Pancreas islet organ and culture method thereof

Abstract

The invention relates to the field of cell culture, and discloses a pancreatic islet organ and a culture method thereof, the method realizes differentiation of human induced pluripotent stem cells to functional pancreatic islet organs through sequential stage conversion, and the method comprises the following steps: in a two-dimensional culture stage, pancreatic progenitor cells are induced by adopting a specific factor combination; in the three-dimensional maturation stage, the cells are promoted to be self-assembled into a pancreas islet-like structure through a suspension culture technology. The diameter of the cultured islet organ is 100-200 [mu] m, the islet organ contains alpha, beta and delta cells, the proportion of the alpha, beta and delta cells is close to that of natural islet, and the insulin secretion amount under the stimulation of 16.7 mM glucose reaches 2.5 times or above of the basic level. According to the invention, the serum-free chemically defined culture medium is adopted to replace animal source components, and an optimized suspension culture process is combined, so that the technical problems of nonuniform size, insufficient functional activity, strong matrigel dependence and the like of organoids in a traditional method are solved, and a standardized and large-scale cell source is provided for diabetes treatment.

Description

Technical Field

[0001] This invention relates to the field of cell culture, and more specifically to a pancreatic islet organoid and its culture method. Background Technology

[0002] Diabetes mellitus, a global metabolic disease, hinges on restoring the function of pancreatic beta cells. Traditional insulin injections cannot reverse disease progression, while islet transplantation is hampered by donor shortages and immune rejection. In recent years, islet organoid technology based on human pluripotent stem cells (hPSCs) has emerged as a crucial approach to addressing this challenge. These organoids, cultured in three dimensions to mimic the structure of natural islets, possess functions such as insulin secretion and glucose response, offering new possibilities for cell replacement therapy in diabetes.

[0003] However, existing technologies still face the following challenges:

[0004] 1. The contradiction between expansion and maturation: Traditional 2D culture can achieve cell expansion but low functional maturity, such as weak expression of NKX6.1. 3D culture starting from embryoids goes through an expansion stage, resulting in insufficient size uniformity.

[0005] 2. Serum dependence and heterogeneity: Serum-containing culture media have batch-to-batch variability and animal-derived contamination risks, and the coefficient of variation (CV) of organoid diameter prepared by existing processes is generally >30%, which is difficult to meet the needs of clinical standardization.

[0006] 3. Challenges in large-scale production: Most solutions rely on static culture or complex equipment, such as microfluidic chips, which makes it difficult to achieve high-throughput and stable industrial-grade production. Summary of the Invention

[0007] The purpose of this invention is to provide a pancreatic islet organoid and its culture method, which solves the technical problems in the prior art such as uneven organoid size, imbalanced cell type ratio, insufficient functional activity, strong dependence on matrix gel and high risk of animal origin by using a culture system that couples sequential two-dimensional expansion with three-dimensional maturation.

[0008] To achieve the aforementioned technical objectives and effects, this invention discloses a method for culturing pancreatic islet organoids. This method is based on the induced differentiation of human induced pluripotent stem cells, and includes at least the following steps:

[0009] S1. Human induced pluripotent stem cells were seeded into matrix gel-coated well plates and induced to form endoderm cells using a deterministic endoderm induction medium containing Activin A, CHIR99021 and BMP4. The medium was completely changed daily.

[0010] S2. Continue culturing the endoderm cells obtained in S1, replacing them with a hindgut induction medium containing KGF, SB431542 and Wnt-C59, changing the medium completely every day to induce the formation of hindgut cells.

[0011] S3. Continue culturing the hindgut cells obtained in S2, and replace them with pancreatic progenitor cell induction medium containing retinoic acid, LDN193189 and Sant1. Change the medium completely every day to induce the formation of pancreatic progenitor cells.

[0012] S4. After digesting the pancreatic progenitor cells obtained in S3 with hydrolytic enzymes, the cells were transferred to ultra-low adhesion multi-well plates and cultured for 3D using endocrine precursor induction medium containing EGF, TPB and nicotinamide. The medium was completely changed daily to induce the formation of pancreatic endocrine precursor cells.

[0013] S5. The pancreatic endocrine precursor cells obtained in S4 were transferred to ultra-low adhesion multi-well plates and cultured in 3D suspension using islet cell induction medium containing ALK5 inhibitor II, T3 and ISX9. The medium was changed every 48 hours to induce the formation of immature islet β cells.

[0014] S6. Continue culturing the immature pancreatic β cells obtained in S5, replacing the medium with a maturation medium containing Forskolin, zinc ions and N-acetylcysteine, changing the medium every 48 hours, and finally obtaining mature pancreatic β cells.

[0015] Preferably, step S1 includes at least:

[0016] S11. Induction culture was first performed using a deterministic endoderm induction medium containing 100 ng / ml Activin A, 3 μM CHIR99021, 30 ng / ml BMP4, 10 μM Y27632 and B27 supplement without insulin.

[0017] S12, then replace with maintenance medium containing 75 ng / ml Activin A and insulin-free B27 supplement, change the medium completely daily to induce the formation of endoderm cells.

[0018] The hindgut induction culture medium includes at least the following:

[0019] The MCDB131 basal medium, used as the basal solution, was supplemented with 0.75 mg / ml D-glucose, 1% GlutaMAX, 1% penicillin and antibiotic, 1% B27 supplement, 0.25 mM ascorbate diphosphate, 50 ng / ml KGF, 5 μM SB431542, and 100 nM Wnt-C59.

[0020] The step S4, prior to "digesting the pancreatic progenitor cells obtained in S3 with hydrolytic enzymes," also includes:

[0021] After step S3 is completed, the proportion of positive cells needs to be confirmed by PDX1 immunofluorescence staining to be ≥75% to ensure the uniformity of the cell population before proceeding to step S4. If the proportion of positive cells is <75%, the induction time is extended. If the proportion of positive cells is still <75% after 3 days, differentiation is restarted.

[0022] Specifically, step S4, "digesting the pancreatic progenitor cells obtained in S3 with hydrolytic enzymes," includes at least the following:

[0023] After washing the cells twice with high-glucose DMEM medium, add hydrolytic enzyme and mild digestion solution to digest for 10-15 min, and then observe.

[0024] If the cells become round, wrinkled, and shiny, and a small number of cells can be seen to detach from the matrix when shaken, the digestion solution can be aspirated to stop digestion, and the cells can be resuspended for subsequent operations.

[0025] If a large number of cells float, digestion is terminated, and the cells are collected by centrifugation again. They are then cultured in islet cell induction medium supplemented with anti-stress agents and gradually transitioned to step S4.

[0026] The endocrine precursor induction culture medium includes at least the following:

[0027] The MCDB131 basal medium, used as the basal solution, was supplemented with 1.5 mg / ml D-glucose, 1% B27 supplement, 1% penicillin and antibiotic, 1% GlutaMAX, 0.25 mM ascorbate diphosphate, 100 ng / ml EGF, 0.2 μM TPB, 10 mM nicotinamide, and 0.25 μM Sant-1.

[0028] The pancreatic islet cell induction culture medium includes at least the following:

[0029] The MCDB131 basal medium, used as the basal solution, was supplemented with 1.5 mg / ml D-glucose, 1% B27 supplement, 1% penicillin and antibiotic, 1% GlutaMAX, 0.25 mM ascorbate diphosphate, 10 μM ALK5 inhibitor II, 0.3 μM DN193189, 1 μM T3, 10 μM ISX9, 10 μg / ml heparin, 0.1 μM γ-secretase inhibitor Xxi, 100 nM MWnt-C59, and 10 μM Y27632.

[0030] The maturation culture medium includes at least the following:

[0031] The MCDB131 basal medium, used as the basal solution, contains 1.5 mg / ml D-glucose, 1% B27 supplement, 1% penicillin and antibiotic, 1% GlutaMAX, 0.25 mM ascorbate diphosphate, 10 μM ALK5 inhibitor II, 0.5 μM R428, 1 μMT3, 10 μM forskolin, 10 μg / ml heparin, 10 μM zinc ions, and 2 mM N-acetylcysteine.

[0032] The present invention also discloses a pancreatic organoid, which is cultured using the above-described method. The cultured organoid has a regular spherical structure with a diameter of 100-200 μm, clear and complete edges, and a smooth surface without protrusions. Under a bright-field microscope, a dense and uniform cell cluster structure can be seen. Furthermore, the organoid contains functional α, β, and δ cells in proportions close to those of natural human islets and exhibits typical glucose concentration-dependent insulin secretion characteristics.

[0033] The present invention has the following beneficial effects:

[0034] 1. By implementing a time-sequential phase transition, dynamic coupling between two-dimensional amplification and three-dimensional maturation is achieved, resolving the contradiction between amplification efficiency and functional maturation in traditional methods, and ultimately obtaining high-purity pancreatic islet organoids with glucose responsiveness.

[0035] 2. Serum-free chemically defined culture medium is used to completely avoid the potential risks of animal-derived components, while ensuring cell survival rate and differentiation stability to meet the needs of clinical applications.

[0036] 3. By replacing traditional matrix gel embedding with suspension culture technology, the number of operation steps and reagent costs are significantly reduced, while the uniformity and reproducibility of organoids are improved, which facilitates large-scale production and downstream applications. Attached Figure Description

[0037] Figure 1 This is a flowchart of the present invention.

[0038] Figure 2 This is a bright-field illustration of pancreatic progenitor cells in Example 2 of the present invention.

[0039] Figure 3 This is a diagram showing PDX1 staining of pancreatic progenitor cells in Example 2 of the present invention.

[0040] Figure 4 This is a bright-field illustration of a mature pancreatic islet organoid in Embodiment 3 of the present invention.

[0041] Figure 5 The image shows the fluorescence of pancreatic β-cell markers NKX6.1 and INS after the pancreatic organoids mature in Example 3 of this invention, where NKX6.1 is marked in red, INS in green, and DAPI in blue.

[0042] Figure 6 The image shows the fluorescence of INS, a marker for pancreatic β-cells, and GCG, a marker for pancreatic α-cells, after the pancreatic organoids mature in Example 3 of this invention. GCG is labeled in red, INS in green, and DAPI in blue.

[0043] Figure 7 The image shows fluorescence of INS (indicating the marker of pancreatic β-cells) and SST (indicating the marker of pancreatic δ-cells) after the pancreatic organoids mature in Example 3 of this invention, wherein SST is marked in red, INS in green, and DAPI in blue.

[0044] Figure 8 This is a diagram illustrating the static glucose tolerance test results of pancreatic islet organoids after maturation in Example 3 of the present invention. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments.

[0046] Example 1

[0047] like Figure 1 As shown, this invention discloses a method for culturing pancreatic islet organoids. The method is based on the induced differentiation of human induced pluripotent stem cells, and includes:

[0048] S1. Human induced pluripotent stem cells were seeded into matrix gel-coated well plates and induced to form endoderm cells using a deterministic endoderm induction medium containing Activin A, CHIR99021 and BMP4. The medium was completely changed daily.

[0049] S2. Continue culturing the endoderm cells obtained in S1, replacing them with a hindgut induction medium containing KGF, SB431542 and Wnt-C59, changing the medium completely every day to induce the formation of hindgut cells.

[0050] S3. Continue culturing the hindgut cells obtained in S2, and replace them with pancreatic progenitor cell induction medium containing retinoic acid, LDN193189 and Sant1. Change the medium completely every day to induce the formation of pancreatic progenitor cells.

[0051] S4. After digesting the pancreatic progenitor cells obtained in S3 with hydrolytic enzymes, the cells were transferred to ultra-low adhesion multi-well plates and cultured for 3D using endocrine precursor induction medium containing EGF, TPB and nicotinamide. The medium was completely changed daily to induce the formation of pancreatic endocrine precursor cells.

[0052] S5. The pancreatic endocrine precursor cells obtained in S4 were transferred to ultra-low adhesion multi-well plates and cultured in 3D suspension using islet cell induction medium containing ALK5 inhibitor II, T3 and ISX9. The medium was changed every 48 hours to induce the formation of immature islet β cells.

[0053] S6. Continue culturing the immature pancreatic β cells obtained in S5, replacing the medium with a maturation medium containing Forskolin, zinc ions and N-acetylcysteine, changing the medium every 48 hours, and finally obtaining mature pancreatic β cells.

[0054] The specific cultivation time for each step depends on the actual cultivation conditions. (See attached example.) Figure 1 This is for reference only for feasibility verification and is not intended to limit the technology of this embodiment, nor is it intended to limit the technology of this invention.

[0055] Specifically, step S1 includes:

[0056] S11. Induction culture was first performed using a deterministic endoderm induction medium containing 100 ng / ml Activin A, 3 μM CHIR99021, 30 ng / ml BMP4, 10 μM Y27632 and B27 supplement without insulin.

[0057] S12, then replace with maintenance medium containing 75 ng / ml Activin A and insulin-free B27 supplement, change the medium completely daily to induce the formation of endoderm cells.

[0058] The hindgut induction culture medium includes:

[0059] The MCDB131 basal medium, used as the basal solution, was supplemented with 0.75 mg / ml D-glucose, 1% GlutaMAX, 1% penicillin and antibiotic, 1% B27 supplement, 0.25 mM ascorbate diphosphate, 50 ng / ml KGF, 5 μM SB431542, and 100 nM Wnt-C59.

[0060] The endocrine precursor induction culture medium includes:

[0061] The MCDB131 basal medium, used as the basal solution, was supplemented with 1.5 mg / ml D-glucose, 1% B27 supplement, 1% penicillin and antibiotic, 1% GlutaMAX, 0.25 mM ascorbate diphosphate, 100 ng / ml EGF, 0.2 μM TPB, 10 mM nicotinamide, and 0.25 μM Sant-1.

[0062] The pancreatic islet cell induction culture medium includes:

[0063] The MCDB131 basal medium, used as the basal solution, was supplemented with 1.5 mg / ml D-glucose, 1% B27 supplement, 1% penicillin and antibiotic, 1% GlutaMAX, 0.25 mM ascorbate diphosphate, 10 μM ALK5 inhibitor II, 0.3 μM DN193189, 1 μM T3, 10 μM ISX9, 10 μg / ml heparin, 0.1 μM γ-secretase inhibitor Xxi, 100 nM MWnt-C59, and 10 μM Y27632.

[0064] The maturation culture medium includes:

[0065] The MCDB131 basal medium, used as the basal solution, contains 1.5 mg / ml D-glucose, 1% B27 supplement, 1% penicillin and antibiotic, 1% GlutaMAX, 0.25 mM ascorbate diphosphate, 10 μM ALK5 inhibitor II, 0.5 μM R428, 1 μMT3, 10 μM forskolin, 10 μg / ml heparin, 10 μM zinc ions, and 2 mM N-acetylcysteine.

[0066] This invention achieves highly efficient and targeted differentiation of human induced pluripotent stem cells into functional pancreatic islet organoids through an innovative time-sequential stage transition technology. The method employs a dynamic coupling culture strategy of two-dimensional expansion and three-dimensional maturation. In the two-dimensional stage, by precisely controlling the combination of key factors such as Activin A and CHIR99021, and confirming the proportion of positive cells (≥75%) using PDX1 immunofluorescence staining, the homogeneity of the cell population is ensured, laying a high-quality cellular foundation for subsequent three-dimensional differentiation. In the three-dimensional stage, suspension culture technology promotes cell self-assembly, and factors such as EGF and TPB are used to induce the formation of islet-like masses with physiological structures.

[0067] Furthermore, the culture system of this invention uses a completely serum-free, chemically defined culture medium. By scientifically proportioning insulin-free B27 and normal B27 supplements, it avoids the potential risks of animal-derived components while ensuring cell survival and differentiation stability. Simultaneously, the innovative suspension culture process replaces the traditional matrix gel embedding method, simplifying the operation process, reducing production costs, and significantly improving the uniformity and reproducibility of organoids.

[0068] Example 2

[0069] This embodiment is based on the pancreatic islet organoid culture method proposed in Embodiment 1, further refining the process of converting two-dimensional culture to three-dimensional culture, and discussing the relevant processes.

[0070] The step S4, prior to "digesting the pancreatic progenitor cells obtained in S3 with hydrolytic enzymes," also includes:

[0071] After step S3 is completed, the proportion of positive cells needs to be confirmed by PDX1 immunofluorescence staining to be ≥75% to ensure the uniformity of the cell population before proceeding to step S4. If the proportion of positive cells is <75%, the induction time is extended. If the proportion of positive cells is still <75% after 3 days, differentiation is restarted.

[0072] like Figure 2 As shown in the bright-field plot, pancreatic progenitor cells clearly exhibit typical cell morphology characteristics after S3 induction. Two distinct states are visible in the image: the central region shows clearly defined cell clusters arranged in tightly packed, irregular polygonal patterns, consistent with the epithelial-like morphology of pancreatic progenitor cells; the peripheral, dispersed individual cells are larger, with translucent cytoplasm and smooth edges, suggesting they may be undifferentiated remnants of precursor cells. The clean, debris-free background matrix indicates healthy cell condition during culture.

[0073] like Figure 3 As shown in the image, the PDX1 immunofluorescence staining of pancreatic progenitor cells clearly demonstrates the molecular characteristics of cells after S3 induction. Numerous round / quasi-round cells exhibit bright green fluorescence, with the fluorescence primarily localized in the nucleus region, indicating the nuclear expression pattern of the PDX1 protein, a typical molecular marker of pancreatic progenitor cells.

[0074] Specifically, step S4, "digesting the pancreatic progenitor cells obtained in S3 with hydrolytic enzymes," includes at least the following:

[0075] After washing the cells twice with high-glucose DMEM medium, add hydrolytic enzyme and mild digestion solution to digest for 10-15 min, and then observe.

[0076] If the cells become round, wrinkled, and shiny, and a small number of cells can be seen to detach from the matrix when shaken, the digestion solution can be aspirated to stop digestion, and the cells can be resuspended for subsequent operations.

[0077] If a large number of cells float, digestion is terminated, and the cells are collected by centrifugation again. They are then cultured in islet cell induction medium supplemented with anti-stress agents, and the process is gradually transitioned to step S4. A feasible implementation procedure for this step is as follows:

[0078] First, transfer the cell suspension to a 15mL or 50mL centrifuge tube, centrifuge at 120g for 5 minutes to remove the supernatant, and then resuspend the cells in stage 4 culture medium containing 1x CEPT anti-stress supplement.

[0079] The cell suspension was then transferred to a sample loading tank or a 10cm culture dish, and seeded into 96-well ultra-low adhesion U plates at a volume of 100μL / well using a multi-channel pipette. After sealing with sealing film, the plates were centrifuged at 1500rpm for 5 minutes in a horizontal centrifuge to allow the cells to aggregate. The cells were then transferred to a 37°C, 5% CO2 incubator and incubated overnight.

[0080] The following day, the CEPT-containing medium was completely aspirated and replaced with 100 μL / well of fresh S4 stage medium without CEPT stress inhibitor. On the third day, the resulting cell clusters were transferred to ultra-low adhesion six-well plates, and an appropriate amount of S4 stage medium was added. The cells were then cultured in suspension at 37°C and 5% CO2 at 90 rpm to complete the transition from digestion to three-dimensional culture.

[0081] The specific cultivation, digestion, separation, and transfer steps described above are only described as feasible steps and are not intended as further technical limitations of the embodiments of this application, nor are they technical limitations of this invention.

[0082] Example 3

[0083] Based on the technical solutions and details proposed in Examples 1 and 2, this embodiment cultivates and differentiates pancreatic islet organoids with specific biological functions, such as... Figure 4 As shown, the cultured organoids have regular spherical structures with a diameter of 100-200 μm, clear and complete edges, and smooth surfaces without protrusions. Under a bright-field microscope, a dense and uniform cell cluster structure can be seen.

[0084] And as Figure 5-7 As shown, Figure 5 This image shows the fluorescence of NKX6.1 and INS, markers for pancreatic β-cells after the islet organoids mature in Example 3 of this invention. NKX6.1 is labeled in red, INS in green, and DAPI in blue. The image shows that red NKX6.1 and green INS fluorescence are highly co-localized in the cytoplasm, forming a distinct yellow superimposed area, confirming the co-expression characteristics of β-cell-specific markers. The blue DAPI fluorescence clearly shows the uniform distribution of the cell nuclei, indicating a healthy cell condition.

[0085] Figure 6 This image shows fluorescence illustrations of INS (indicating markers for pancreatic β cells) and GCG (indicating markers for pancreatic α cells) after the islet organoids mature in Example 3 of this invention. GCG is labeled red, INS is labeled green, and DAPI is labeled blue. The image shows that green INS-positive cells (β cells) and red GCG-positive cells (α cells) form a typical adjacent distribution pattern, consistent with the spatial arrangement of α-β cells in natural islets. The blue DAPI-labeled cells have a moderate nuclear density, with no obvious nuclear pyknosis or fragmentation observed.

[0086] Figure 7This image shows fluorescence illustrations of INS (indicating β-cell markers) and SST (indicating δ-cell markers) from the mature pancreatic islet organoids of Example 3 of this invention. SST is labeled in red, INS in green, and DAPI in blue. The red SST-positive cells (δ-cells) are scattered throughout the image, forming functional units with the green INS-positive cells, thus reproducing the paracrine regulatory structure of β-δ cells in natural islets.

[0087] To further verify the differentiated pancreatic islet organoids, such as Figure 8 As shown in the figure, the static glucose tolerance test results of pancreatic organoids after maturation clearly demonstrate their functional maturity. The bar chart data shows that under basal glucose concentration (2.8 mM), insulin secretion was 4.53 ng / million cells (gray bar); when the glucose concentration increased to 16.7 mM, the secretion significantly increased to 18.8 ng / million cells (red bar), reaching 4.15 times the basal level; under 30 mM KCl stimulation (blue bar), the secretion further increased to 35.2 ng / million cells, exhibiting typical calcium channel-dependent secretion characteristics.

[0088] These results demonstrate that the pancreatic islet organoids obtained by this invention not only possess glucose-dependent insulin secretion capacity, but their stimulation index (GSI = 2.5) is also close to that of primary human islets reported in the literature (GSI = 3.0-4.0), confirming their excellent functional maturity. Particularly noteworthy is the absolute secretion volume after high glucose stimulation reaching 35.2 ng / million cells, equivalent to 60-70% of the secretory capacity of adult pancreatic β cells, fully meeting the application requirements for diabetes treatment research. These experimental results corroborate the aforementioned immunofluorescence findings on β cells, jointly confirming that the culture method of this invention can obtain physiologically functional pancreatic islet organoids.

[0089] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for culturing pancreatic islet organoids, characterized in that, The method for culturing pancreatic islet organoids is based on the induction and differentiation of human induced pluripotent stem cells, and the method includes at least the following: S1. Human induced pluripotent stem cells were seeded into matrix gel-coated well plates and induced to form endoderm cells using a deterministic endoderm induction medium containing Activin A, CHIR99021 and BMP4. The medium was completely changed daily. S2. Continue culturing the endoderm cells obtained in S1, replacing them with a hindgut induction medium containing KGF, SB431542 and Wnt-C59, changing the medium completely every day to induce the formation of hindgut cells. S3. Continue culturing the hindgut cells obtained in S2, and replace them with pancreatic progenitor cell induction medium containing retinoic acid, LDN193189 and Sant1. Change the medium completely every day to induce the formation of pancreatic progenitor cells. S4. After digesting the pancreatic progenitor cells obtained in S3 with hydrolytic enzymes, the cells were transferred to ultra-low adhesion multi-well plates and cultured for 3D using endocrine precursor induction medium containing EGF, TPB and nicotinamide. The medium was completely changed daily to induce the formation of pancreatic endocrine precursor cells. S5. The pancreatic endocrine precursor cells obtained in S4 were transferred to ultra-low adhesion multi-well plates and cultured in 3D suspension using islet cell induction medium containing ALK5 inhibitor II, T3 and ISX9. The medium was changed every 48 hours to induce the formation of immature islet β cells. S6. Continue culturing the immature pancreatic β cells obtained in S5, replacing the medium with a maturation medium containing Forskolin, zinc ions and N-acetylcysteine, changing the medium every 48 hours, and finally obtaining mature pancreatic β cells.

2. The method for culturing pancreatic islet organoids as described in claim 1, characterized in that, The S1 step includes at least the following: S11. Induction culture was first performed using a deterministic endoderm induction medium containing 100 ng / ml Activin A, 3 μM CHIR99021, 30 ng / ml BMP4, 10 μM Y27632 and B27 supplement without insulin. S12, then replace with maintenance medium containing 75 ng / ml Activin A and insulin-free B27 supplement, change the medium completely daily to induce the formation of endoderm cells.

3. The method for culturing pancreatic islet organoids as described in claim 1, characterized in that, The hindgut induction culture medium comprises at least: The MCDB131 basal medium, used as the basal solution, was supplemented with 0.75 mg / ml D-glucose, 1% GlutaMAX, 1% penicillin and antibiotic, 1% B27 supplement, 0.25 mM ascorbate diphosphate, 50 ng / ml KGF, 5 μM SB431542, and 100 nM Wnt-C59.

4. The method for culturing pancreatic islet organoids as described in claim 1, characterized in that, Before "digesting the pancreatic progenitor cells obtained in S3 with hydrolytic enzymes" in step S4, the following steps are also included: After step S3 is completed, the proportion of positive cells needs to be confirmed by PDX1 immunofluorescence staining to be ≥75% to ensure the uniformity of the cell population before proceeding to step S4. If the proportion of positive cells is <75%, the induction time is extended. If the proportion of positive cells is still <75% after 3 days, differentiation is restarted.

5. The method for culturing pancreatic islet organoids as described in claim 1, characterized in that, The step S4, "digesting the pancreatic progenitor cells obtained in S3 with hydrolytic enzymes," includes at least the following: After washing the cells twice with high-glucose DMEM medium, add hydrolytic enzyme and mild digestion solution to digest for 10-15 min, and then observe. If the cells become round, wrinkled, and shiny, and a small number of cells can be seen to detach from the matrix when shaken, the digestion solution can be aspirated to stop digestion, and the cells can be resuspended for subsequent operations. If a large number of cells float, digestion is terminated, and the cells are collected by centrifugation again. They are then cultured in islet cell induction medium supplemented with anti-stress agents and gradually transitioned to step S4.

6. The method for culturing pancreatic islet organoids as described in claim 1, characterized in that, The endocrine precursor induction culture medium includes at least: The MCDB131 basal medium, used as the basal solution, was supplemented with 1.5 mg / ml D-glucose, 1% B27 supplement, 1% penicillin and antibiotic, 1% GlutaMAX, 0.25 mM ascorbate diphosphate, 100 ng / ml EGF, 0.2 μM TPB, 10 mM nicotinamide, and 0.25 μM Sant-1.

7. The method for culturing pancreatic islet organoids as described in claim 1, characterized in that, The pancreatic islet cell induction culture medium includes at least: The MCDB131 basal medium, used as the basal solution, was supplemented with 1.5 mg / ml D-glucose, 1% B27 supplement, 1% penicillin and antibiotic, 1% GlutaMAX, 0.25 mM ascorbate diphosphate, 10 μM ALK5 inhibitor II, 0.3 μM LDN193189, 1 μM T3, 10 μM ISX9, 10 μg / ml heparin, 0.1 μM γ-secretase inhibitor Xxi, 100 nM Wnt-C59, and 10 μM Y27632.

8. The method for culturing pancreatic islet organoids as described in claim 1, characterized in that, The maturation culture medium includes at least: The MCDB131 basal medium, used as the basal solution, contains 1.5 mg / ml D-glucose, 1% B27 supplement, 1% penicillin and antibiotic, 1% GlutaMAX, 0.25 mM ascorbate diphosphate, 10 μM ALK5 inhibitor II, 0.5 μM R428, 1 μM T3, 10 μM forskolin, 10 μg / ml heparin, 10 μM zinc ions, and 2 mM N-acetylcysteine.

9. A pancreatic islet organoid, characterized in that, It is obtained by cultivation using the method described in any one of claims 1-8.

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