjust the phosphate concentration and maintain acid-base balance. Finally, third differentiation medium was added to bring the volume to a final volume, yielding a 0.8% (w / v) CdECM gel solution, i.e., 100 mg of CdECM was brought to a final volume of 12.5 mL using third differentiation medium. This CdECM hydrogel solution cured into a gel at 37℃. A schematic diagram of the CdECM manufacturing process and the gelation results are shown below. Figure 1 As shown.

[0038] 2. Induction of primary human kidney organoids before coating: 1) Culture hPSCs in BPEL medium until embryoid bodies are formed: After the hPSC density in the 10 cm culture dish reached 60%-80%, 4 mL of Dispase digestion solution (Gibco, 17105041) was added and digested at 37°C for 6 min. Sterile PBS (SmartBuffers, PBS-1050) was added to the culture dish to stop digestion. The supernatant was discarded, and 6 mL of the first differentiation medium was added. The digested hPSC clones were scraped off with a cell scraper (Corning, 3008) and agitated into uniform cell clusters. This uniform cell cluster suspension was seeded at 1 mL per well into 6-well ultra-low adhesion plates (Corning, CLS3471-24EA) for suspension culture. (At this point, the hPSC clones are small cell clusters after agitation; approximately 150-200 cell clusters are present in 1 mL of resuspension). Then, the first-stage differentiation medium BPEL was added to 3 mL per well. This was recorded as day 0 of differentiation (D0). The cells were incubated statically at 37°C for 48 hours. After h (D2), embryoids are formed. At this time, the culture medium has turned yellowish and half of the medium needs to be replaced. Remove 1.5 mL of supernatant from each well and add 1.5 mL of fresh BPEL medium (with Y-27632 and β-mercaptoethanol removed) to continue culturing for 24 h (D3).

[0039] 2) Culture embryoids in Stage II medium until they form 3D primary kidney organoids: On day 3 of differentiation (D3), the embryoids were discarded and washed once with DMEM basal medium (differentiation medium II). Then, the medium was replaced with Stage II differentiation medium. Half of the medium was changed every other day until day 8 of differentiation (D8), when the primary kidney organoids differentiated into early renal tubules and glomerular podocytes. At this time, the diameter of the kidney organoids was 300-500 μm (see...). Figure 5 (B in the middle).

[0040] 3. CdECM matrix gel encapsulates primary kidney organoids: Dispense the primary kidney organoids differentiated to day 8 in step 2 into sterile 1.5 mL EP tubes, 8 tubes per tube (5-10 tubes are acceptable), and pre-chill on ice. Add 15 μL of pre-chilled 0.8% CdECM matrix gel solution to each tube of primary kidney organoids and mix well. Quickly drop the solution onto a sterile sealing film (Sigma-Aldrich, HS120758-1EA). Place the sealing film containing the primary kidney organoid droplets in a 37°C cell culture incubator and incubate for 12 min (10-15 min is acceptable). After the CdECM droplets have solidified, wash the cells with third differentiation medium into the cell culture plate, add third differentiation medium to 3 mL per well, and incubate at 37°C.

[0041] Subsequently, half of the medium was replaced with the third differentiation medium every other day. Microscopic observation was performed to observe the continued differentiation of the kidney organoids into more complex cell spheroids. After 7 days of culture (denoted as D8+7), vascularized human kidney organoids were successfully induced. A flowchart of step 3 is shown below. Figure 2 As shown.

[0042] Test case This experiment tested the induction effect of the 3D culture method for human kidney organoids provided in Example 1 using in vitro and in vivo experiments. The optimal culture conditions and time were also screened. Specific experimental methods and results are as follows: 1. To investigate the optimal CdECM concentration for culturing kidney organoids: Based on the method provided in Example 1, three different CdECM concentrations were set in step 3, namely 0.2% (w / v), 0.5% (w / v) and 0.8% (w / v), and the remaining steps were performed in accordance with the description in Example 1. Human kidney organoid samples were collected and fixed in 4% paraformaldehyde (LEAGENE, DF0135) solution for 45 min. The solution was then replaced with 30% sucrose solution (Macklin, S818045) for dehydration for 24 h. The dehydrated samples were then embedded in OCT frozen section embedding medium (SAKURA, 4583), frozen at -80°C until the embedding medium solidified, and frozen at -20°C to 16 μm sections. Immunofluorescence staining was performed. Human CD31 (PECAM1) antibody (R&D Systems, AF806) was used to characterize mature vascular endothelium, PODXL antibody (R&D Systems, MAB1658) was used to characterize glomerular podocytes, and CD146 antibody (Abcam, AB75769) was used to characterize vascular endothelium before CD31 maturation. The results are as follows: Figure 3 As shown in A in the diagram.

[0043] Depend on Figure 3 As shown in A, a 0.8% (w / v) concentration of CdECM can better solidify and support the continued differentiation of kidney organoids at 37°C.

[0044] 2. Investigating the differentiation culture conditions after CdECM encapsulation Based on the method provided in Example 1, the culture medium system in step 3 was set into two groups: Stage II medium without FBS and Stage II medium containing 1.5% (v / v) FBS. All other steps were performed according to the description in Example 1. Human kidney organoid samples were collected, fixed with 4% paraformaldehyde for 45 min, dehydrated with 30% sucrose solution for 24 h, embedded in OCT frozen section embedding medium, and frozen at -80℃ until the embedding medium solidified. Frozen sections were prepared and immunofluorescence staining was performed. Human CD31 antibody characterized mature vascular endothelial differentiation, PODXL antibody characterized glomerular podocytes, and CD146 antibody characterized vascular endothelium before CD31 maturation. The results are as follows: Figure 3 As shown in B in the diagram.

[0045] Angio Tool was used to quantify the percentage of vessel area, vessel connectivity density, and average vessel length using immunofluorescence mapping. qPCR was used to quantify the expression level of CD31 (PECAM1) in the same batch of samples; the results are shown below. Figure 3 As shown in C.

[0046] Depend on Figure 3 As shown in B and C, after CdECM encapsulation, using Stage II medium supplemented with FBS can better promote vascular endothelial cell differentiation.

[0047] 3. Investigating the optimal differentiation time after CdECM encapsulation Based on the method provided in Example 1, the culture time in step 3 was set to 3, 5, and 7 days, respectively. Primary kidney organoids differentiated to day 8 were encapsulated in 0.8% (w / v) CdECM. All steps were performed according to the description in Example 1. Human kidney organoid samples were collected, fixed with 4% paraformaldehyde for 45 min, dehydrated with 30% sucrose solution for 24 h, embedded in OCT cryopreservation medium, and frozen at -80℃. Frozen sections were prepared and immunofluorescence staining was performed. Human CD31 cells represented vascular endothelial differentiation, and PODXL represented podocytes of the kidney organoids. The percentage of vascular area, vascular connectivity density, and average vascular length were quantified using the Angio Tool. The results are shown below. Figure 4 As shown.

[0048] Depend on Figure 4It was observed that primary kidney organoids encapsulated in CdECM continued to differentiate and their vascular networks gradually became richer. However, bright-field microscopy revealed that extending the culture time in CdECM to day 9 resulted in a large number of off-target differentiated cells; these cells lacked kidney organoid structures, and some exhibited rhythmic beating similar to cardiac muscle, indicating that they were no longer the cell types of kidney tissue. The optimal culture time in CdECM was day 7, i.e., a total of 8+7 days for kidney organoid differentiation (D8+7), with fewer off-target cells and a richer vascular network. Immunofluorescence imaging showed that the green fluorescent vascular marker CD31 increased with increasing culture time in CdECM. Quantitative results indicated a slight decrease on day 5 because the primary kidney organoids rapidly proliferated and differentiated after being encapsulated in CdECM, increasing the organoid area and relatively decreasing the proportion of vascular area. Combining immunofluorescence images and quantitative results, the vascular area, vascular connectivity density, and average vascular length of kidney organoids encapsulated in CdECM all reached the optimal vascularization effect on day 7 of CdECM culture, i.e., day 8+7 of kidney organoid differentiation.

[0049] 4. Investigate the effects of different coating matrices on vascularization of human kidney organoids. Based on the method provided in Example 1, the coating matrix in step 3 was set as follows: commercially available collagen (Corning, 354249) and mouse base gel Matrigel (BioCoat, 354277) were used as control groups, and porcine renal cortical extracellular matrix (CdECM) was used as the experimental group. CdECM was diluted to a concentration of 0.8% (w / v) with third-order differentiation medium, i.e., 100 mg of CdECM dissolved in third-order differentiation medium to a total volume of 12.5 mL of gel solution (which can also be denoted as 8 mg / mL). Collagen, as a control, typically ranged from 8 to 11 mg / mL depending on the batch number purchased, and was diluted to 8 mg / mL with third-order differentiation medium. Matrigel, purchased at different batches, ranged from 8 to 12 mg / mL and was diluted to 8 mg / mL with third-order differentiation medium. The remaining steps were performed as described in Example 1. Human kidney organoid samples were collected and first recorded under bright field, then fixed with 4% paraformaldehyde for 45 min, dehydrated with 30% sucrose solution for 24 h, embedded in OCT embedding medium, and frozen at -80℃. Frozen sections were prepared and immunofluorescence staining was performed. Human CD31 antibody was used to characterize vascular endothelial differentiation, PODXL to characterize podocytes of kidney organoids, and CD146 to characterize vascular endothelium before CD31 maturation. Angiotool was used to quantify the percentage of vascular area, vascular connectivity density, and average vascular length. qPCR was used to quantify the expression level of CD31 (PECAM1) in the same batch of samples. The results are shown below. Figure 5 As shown.

[0050] Depend on Figure 5 It can be seen that, by comparing the differentiation effects of different matrix gels on kidney organoids using bright field and immunofluorescence, the vascular endothelium encapsulated in CdECM is more abundant, and a large number of vascular endothelial cells invade the glomerular podocytes, similar to the vascular-encapsulating structures of the glomeruli in vivo. Figure 5 In the control group (B), the vascular area, vascular connectivity density, and average vascular length of the human kidney organoids encapsulated by CdECM were all higher than those in the control group, and the expression of the vascular maturation marker CD31 (PECAM1) was also higher than that in the control group. Figure 5 In the C), immunofluorescence staining showed obvious vascular ingress into the glomerulus. According to three-dimensional imaging analysis, in images with a 2 μm difference between each layer, the vascular precursor marker CD146 (MCAM) arrived at the center of the glomerular podocytes before the vascular maturation marker CD31 (PECAM1), which is consistent with the process of vascular precursors being generated before mature vascular endothelium. Figure 5 (D in the image). The changes in the continuous monolayer images show that the vascular maturation marker CD31 (PECAM1) gradually infiltrates into the red fluorescently labeled glomerular podocytes. Furthermore, in the central locations between podocytes where CD31 has not yet reached, the vascular precursor marker CD146 (MCAM) is expressed. This indicates that the vascularization process of the kidney organoids encapsulated in the CdECM involves angiogenesis, a process similar to the formation of new blood vessels through the proliferation and migration of existing vascular endothelial cells in vivo.

[0051] 5. Validation of transplantation of vascularized human kidney organoids differentiated in CdECM into the subcapsular region of mouse kidneys: Eight to ten primary kidney organoids encapsulated in CdECM were transplanted under the renal capsule of immunodeficient mice. One week later, the transplanted kidneys were dissected and removed. After fixation with 4% paraformaldehyde for 24 h and dehydration with 30% sucrose solution for 48 h, the kidneys were embedded in OCT cryopreservation medium and frozen at -80°C to prepare frozen sections.

[0052] A portion of the slide was stained with hematoxylin and eosin (HE) using an HE staining kit (Beyotime, C0105M). The specific method was as follows: first, hematoxylin staining solution was added to the slide to cover the sample and stain for about 1 minute. After rinsing with tap water and soaking for 15 minutes, eosin staining solution was added to cover the sample for 30 seconds. After rinsing with tap water, the slide was soaked in tap water for 2-5 minutes. The stained slide was then passed sequentially through 30% ethanol for 2-3 seconds, 50% ethanol for 2-3 seconds, 70% ethanol for 2-3 seconds, 80% ethanol for 2-3 seconds, 90% ethanol for 2-3 seconds, anhydrous ethanol (Cologne, KL-11011) for 2-3 seconds, anhydrous ethanol for 1 minute, xylene (CNW, CFEQ-4-120173) for 2 minutes, and xylene for 2 minutes. A coverslip was then placed on top, and the slide was air-dried in a fume hood before being photographed using a slide scanner. The results are shown below. Figure 6 As shown in A in the diagram.

[0053] Another portion of the slides was subjected to immunofluorescence staining to observe the structure of the human kidney organoid at the transplantation site. Human CD31 antibody characterized the differentiation of mature vascular endothelium, PODXL antibody characterized glomerular podocytes, and CD146 antibody characterized the vascular endothelium before CD31 maturation. The results are as follows: Figure 6 As shown in B in the diagram.

[0054] Depend on Figure 6 HE staining revealed the presence of glomerular podocytes and renal tubules at the transplant site, while immunofluorescence staining showed that vascular endothelium had invaded the glomerular podocytes, forming a structure similar to the in vivo glomerular-enclosed blood vessel structure. The transplant results indicate that the transplanted blood vessels can be correctly located within the glomerular podocyte clusters (labeled with PODXL) in vivo, forming a structure similar to the in vivo podocyte-enclosed capillary structure.

[0055] Existing methods for promoting renal organoid vascularization using ECM involve layering porcine kidney dECM at the bottom of a culture plate. However, this only allows cells to contact the dECM from the bottom, resulting in incomplete signal reception. Adding the angiogenic factor VEGF is necessary to enhance the interaction between the renal organoids and the dECM, thereby improving vascular endothelial differentiation efficiency. Alternatively, simply adding a low concentration of porcine kidney dECM to gelatin and fibrinogen to encapsulate renal progenitor cells does not achieve vascularization in the resulting renal organoids. The method provided by this invention not only offers low material costs and ease of standardization, but the 3D encapsulation also provides renal organoids with physical support closer to the in vivo environment. This facilitates the reception of signaling factors from various directions within the decellularized porcine kidney matrix during organoid differentiation, thus promoting more efficient vascularization and differentiation maturation. In vitro and in vivo transplantation validation results demonstrate that this 3D culture method can stably obtain vascularized human renal organoids with rich structures both in vitro and in vivo, providing a stable in vitro and in vivo model for disease model construction, drug screening, and toxicity testing.

[0056] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A 3D culture method of vascularized human kidney organoids, characterized by, The 3D culture method of the human kidney organoid comprises the following steps: S1: Take the porcine renal cortical part for decellularization treatment to obtain a decellularized matrix, freeze-dry and then digest and resuspend to obtain a porcine renal acellular extracellular matrix gel solution; S2: Resuspend and induce differentiation of human pluripotent stem cells using a first differentiation medium for 36-60 h, replace half of the medium and continue to culture for 12-36 h to obtain a blastocyst; then replace the culture system with a second differentiation medium for 3-7 days, and replace half of the medium every other day, thereby obtaining a primary human kidney organoid; S3: Mix the primary human kidney organoid obtained in step S2 and the porcine renal acellular extracellular matrix gel solution obtained in step S1, and stand for 10-15 min, then transfer to a cell culture plate for culture for 6-8 days, and replace half of the medium every other day, thereby obtaining a vascularized human kidney organoid.

2. The 3D culture method of vascularized human kidney organoids according to claim 1, characterized in that, The decellularization treatment in step S1 comprises the following steps: A1: Chop the porcine renal cortical part, rinse with ddH2O, extract with 2%-4% Triton X-100 aqueous solution for 10-15 h, then transfer to 2%-6% sodium deoxycholate aqueous solution for 20-30 h of room temperature shaking for decellularization, and finally rinse 2-4 times in ddH2O, which is one cycle, and two cycles are performed in total and the decellularized cortical fragments are collected; A2: Add pepsin to the decellularized cortical fragments obtained in step A1 for room temperature digestion for 6-24 h to obtain a decellularized matrix hydrogel, and the mass ratio of pepsin to cortical fragments is 1: (8-12).

3. The 3D culture method of vascularized human kidney organoids according to claim 1, characterized in that, The resuspension solvent in step S1 comprises 10×PBS buffer.

4. The 3D culture method of vascularized human kidney organoids according to claim 1, characterized in that, The first differentiation medium in step S2 comprises a basic medium and additives, the basic medium comprises IMDM medium, F12 medium and PFHM-II medium, and the additives in the first differentiation medium comprise the following components at the following final concentrations: 2.2-2.8 mg / mL bovine serum albumin, 0.05-0.2% (v / v) insulin-transferrin-selenium-aminethanol, 0.002%-0.005% 1-thioglycerol, 0.03-0.07 mg / mL ascorbic acid-2-phosphate, 0.5-2% (v / v) Glutamax, 0.5-2% (v / v) lipid concentrate, 1.7-3.3 mg / mL polyvinyl alcohol, 1-10 μM GSK-3 inhibitor, 2-10 μM ROCK inhibitor and 0.7-1.3 mM β-mercaptoethanol.

5. The 3D culture method of vascularized human kidney organoids according to claim 1, characterized in that, The second differentiation medium in step S2 comprises a basic medium and additives, the basic medium comprises DMEM medium, and the additives in the second differentiation medium comprise the following components at the following final concentrations: 10%-20% KnockOut serum replacement, 0.5-2% (v / v) non-essential amino acid solution, 0.5-2% (v / v) Glutamax, 0.5-2% (v / v) HEPES and 1.7-3.3 mg / mL polyvinyl alcohol.

6. The 3D culture method of vascularized human kidney organoids according to claim 1, characterized in that, The porcine kidney acellular extracellular matrix gel solution in step S3 is diluted to a concentration of 0.6-1% (w / v) with a third differentiation medium before being mixed with the primary human kidney organoids.

7. The 3D culture method of vascularized human kidney organoids according to claim 6, characterized in that, The third differentiation medium is the second differentiation medium added with 1-3% fetal bovine serum.

8. The 3D culture method of vascularized human kidney organoids according to claim 6, characterized in that, 15 μL of the diluted porcine kidney acellular extracellular matrix gel solution is added for every 5-10 primary human kidney organoids.

9. The vascularized human kidney organoid prepared by the 3D culture method of the vascularized human kidney organoid according to any one of claims 1-8.

10. The vascularized human kidney organoid of claim 9 for use in disease modeling, drug screening, or toxicity testing.

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