Method for increasing yield of pancreas islet organoids differentiated by pluripotent stem cells and improving stability

Abstract

The invention belongs to the technical field of biomedicine, and relates to a method for increasing the yield of pancreas islet organs differentiated by pluripotent stem cells and improving the stability. The method comprises the following steps: (1) inoculating pluripotent stem cells, and culturing by adopting a basic culture medium containing Y-27632; (2) changing the liquid, and continuously culturing by adopting a basic culture medium; (3) culturing for 1 day by using an S1D1 culture medium, and then culturing for 2-3 days by using an S1D2 culture medium; (4) changing the liquid, and culturing for 2-3 days by adopting the S2 culture medium; (5) changing the liquid, and culturing for 1-2 days by adopting an S3 culture medium; (6) changing the liquid, and culturing for 4-7 days by adopting a culture medium S4; (7) changing the liquid, and culturing for 3-10 days by adopting an S5 culture medium; and (8) carrying out cell sphere repolymerization, and culturing for 3-21 days by using an S6 culture medium to obtain the pancreas islet organ. The present invention increases the yield and differentiation efficiency at each differentiation stage and increases the scale yield at the cellular level.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a method for increasing the yield and stability of pancreatic islet organoids differentiated from pluripotent stem cells. Background Technology

[0002] There are several existing methods for differentiating pancreatic β cells from embryonic stem cells or induced pluripotent stem cells (iPSCs). These methods all use a combination of compounds to simulate the development process of pancreatic β cells in a specific order, differentiating embryonic stem cells sequentially into several steps: definitive endoderm, primitive gut tube, posterior foregut, pancreatic progenitors (PP cells), endocrine progenitors, and mature islet organoids.

[0003] Pancreatic islet cells, especially beta cells, are crucial for blood glucose control. For some diabetic patients, particularly type 1 diabetes, islet transplantation is an effective treatment. However, islet donors are extremely limited. Obtaining islet organoids for transplantation through stem cell differentiation is a promising alternative. However, to utilize stem cell-differentiated islet bodies as a transplant source, a large quantity of functional islet organoids, approximately 1 x 10^6, is required. 9 Cell count.

[0004] Existing differentiation technologies have several drawbacks: First, they involve long differentiation cycles and high costs; second, they have low differentiation efficiency (typically, β cells should account for more than 30% of the final pancreatic islet organoid cells). Furthermore, many differentiation methods are based on 2D iPSC cell culture for differentiating pancreatic progenitor cells and islet cells. While this method is simple to operate and easy to culture artificially, scaling up to large-scale production and cell expansion and differentiation is difficult, making it impossible to reduce production costs and increase yield. Moreover, some differentiation methods presented in the literature cannot guarantee differentiation consistency and production process stability under different differentiation systems and operating techniques, significantly limiting the feasibility of translating pancreatic islet organoids from research to industrial applications. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for increasing the yield and stability of pancreatic islet organoids differentiated from pluripotent stem cells. By optimizing 3D culture, the production process and differentiation stability are improved during the differentiation of pluripotent stem cells into pancreatic islet organoids, resulting in more mature pancreatic progenitor cells and increasing the future differentiation yield into islet cells, providing an innovative strategy for future large-scale production.

[0006] This invention is achieved through the following technical solution:

[0007] A method for increasing the yield and stability of pancreatic islet organoids differentiated from pluripotent stem cells, the method comprising:

[0008] (1) Seeding and culturing pluripotent stem cells: Seeding pluripotent stem cells and culturing them in a basic culture medium containing Y-27632;

[0009] (2) Culture in a 3D environment: change the medium and continue culturing using basal culture medium;

[0010] (3) Perform S1 stage differentiation: first culture in S1D1 medium for 1 day, then culture in S1D2 medium for 2-3 days;

[0011] (4) Perform S2 stage differentiation: Change the medium and culture in S2 medium for 2-3 days;

[0012] (5) Perform S3 stage differentiation: Change the medium and culture in S3 medium for 1-2 days;

[0013] (6) Perform S4 stage differentiation: Change the medium and culture in S4 medium for 4-7 days;

[0014] (7) Perform S5 stage differentiation: Change the medium and culture in S5 medium for 3-10 days;

[0015] (8) Reaggregate and continue to differentiate: First, reaggregate the cell spheres and culture them in S6 medium for 3-21 days to obtain pancreatic islet organoids.

[0016] Further, step (1) specifically involves: selecting pluripotent stem cells with a confluence of 60-95%, digesting them for 1-10 minutes, terminating the digestion with basal culture medium, centrifuging and discarding the supernatant, resuspending them in basal culture medium containing Y-27632, filtering the cells through a 20-100 μm filter, and then counting the cells.

[0017] Pluripotent stem cells were seeded into 100 mL bioreactor flasks at a seeding rate of 1E3 / mL–1E7 / mL, using basal medium containing Y-27632, and cultured at a rotation speed of 30–100 rpm for 1 day.

[0018] The basal culture medium includes any one of mTeSR1 medium, E8 medium, mTeSR Plus medium, mTeSRAOF medium, and NutriStem hPSC XF Medium.

[0019] The concentration of Y-27632 in the basal culture medium containing Y-27632 is 2-50 μM.

[0020] Further, step (2) specifically involves replacing 70-90 mL of culture medium in each bioreactor bottle with basic culture medium and continuing to culture for 0-2 days;

[0021] The basal culture medium includes any one of mTeSR1 medium, E8 medium, mTeSR Plus medium, mTeSRAOF medium, and NutriStem hPSC XF Medium.

[0022] Furthermore, step (3) includes:

[0023] (3.1) Wash the cells cultured in step (2) once with DPBS washing solution and replace with S1D1 medium for 1 day;

[0024] The S1D1 medium uses MCDB131 or RPMI1640 as the basal medium, and the basal medium is supplemented with sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin (BSA), human serum albumin (HSA), B-27, sodium bicarbonate (NaHCO3), GlutaMax, L-glutamine, glucose, vitamin C, Activin A, CHIR99021 and bFGF;

[0025] In the S1D1 medium, the concentrations are as follows: sodium pyruvate 0-2.0 mM, HEPES buffer 0-50 mM, antibiotic 1-50 g / L, bovine serum albumin 0.5-100 g / L, B-27 0.1-10% (v / v); human serum albumin 0.5-100 g / L, sodium bicarbonate 0.5-2 g / L, GlutaMax 0.1-10% (v / v); glutamine 100-300 mg / L; glucose 0-4 g / L, vitamin C 0-100 mg / L, Activin A 50-200 ng / mL, CHIR99021 0-5 μM, and bFGF 0-50 ng / mL; the antibiotics used are penicillin and / or streptomycin.

[0026] (3.2) After changing to S1D2 medium, culture for 2-3 days;

[0027] The S1D2 medium uses MCDB131 or RPMI1640 as the basal medium, and the basal medium is supplemented with sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, Activin A and bFGF.

[0028] In the S1D2 medium, the concentrations of sodium pyruvate (0-2.0 mM), HEPES buffer (0-50 mM), antibiotic (1-50 g / L), bovine serum albumin (0.5-100 g / L), B-27 (0.1-10% v / v), human serum albumin (0.5-100 g / L), sodium bicarbonate (0.5-2 g / L), GlutaMax (0.1-10% v / v), glutamine (100-300 mg / L), glucose (0-4 g / L), vitamin C (0-100 mg / L), Activin A (50-200 ng / mL), and bFGF (0-50 ng / mL) are specified. The antibiotics used are penicillin and / or streptomycin.

[0029] Further, in step (4), the S2 medium uses MCDB131 or RPMI1640 as the basal medium, and sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, insulin-transferrin-selenoethanol (ITS-X) and fibroblast growth factor 7 (FGF7) are added to the basal medium.

[0030] In the S2 medium, the concentrations are as follows: sodium pyruvate 0-2.0 mM, HEPES buffer 0-50 mM, antibiotic 1-50 g / L, bovine serum albumin 0.5-100 g / L, B-27 0.1-10% (v / v), human serum albumin 0.5-100 g / L, sodium bicarbonate 0.5-2.0 g / L, GlutaMax 0.1-10% (v / v), glutamine 100-300 mg / L, glucose 0-4 g / L, vitamin C 0-100 mg / L, insulin-transferrin-selenoethanol 0-1.5% (v / v), and fibroblast growth factor 7 20-200 ng / mL; the antibiotics used are penicillin and / or streptomycin.

[0031] Further, in step (5), the S3 culture uses MCDB131 or DMEM medium as the basal medium, and sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, insulin-transferrin-selenoethanol, fibroblast growth factor 7, SANT1, retinoic acid, LDN-193189, TPPB, PdBu, Wnt-C59, Y-27632 and KAAD-Cyclopamine are added to the basal medium.

[0032] In the S3 medium, the concentrations of sodium pyruvate (0-2.0 mM), HEPES buffer (0-50 mM), antibiotics (1-50 g / L), bovine serum albumin (0.5-100 g / L), B-27 (0.1-10% v / v), human serum albumin (0.5-100 g / L), sodium bicarbonate (0.5-2.0 g / L), GlutaMax (0.1-10% v / v), glutamine (100-300 mg / L), glucose (0-4 g / L), vitamin C (0-100 mg / L), and insulin-converting... The concentrations of ferritin-selenium-aminoethanol were 0-1.5% (v / v), fibroblast growth factor 7 was 20-200 ng / mL, SANT1 was 0.05-0.3 μM, retinoic acid was 1-5 μM, LDN-193189 was 50-250 nM, TPPB was 50-750 nM, PdBu was 0-0.75 μM, Wnt-C59 was 0-0.2 μM, Y-27632 was 0-25 μM, and KAAD-Cyclopamine was 0-0.45 μM; the antibiotics used were penicillin and / or streptomycin.

[0033] Further, in step (6), the S4 medium uses MCDB131 or DMEM medium as the basal medium, and the basal medium is supplemented with sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, sodium heparin, insulin-transferrin-selenoethanol, fibroblast growth factor 7, epidermal growth factor (EGF), SANT1, retinoic acid, LDN-193189, TPPB, PdBu, nicotinamide, Activin A, Y-27632 and aphidicolin;

[0034] In the S4 medium, the concentrations of sodium pyruvate (0-2.0 mM), HEPES buffer (0-50 mM), antibiotics (1-50 g / L), bovine serum albumin (0.5-100 g / L), B-27 (0.1-10% v / v), human serum albumin (0.5-100 g / L), sodium bicarbonate (0.5-2.0 g / L), GlutaMax (0.1-10% v / v), glutamine (100-300 mg / L), glucose (0-4 g / L), vitamin C (0-100 mg / L), heparin sodium (0-50 μg / mL), and insulin-transferrin-seleno-ammonia... The concentrations of the following antibiotics are as follows: ethanol 0-1.5% (v / v), fibroblast growth factor 7 20-200 ng / mL, epidermal growth factor 0-250 ng / mL, SANT1 0.05-0.3 μM, retinoic acid 0.05-0.45 μM, LDN-193189 50-250 nM, TPPB 50-750 nM, PdBu 0-0.75 μM, nicotinamide 0-20 mM, Activin A 50-200 ng / mL, Y-27632 0-25 μM, and afedipine 0-5 μM; the antibiotics used are penicillin and / or streptomycin.

[0035] Further, in step (7), the S5 culture medium uses MCDB131 or RPMI1640 as the basal culture medium, and antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, sodium heparin, zinc sulfate (ZnSO4), SANT1, retinoic acid, LDN-193189, Y-27632, Alk5 inhibitor II, T3 thyroxine, γ-secretase inhibitor (GSI-IX), Betacellulin, ISX9, afedipine and Wnt-C59 are added to the basal culture medium;

[0036] In the S5 medium, the concentrations of antibiotics were 1-50 g / L, bovine serum albumin 0.5-100 g / L, B-27 0.1-10% (v / v), human serum albumin 0.5-100 g / L, sodium bicarbonate 0.5-2.0 g / L, GlutaMax 0.1-10% (v / v), glutamine 100-300 mg / L, glucose 0-4 g / L, vitamin C 0-100 mg / L, heparin sodium 0-50 μg / mL, zinc sulfate 0-100 μM, and SANT1... The concentrations of the following antibiotics are specified: 0-0.3 μM for retinoic acid, 0.01-0.10 μM for LDN-193189, 50-250 nM for Y-27632, 0-25 μM for Alk5 inhibitor II, 0-20 μM for T3 thyroxine, 0.1-2 μM for γ-secretase inhibitor XX, 0-0.1 μM for Betacellulin, 0-25 ng / mL for ISX9, 0-10 μM for afedipine, 0-5 μM for Wnt-C59, and 0-0.2 μM for Wnt-C59. The antibiotics used are penicillin and / or streptomycin.

[0037] Further, in step (8), the cell spheroid reaggregation specifically includes: centrifuging and discarding the supernatant, collecting cell spheroids, adding cell washing solution and centrifuging to wash, discarding the supernatant and adding TrypLE, Accutase, Gentle Cell Dissociation Reagent (GCDR) or Trypsin to digest the cells, using a pipette to disperse the cells until cell spheroids are visible to the naked eye, adding S6 medium to stop digestion; filtering with a cell filter, centrifuging and resuspending the cells in S6 medium and counting; diluting the cells and seeding them in a cell culture plate to reaggregate the cell spheroids;

[0038] The S6 medium uses MCDB131 or RPMI1640 as the basal medium, and antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, sodium heparin, zinc sulfate, Y-27632, Alk5 inhibitor II, T3 thyroxine, non-essential amino acids (NEAA), TraceA Element, Trace B Element, Trolox, acetylcysteine ​​(NAC), R-428, and ZM-447439 are added to the basal medium.

[0039] In the S6 medium, the concentrations of antibiotics were 1-50 g / L, bovine serum albumin 0.5-100 g / L, B-27 0.1-10% (v / v), human serum albumin 0.5-100 g / L, sodium bicarbonate 0.5-2.0 g / L, GlutaMax 0.1-10% (v / v), glutamine 100-300 mg / L, and glucose 0-4 g / L. The concentrations of the following antibiotics are as follows: L, Vitamin C (0-100 mg / L), Sodium Heparin (0-50 μg / mL), Zinc Sulfate (0-100 μM), Y-27632 (0-25 μM), Alk5 Inhibitor II (0-20 μM), T3 Thyroxine (0.1-2 μM), Non-essential Amino Acids (0-1.0% (v / v), Trace A Element (0-1.0% (v / v), Trace B Element (0-1.0% (v / v), Trolox (0-25 μM), Acetylcysteine ​​(0-2 mM), R-428 (0-10 μM), and ZM-447439 (0-1 μM); the antibiotics used are penicillin and / or streptomycin.

[0040] Furthermore, during the cell spheroid reassembly process, TrypLE was added to digest the cells.

[0041] Furthermore, in step (8), the culture is carried out in S6 medium for 9 days.

[0042] Furthermore, when cultured in S6 medium for 9 days, the GSIS value was >1, and the CP / NKX6.1 pancreatic β-cell positivity rate reached 53%.

[0043] Beneficial technical effects of the present invention:

[0044] This invention relates to a method for increasing the yield and stability of pancreatic islet organoids differentiated from pluripotent stem cells, by inducing human induced pluripotent stem cells (iPSCs) to differentiate into pancreatic islet organoids.

[0045] This invention ensures sufficient differentiation at each stage by controlling the number of culture days and the composition of the culture medium, without causing over-differentiation. At the cellular level, it maximizes the differentiation effect at each stage, increases the yield and differentiation efficiency of each differentiation stage (including the endoderm FOXA2 / SOX17, hindbrain PDX1, pancreatic progenitor PDX1 / NKX6.1, endocrine progenitor cells CHGA, and pancreatic islet organoid C-Peptide / NKX6.1 stages), and increases the scale of production.

[0046] Compared to 2D culture, this invention uses 3D differentiation, which makes it easier to scale up. In addition, 2D differentiation systems are prone to cell nutrient deficiency, uneven cell growth, uneven substrate layering, inconvenient operation, and inability to perform fully enclosed culture. This invention uses 3D culture, which can effectively solve these problems of 2D differentiation systems and has greater advantages for industrialization. Attached Figure Description

[0047] Figure 1 The flow cytometry results of DE-directed endoderm differentiation (S1 stage) of pluripotent stem cells are shown as FOXA2 / SOX17.

[0048] Figure 2 PDX data of flow cytometry analysis of pluripotent stem cells after Posterior Foregut differentiation (S2-S3 stage);

[0049] Figure 3 The flow cytometry results of the pancreatic progenitor pluripotent stem cell differentiation (S4 stage) PDX1 / NKX6.1 are shown.

[0050] Figure 4 The flow cytometry results of endocrine cells from pluripotent stem cells undergoing endocrine differentiation (S5 stage) are shown, with C-Peptide / CHGA ratio.

[0051] Figure 5 The flow cytometry results of pancreatic β cells from the endocrine differentiation (S5 stage) of pluripotent stem cells (Endocrine) are shown as C-Peptide / NKX6.1.

[0052] Figure 6 The flow cytometry results of pancreatic islet α cells from pluripotent stem cell Endocrine endocrine differentiation (S5 stage) are shown, with C-Peptide / Glucagon.

[0053] Figure 7 The flow cytometry results of pancreatic islet delta cells from pluripotent stem cell Endocrine endocrine differentiation (S5 stage) are shown, with C-Peptide / SST ratio.

[0054] Figure 8 The flow cytometry results of pancreatic β cells differentiated from pluripotent stem cells S6D9-S6D14 are shown, with C-Peptide / NKX6.1.

[0055] Figure 9 The flow cytometry results of pancreatic islet α cells differentiated from pluripotent stem cells S6D9-S6D14 are shown, with C-Peptide / Glucagon.

[0056] Figure 10 The results of flow cytometry analysis of pancreatic islet endocrine cells differentiated from pluripotent stem cells (S6D9-S6D14) are shown, with C-Peptide / CHGA.

[0057] Figure 11 Glucose-stimulated insulin release GSIS function assay of pancreatic islets differentiated from pluripotent stem cells S6D9-S6D14;

[0058] Figure 12 Immunofluorescence staining of pancreatic islet organoids differentiated from pluripotent stem cells S6D9-S6D14 islet groups was shown. Detailed Implementation

[0059] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0060] Conversely, this invention encompasses any substitutions, modifications, equivalent methods, and solutions made within the spirit and scope of the invention as defined in the claims. Furthermore, to provide a better understanding of the invention, certain specific details are described in detail below. However, those skilled in the art will fully understand the invention even without these detailed descriptions.

[0061] This invention provides an embodiment of a method for increasing the yield and stability of pancreatic islet organoids differentiated from pluripotent stem cells, the method comprising:

[0062] (1) Seeding and culturing pluripotent stem cells: Seeding pluripotent stem cells and culturing them in a basic culture medium containing Y-27632;

[0063] (2) Culture in a 3D environment: change the medium and continue culturing using basal culture medium;

[0064] (3) Perform S1 stage differentiation: first culture in S1D1 medium for 1 day, then culture in S1D2 medium for 2-3 days;

[0065] (4) Perform S2 stage differentiation: Change the medium and culture in S2 medium for 2-3 days;

[0066] (5) Perform S3 stage differentiation: Change the medium and culture in S3 medium for 1-2 days;

[0067] (6) Perform S4 stage differentiation: Change the medium and culture in S4 medium for 4-7 days;

[0068] (7) Perform S5 stage differentiation: Change the medium and culture in S5 medium for 3-10 days;

[0069] (8) Reaggregate and continue to differentiate: First, reaggregate the cell spheres and culture them in S6 medium for 3-21 days to obtain pancreatic islet organoids.

[0070] In this invention, step (1) can be:

[0071] Pluripotent stem cells with a confluence of 60-95% were selected, digested for 1-10 minutes, digestion was terminated with basal medium, the supernatant was discarded after centrifugation, and the cells were resuspended in basal medium containing Y-27632. After filtering the cells through a 20-100μm filter, cell counting was performed.

[0072] Pluripotent stem cells were seeded into 100mL bioreactor flasks at a seeding rate of 1E3 / mL–1E7 / mL, using basal culture medium containing Y-27632, and cultured for 1 day at a rotation speed of 30-100rpm. Specifically, in this invention, 100mL bioreactor flasks were used for each step.

[0073] The basal culture medium includes any one of mTeSR1 medium, E8 medium, mTeSR Plus medium, mTeSRAOF medium, and NutriStem hPSC XF Medium.

[0074] The concentration of Y-27632 in the basal culture medium containing Y-27632 is 2-50 μM.

[0075] In a specific embodiment:

[0076] Step (1) specifically involves selecting pluripotent stem cells with a confluence of 75% (optional, such as 60%, 80%, 95%, etc.), digesting them for 5 minutes (optional, such as 1 minute, 8 minutes, 10 minutes, etc.), terminating the digestion with basal culture medium, centrifuging and discarding the supernatant, resuspending them in basal culture medium containing Y-27632, filtering the cells through a 20 μM (optional, such as 20 μM, 60 μM, 100 μM, etc.) filter, and then counting the cells.

[0077] Pluripotent stem cells were seeded into bioreactor flasks at a cell number of 1E4 / mL (optional, such as 5E3 / mL, 1E6 / mL, 1E7 / mL), using basal medium containing Y-27632, and cultured at a rotation speed of 45 rpm (optional, such as 30 rpm, 60 rpm, 100 rpm, etc.) for 1 day.

[0078] The basal culture medium is mTeSR1 medium (optionally, any one of E8 medium, mTeSR Plus medium, mTeSRAOF medium, or NutriStem hPSC XF Medium).

[0079] The Y-27632-containing basal culture medium has a Y-27632 concentration of 20 μM (optionally, for example, 2 μM, 30 μM, 50 μM, etc.).

[0080] Specifically, the digestion described in this invention refers to digesting pluripotent stem cells using any one of Accutase, Trypsin, TrypLE, EDTA, ReLeSR (StemCell product), or GCDR (StemCell product). Furthermore, in the embodiments, Accutase is preferred.

[0081] Specifically, EB03 cell line pluripotent stem cells can be used in this invention. In this embodiment, the total volume of the pluripotent stem cell resuspension solution plus the volume of mTeSR+Y-27632 culture medium in the bioreactor bottle is 100 mL;

[0082] In this invention, step (2) can be:

[0083] In this embodiment, step (2) specifically involves replacing 70-90 mL of culture medium in each bioreactor bottle with basic culture medium and continuing the culture for 0-2 days.

[0084] The basal culture medium includes any one of mTeSR1 medium, E8 medium, mTeSR Plus medium, mTeSRAOF medium, and NutriStem hPSC XF Medium.

[0085] In a specific embodiment,

[0086] Step (2) specifically involves replacing 70-90 mL (optional, such as 70, 75, 80, 85 or 90 mL) of culture medium in each bioreactor bottle with basic culture medium and continuing to culture for 1 day (optional, such as 1.5 days, 2 days, etc.).

[0087] The basal culture medium is mTeSR1 medium (optionally, any one of E8 medium, mTeSR Plus medium, mTeSRAOF medium, or NutriStem hPSC XF Medium).

[0088] Step (3) in this embodiment includes:

[0089] (3.1) Wash the cells cultured in step (2) once with DPBS washing solution and replace with S1D1 medium for 1 day;

[0090] The S1D1 medium uses MCDB131 or RPMI1640 as the basal medium, and the basal medium is supplemented with sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin (BSA), human serum albumin (HSA), B-27, sodium bicarbonate (NaHCO3), GlutaMax, L-glutamine, glucose, vitamin C, Activin A, CHIR99021 and bFGF;

[0091] In this invention, the S1D1 culture medium may contain the following concentrations: sodium pyruvate (0-2.0 mM), HEPES buffer (0-50 mM), antibiotic (1-50 g / L), bovine serum albumin (0.5-100 g / L), B-27 (0.1-10% v / v); human serum albumin (0.5-100 g / L), sodium bicarbonate (0.5-2 g / L), GlutaMax (0.1-10% v / v); glutamine (100-300 mg / L); glucose (0-4 g / L), vitamin C (0-100 mg / L), Activin A (50-200 ng / mL), CHIR99021 (0-5 μM), and bFGF (0-50 ng / mL); the antibiotic may be penicillin or / and streptomycin.

[0092] In a specific embodiment: In the S1D1 culture medium, the concentration of sodium pyruvate is 1.5 mM (optionally, for example, 0.1 mM, 1.0 mM, 1.5 mM, 2.0 mM, etc.), the concentration of HEPES buffer is 20 mM (optionally, for example, 5 mM, 10 mM, 30 mM, 40 mM, 50 μM, etc.), the concentration of antibiotic is 30 g / L (optionally, for example, 1 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, etc.), and the concentration of bovine serum albumin is 5 g / L (optionally...). For example, 0.5 g / L, 30 g / L, 100 g / L, etc.); B-27 concentration of 1% (v / v) (optional, for example, 0.1% (v / v), 5% (v / v), 10% (v / v), etc.); human serum albumin concentration of 5 g / L (optional, for example, 0.5 g / L, 30 g / L, 100 g / L, etc.); sodium bicarbonate concentration of 1 g / L (optional, for example, 0.5 g / L, 1.5 g / L, 2 g / L, etc.); GlutaMax concentration of 1% (v / v) (optional, for example, 0.1% (v / v), 5% (v / v), 10% (v / v), etc.). For example, 0.1% (v / v), 3% (v / v), 10% (v / v), etc.; glutamine concentration of 100 mg / L (optional, such as 50 mg / L, 150 mg / L, 200 mg / L, 300 mg / L, etc.); glucose concentration of 2 g / L (optional, such as 0.1 g / L, 1 g / L, 3 g / L, 4 g / L, etc.); vitamin C concentration of 50 mg / L (optional, such as 10 mg / L, 30 mg / L, 50 mg / L, 70 mg / L, 100 mg / L, etc.). The concentrations of Activin A (e.g., 50 ng / ml, 100 ng / ml, 150 ng / ml, 200 ng / ml, etc.) and CHIR99021 (e.g., 0.1 μM, 1 μM, 2 μM, 4 μM, 5 μM, etc.) and bFGF (e.g., 10 ng / mL, 30 ng / mL, 50 ng / mL, etc.) are used; the antibiotic used is penicillin (e.g., streptomycin, penicillin, and streptomycin).

[0093] (3.2) After changing to S1D2 medium, culture for 2-3 days;

[0094] The S1D2 medium uses MCDB131 or RPMI1640 as the basal medium, and the basal medium is supplemented with sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, Activin A, and bFGF.

[0095] In the S1D2 medium, the concentrations of sodium pyruvate (0-2.0 mM), HEPES buffer (0-50 mM), antibiotic (1-50 g / L), bovine serum albumin (0.5-100 g / L), B-27 (0.1-10% v / v), human serum albumin (0.5-100 g / L), sodium bicarbonate (0.5-2 g / L), GlutaMax (0.1-10% v / v), glutamine (100-300 mg / L), glucose (0-4 g / L), vitamin C (0-100 mg / L), Activin A (50-200 ng / mL), and bFGF (0-50 ng / mL) are specified. The antibiotics used are penicillin and / or streptomycin.

[0096] In a specific embodiment:

[0097] In the S1D2 medium, the concentration of sodium pyruvate is 1.5 mM (optionally, 0.1 mM, 1.0 mM, 1.5 mM, 2.0 mM, etc.), the concentration of HEPES buffer is 20 mM (optionally, 5 mM, 10 mM, 30 mM, 40 mM, 50 μM, etc.), the concentration of antibiotic is 30 g / L (optionally, 1 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, etc.), and the concentration of bovine serum albumin is 5 g / L. (Optional concentrations include, for example, 0.5 g / L, 30 g / L, 100 g / L, etc.); B-27 concentration is 1% (v / v) (optional concentrations include, for example, 0.1% (v / v), 5% (v / v), 10% (v / v), etc.); human serum albumin concentration is 5 g / L (optional concentrations include, for example, 0.5 g / L, 30 g / L, 100 g / L, etc.); sodium bicarbonate concentration is 1 g / L (optional concentrations include, for example, 0.5 g / L, 1.5 g / L, 2 g / L, etc.); Gluta Max concentration is 1% (v / v) (options include, for example, 0.1% (v / v), 3% (v / v), 10% (v / v), etc.); glutamine concentration is 100 mg / L (options include, for example, 50 mg / L, 150 mg / L, 200 mg / L, 300 mg / L, etc.); glucose concentration is 2 g / L (options include, for example, 0.1 g / L, 1 g / L, 3 g / L, 4 g / L, etc.); vitamin C concentration is 50 mg / L (options include, for example, 1 g / L, 3 g / L, 4 g / L, etc.). The concentrations are 10 mg / L, 30 mg / L, 50 mg / L, 70 mg / L, 100 mg / L, etc.), Activin A concentration is 50 ng / mL (optional, such as 100 ng / mL, 150 ng / mL, 200 ng / mL, etc.), and bFGF concentration is 20 ng / mL (optional, such as 10 ng / mL, 30 ng / mL, 50 ng / mL, etc.); the antibiotic used is penicillin (optional, streptomycin, penicillin, and streptomycin).

[0098] In this invention, in step (4), the S2 culture medium uses MCDB131 or RPMI1640 as the basal culture medium, and sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin (BSA), human serum albumin (HSA), B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, insulin-transferrin-selenoethanol (ITS-X), and fibroblast growth factor 7 (FGF7) are added to the basal culture medium.

[0099] In the S2 medium, the concentrations are as follows: sodium pyruvate 0-2.0 mM, HEPES buffer 0-50 mM, antibiotic 1-50 g / L, bovine serum albumin 0.5-100 g / L, B-27 0.1-10% (v / v), human serum albumin 0.5-100 g / L, sodium bicarbonate 0.5-2.0 g / L, GlutaMax 0.1-10% (v / v), glutamine 100-300 mg / L, glucose 0-4 g / L, vitamin C 0-100 mg / L, insulin-transferrin-selenoethanol 0-1.5% (v / v), and fibroblast growth factor 7 20-200 ng / mL; the antibiotics used are penicillin and / or streptomycin.

[0100] In a specific embodiment:

[0101] In the S2 medium, the concentration of sodium pyruvate is 1.5 mM (optionally, 0.1 mM, 1.0 mM, 1.5 mM, 2.0 mM, etc.), the concentration of HEPES buffer is 20 mM (optionally, 5 mM, 10 mM, 30 mM, 40 mM, 50 μM, etc.), the concentration of antibiotic is 30 g / L (optionally, 1 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, etc.), and the concentration of bovine serum albumin is 5 g / L (optionally, 0.5 g / L, 3 g / L, 40 g / L, 50 μM, etc.). The following concentrations are available: 0 g / L, 100 g / L, etc.); B-27 concentration of 1% (v / v) (optional, e.g., 0.1% (v / v), 5% (v / v), 10% (v / v), etc.); human serum albumin concentration of 5 g / L (optional, e.g., 0.5 g / L, 30 g / L, 100 g / L, etc.); sodium bicarbonate concentration of 1 g / L (optional, e.g., 0.5 g / L, 1.5 g / L, 2 g / L, etc.); GlutaMax concentration of 1% (v / v) (optional, e.g., 0.1% (v / v)). 3% (v / v), 10% (v / v), etc.); glutamine concentration of 100 mg / L (optional, such as 50 mg / L, 150 mg / L, 200 mg / L, 300 mg / L, etc.); glucose concentration of 2 g / L (optional, such as 0.1 g / L, 1 g / L, 3 g / L, 4 g / L, etc.); vitamin C concentration of 50 mg / L (optional, such as 10 mg / L, 30 mg / L, 50 mg / L, 70 mg / L, 100 mg / L, etc.); insulin-transferrin- The concentration of selenium-aminoethanol is 1% (v / v) (optionally, for example, 0.1% (v / v), 0.5% (v / v), 0.8% (v / v), 1.2% (v / v), 1.5% (v / v), etc.), and the concentration of fibroblast growth factor 7 is 100 ng / mL (optionally, for example, 20 ng / mL, 50 ng / mL, 80 ng / mL, 120 ng / mL, 150 ng / mL, 200 ng / mL, etc.); the antibiotic is penicillin (optionally, streptomycin, penicillin, and streptomycin).

[0102] In step (5) of this embodiment, the S3 culture uses MCDB131 or DMEM medium as the basic medium, and sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, insulin-transferrin-selenoethanol, fibroblast growth factor 7, SANT1, retinoic acid, LDN-193189, TPPB, PdBu, Wnt-C59, Y-27632, and KAAD-Cyclopamine are added to the basic medium.

[0103] In the S3 medium, the concentrations of sodium pyruvate (0-2.0 mM), HEPES buffer (0-50 mM), antibiotics (1-50 g / L), bovine serum albumin (0.5-100 g / L), B-27 (0.1-10% v / v), human serum albumin (0.5-100 g / L), sodium bicarbonate (0.5-2.0 g / L), GlutaMax (0.1-10% v / v), glutamine (100-300 mg / L), glucose (0-4 g / L), and vitamin C (0-100 mg / L) were as follows: / L, insulin-transferrin-seleno-aminoethanol concentration is 0-1.5% (v / v), fibroblast growth factor 7 concentration is 20-200 ng / mL, SANT1 concentration is 0.05-0.3 μM, retinoic acid concentration is 1-5 μM, LDN-193189 concentration is 50-250 nM, TPPB concentration is 50-750 nM, PdBu concentration is 0-0.75 μM, Wnt-C59 concentration is 0-0.2 μM, Y-27632 concentration is 0-25 μM, KAAD-Cyclopamine concentration is 0-0.45 μM.

[0104] In a specific embodiment:

[0105] In the S3 medium, the concentration of sodium pyruvate is 1.5 mM (optionally, 0.1 mM, 1.0 mM, 1.5 mM, 2.0 mM, etc.), the concentration of HEPES buffer is 20 mM (optionally, 5 mM, 10 mM, 30 mM, 40 mM, 50 μM, etc.), the concentration of antibiotic is 30 g / L (optionally, 1 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, etc.), the concentration of bovine serum albumin is 5 g / L (optionally, 0.5 g / L, 30 g / L, 100 g / L, etc.), and the concentration of B-27 is 1% (v / v) (optionally, 0.1% (v / v), 5% (v / v), 10% (v / v), etc.). / v) etc.; human serum albumin concentration of 5g / L (optional, such as 0.5g / L, 30g / L, 100g / L, etc.), sodium bicarbonate concentration of 1g / L (optional, such as 0.5g / L, 1.5g / L, 2g / L, etc.), GlutaMax concentration of 1% (v / v) (optional, such as 0.1% (v / v), 3% (v / v), 10% (v / v), etc.); glutamine concentration of 100mg / L (optional, such as 50mg / L, 150mg / L, 200mg / L, 300mg / L, etc.); glucose concentration of 2g / L (optional, such as 0.1g / L, 1g / L, 3g / L, 4g / L, etc.), vitamin Vitamin C concentration is 50 mg / L (options include, for example, 10 mg / L, 30 mg / L, 50 mg / L, 70 mg / L, 100 mg / L, etc.), insulin-transferrin-selenoethanol concentration is 1.0% (v / v) (options include, for example, 0.1% (v / v), 0.5% (v / v), 0.8% (v / v), 1.2% (v / v), 1.5% (v / v), etc.), fibroblast growth factor 7 concentration is 100 ng / mL (options include, for example, 20 ng / mL, 50 ng / mL, 80 ng / mL, 120 ng / mL, 150 ng / mL, 200 ng / mL, etc.), SANT1 concentration is 0.2 μM (options include, for example, 50 mg / L, 30 mg / L, 50 mg / L, 70 mg / L, 100 mg / L, etc.), and SANT1 concentration is 0.2 μM (options include, for example, 10 mg / L, 30 mg / L, 50 mg / L, 70 mg / L, 100 mg / L, etc.). For example, concentrations of 0.05μM, 0.1μM, 0.15μM, 0.2μM, 0.25μM, 0.3μM, etc.); retinoic acid concentration of 3μM (optional, such as 1μM, 2μM, 4μM, 5μM, etc.); LDN-193189 concentration of 100nM (optional, such as 50nM, 80nM, 120nM, 150nM, 200nM, 250nM, etc.); TPPB concentration of 500nM (optional, such as 50nM, 200nM, 300nM, 400nM, 600nM, 750nM, etc.); and PdBu concentration of 0.5μM (optional, such as 0.1μM, 0.2μM, 0.4μM, 0.6μM, 0.5μM, etc.).The concentrations of various active ingredients are as follows: 75 μM, Wnt-C59 at 0.1 μM (optional concentrations include 0.05 μM, 0.08 μM, 0.12 μM, 0.15 μM, 0.2 μM, etc.), Y-27632 at 20 μM (optional concentrations include 0.1 μM, 0.2 μM, 1 μM, 10 μM, 25 μM, etc.), and KAAD-Cyclopamine at 0.15 μM (optional concentrations include 0.1 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.4 μM, 0.45 μM, etc.).

[0106] In step (6) of this embodiment, the S4 medium uses MCDB131 or DMEM medium as the basic medium, and sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, sodium heparin, insulin-transferrin-selenoethanolamine (ITS-X), fibroblast growth factor 7 (FGF7), epidermal growth factor (EGF), SANT1, retinoic acid, LDN-193189, TPPB, PdBu, nicotinamide, Activin A, Y-27632, and afedipine are added to the basic medium.

[0107] In the S4 medium, the concentrations of sodium pyruvate (0-2.0 mM), HEPES buffer (0-50 mM), antibiotics (1-50 g / L), bovine serum albumin (0.5-100 g / L), B-27 (0.1-10% v / v), human serum albumin (0.5-100 g / L), sodium bicarbonate (0.5-2.0 g / L), GlutaMax (0.1-10% v / v), glutamine (100-300 mg / L), glucose (0-4 g / L), vitamin C (0-100 mg / L), heparin sodium (0-50 μg / mL), and insulin-... The concentrations of transferrin-selenium-aminoethanol were 0-1.5% (v / v), fibroblast growth factor 7 was 20-200 ng / mL, epidermal growth factor was 0-250 ng / mL, SANT1 was 0.05-0.3 μM, retinoic acid was 0.05-0.45 μM, LDN-193189 was 50-250 nM, TPPB was 50-750 nM, PdBu was 0-0.75 μM, nicotinamide was 0-20 mM, Activin A was 50-200 ng / mL, Y-27632 was 0-25 μM, and afedipine was 0-5 μM.

[0108] In a specific embodiment:

[0109] In the S4 medium, the concentration of sodium pyruvate is 1.5 mM (optionally, 0.1 mM, 1.0 mM, 1.5 mM, 2.0 mM, etc.), the concentration of HEPES buffer is 20 mM (optionally, 5 mM, 10 mM, 30 mM, 40 mM, 50 μM, etc.), the concentration of antibiotic is 30 g / L (optionally, 1 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, etc.), the concentration of bovine serum albumin is 5 g / L (optionally, 0.5 g / L, 30 g / L, 100 g / L, etc.), and the concentration of B-27 is 1% (v / v) (optionally, 0.1% (v / v), 5% (v / v). 10% (v / v), etc.); human serum albumin concentration of 5 g / L (options include, for example, 0.5 g / L, 30 g / L, 100 g / L, etc.); sodium bicarbonate concentration of 1 g / L (options include, for example, 0.5 g / L, 1.5 g / L, 2 g / L, etc.); GlutaMax concentration of 1% (v / v), etc. (options include, for example, 0.1% (v / v), 3% (v / v), 10% (v / v), etc.); glutamine concentration of 100 mg / L (options include, for example, 50 mg / L, 150 mg / L, 200 mg / L, 300 mg / L, etc.); glucose concentration of 2 g / L (options include, for example, 0.1 g / L, 1 g / L, 3 g / L, etc.). The concentrations of vitamin C and heparin sodium are as follows: 50 mg / L (optional, e.g., 10 mg / L, 30 mg / L, 50 mg / L, 70 mg / L, 100 mg / L, etc.), 25 μg / mL (optional, e.g., 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, etc.), 1% (v / v) of insulin-transferrin-selenoethanol (optional, e.g., 0.1% (v / v), 0.5% (v / v), 0.8% (v / v), 1.2% (v / v), 1.5% (v / v), etc.), and 100 ng / mL of fibroblast growth factor 7 (optional, e.g., 100 ng / mL, etc.). For example, concentrations of 20 ng / mL, 50 ng / mL, 80 ng / mL, 120 ng / mL, 150 ng / mL, 200 ng / mL, etc. are available. Epidermal growth factor concentration is 150 ng / mL (optional, such as 20 ng / mL, 50 ng / mL, 80 ng / mL, 120 ng / mL, 200 ng / mL, 250 ng / mL, etc. are also available). SANT1 concentration is 0.2 μM (optional, such as 0.05 μM, 0.1 μM, 0.15 μM, 0.18 μM, 0.25 μM, 0.3 μM, etc.), and retinoic acid concentration is 0.2 μM (optional, such as 0.05 μM, 0.15 μM, 0.3 μM, 0.2 μM, etc.).The concentrations of various active ingredients (e.g., 45 μM), LDN-193189 at 100 nM (optional, such as 50 nM, 80 nM, 120 nM, 180 nM, 250 nM), TPPB at 500 nM (optional, such as 50 nM, 200 nM, 300 nM, 400 nM, 600 nM, 750 nM), and PdBu at 0.1 μM (optional, such as 0.05 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM) are available. The following concentrations are available: 5 μM (e.g., nicotinamide), 10 mM (optional, e.g., 5 mM, 8 mM, 15 mM, 20 mM), 50 ng / ml Activin A (optional, e.g., 100 ng / ml, 150 ng / ml, 200 ng / ml), 20 μM Y-27632 (optional, e.g., 5 μM, 10 μM, 15 μM, 25 μM), and 3 μM afenidimycin (optional, e.g., 1 μM, 2 μM, 4 μM, 5 μM).

[0110] In step (7) of this embodiment, the S5 culture medium uses MCDB131 or RPMI1640 as the basic culture medium, and antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, sodium heparin, zinc sulfate, SANT1, retinoic acid, LDN-193189, Y-27632, Alk5 inhibitor II, T3 thyroxine, γ secretase inhibitor (GSI-IX), Betacellulin, ISX9, aphidicolin, and Wnt-C59 are added to the basic culture medium.

[0111] In the S5 medium, the concentrations of antibiotics were 1-50 g / L, bovine serum albumin 0.5-100 g / L, B-27 0.1-10% (v / v), human serum albumin 0.5-100 g / L, sodium bicarbonate 0.5-2.0 g / L, GlutaMax 0.1-10% (v / v), glutamine 100-300 mg / L, glucose 0-4 g / L, vitamin C 0-100 mg / L, heparin sodium 0-50 μg / mL, and zinc sulfate 0-100 μg / mL. μM, SANT1 concentration 0-0.3μM, retinoic acid concentration 0.01-0.10μM, LDN-193189 concentration 50-250nM, Y-27632 concentration 0-25μM, Alk5 inhibitor II concentration 0-20μM, T3 thyroxine concentration 0.1-2μM, γ-secretase inhibitor XX concentration 0-0.1μM, Betacellulin concentration 0-25ng / mL, ISX9 concentration 0-10μM, afedipine concentration 0-5μM, Wnt-C59 concentration 0-0.2μM.

[0112] In a specific embodiment:

[0113] In the S5 culture medium, the antibiotic concentration is 30 g / L (optionally, for example, 10 g / L, 20 g / L, 50 g / L, etc.), the bovine serum albumin concentration is 50 g / L (optionally, for example, 0.5 g / L, 30 g / L, 100 g / L, etc.), the B-27 concentration is 1% (v / v) (optionally, for example, 0.1% (v / v), 2% (v / v), 4% (v / v), 6% (v / v), 8% (v / v), 10% (v / v), etc.); the human serum albumin concentration is 50 g / L (optionally, for example, 0.5 g / L, 30 g / L, 100 g / L, etc.), and the sodium bicarbonate concentration is 1 g / L (optionally, for example, 0.5 g / L). The concentrations of GlutaMax are as follows: 1.5 g / L, 2 g / L, etc.; 1% (v / v) (optional, e.g., 0.1% (v / v), 3% (v / v), 10% (v / v), etc.); 100 mg / L glutamine concentration (optional, e.g., 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, 300 mg / L), 2 g / L glucose concentration (optional, e.g., 0.1 g / L, 1 g / L, 3 g / L, 4 g / L), 50 mg / L vitamin C concentration (optional, e.g., 15 mg / L, 80 mg / L, 100 mg / L), and heparin sodium concentration. The concentrations of various active ingredients are as follows: 25 μg / mL (optional, such as 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, etc.), zinc sulfate concentration of 50 μM (optional, such as 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, etc.), SANT1 concentration of 0.15 μM (optional, such as 0.1 μM, 0.2 μM, 0.25 μM, 0.3 μM, etc.), retinoic acid concentration of 0.05 μM (optional, such as 0.01 μM, 0.02 μM, 0.04 μM, 0.06 μM, 0.08 μM, 0.1 μM, etc.), and LDN-193189 concentration of 150 nM (optional). The selected concentrations are, for example, 50nM, 100nM, 120nM, 180nM, 200nM, 250nM, etc.); Y-27632 concentration is 10μM (selectable concentrations are, for example, 1μM, 5μM, 8μM, 15μM, 25μM, etc.); Alk5 inhibitor II concentration is 10μM (selectable concentrations are, for example, 1μM, 5μM, 8μM, 15μM, 20μM, etc.); T3 thyroxine concentration is 1μM (selectable concentrations are, for example, 0.1μM, 0.5μM, 0.8μM, 1.5μM, 2μM, etc.); γ secretase inhibitor XX concentration is 0.1μM (selectable concentrations are, for example, 0.2μM, 0.5μM, 0.8μM, 1μM, 1.The following concentrations are available: 5 μM, 2 μM, etc.; Betacellulin concentration of 10 ng / mL (optional, e.g., 1 ng / mL, 5 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, etc.); ISX9 concentration of 5 μM (optional, e.g., 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, etc.); Afenidemycin concentration of 3 μM (optional, e.g., 1 μM, 2 μM, 4 μM, 5 μM, etc.); Wnt-C59 concentration of 0.1 μM (optional, e.g., 0.05 μM, 0.15 μM, 0.2 μM, etc.).

[0114] In step (8) of this embodiment, the cell spheroid reaggregation specifically includes: centrifuging and discarding the supernatant, collecting cell spheroids, adding cell washing solution and centrifuging to wash, discarding the supernatant and adding TrypLE, Accutase, Gentle Cell Dissociation Reagent (GCDR) or trypsin to digest the cells, using a pipette to disperse the cells until no cell spheroids are visible to the naked eye, adding S6 medium to stop digestion; filtering with a cell filter, centrifuging and resuspending the cells in S6 medium and counting; diluting the cells and seeding them in a cell culture plate to reaggregate the cell spheroids;

[0115] The S6 medium uses MCDB131 or RPMI1640 as the basal medium, and antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, sodium heparin, zinc sulfate, Y-27632, Alk5 inhibitor II, T3 thyroxine, non-essential amino acids (NEAA), TraceA Element, Trace B Element, Trolox, acetylcysteine ​​(NAC), R-428, and ZM-447439 are added to the basal medium.

[0116] In the S6 medium, the concentrations of antibiotics were 1-50 g / L, bovine serum albumin 0.5-100 g / L, B-27 0.1-10% (v / v), human serum albumin 0.5-100 g / L, sodium bicarbonate 0.5-2.0 g / L, GlutaMax 0.1-10% (v / v), glutamine 100-300 mg / L, and glucose 0-4 g / L. The concentrations of the following antibiotics are as follows: L, Vitamin C (0-100 mg / L), Sodium Heparin (0-50 μg / mL), Zinc Sulfate (0-100 μM), Y-27632 (0-25 μM), Alk5 Inhibitor II (0-20 μM), T3 Thyroxine (0.1-2 μM), Non-essential Amino Acids (0-1.0% (v / v), Trace A Element (0-1.0% (v / v), Trace B Element (0-1.0% (v / v), Trolox (0-25 μM), Acetylcysteine ​​(0-2 mM), R-428 (0-10 μM), and ZM-447439 (0-1 μM); the antibiotics used are penicillin and / or streptomycin.

[0117] In a specific embodiment:

[0118] In the S6 culture medium, the antibiotic concentration is 30 g / L (optionally, for example, 10 g / L, 20 g / L, 50 g / L, etc.), the bovine serum albumin concentration is 50 g / L (optionally, for example, 0.5 g / L, 30 g / L, 100 g / L, etc.), the B-27 concentration is 1% (v / v) (optionally, 0.1% (v / v), 2% (v / v), 4% (v / v), 6% (v / v), 8% (v / v), 10% (v / v), etc.); and the human serum albumin concentration is 50 g / L (optionally, for example, 0.5 g / L, 30 g / L, etc.). The concentrations of sodium bicarbonate and glucose are as follows: 100 g / L, 1 g / L (optional, such as 0.5 g / L, 1.5 g / L, 2 g / L, etc.); GlutaMax concentration is 1% (v / v) (optional, such as 0.1% (v / v), 3% (v / v), 10% (v / v), etc.); glutamine concentration is 100 mg / L (optional, such as 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, 300 mg / L, etc.); glucose concentration is 2 g / L (optional, such as 0.1 g / L, 10 ... 1g / L, 3g / L, 4g / L, etc.), Vitamin C concentration of 50mg / L (optional, such as 15mg / L, 80mg / L, 100mg / L, etc.), Heparin sodium concentration of 25μg / mL (optional, such as 10μg / mL, 20μg / mL, 30μg / mL, 40μg / mL, 50μg / mL, etc.), Zinc sulfate concentration of 50μM (optional, such as 20μM, 40μM, 60μM, 80μM, 100μM, etc.), Y-27632 concentration of 15μM (optional, such as 5μM, 10μM, etc.), Alk5 inhibitor II concentration of 10 μM (optional, e.g., 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, etc.), T3 thyroxine concentration of 1 μM (optional, e.g., 0.1 μM, 0.5 μM, 0.8 μM, 1.5 μM, 2 μM, etc.), non-essential amino acid concentration of 0.5% (v / v) (optional, e.g., 0.1% (v / v), 0.2% (v / v), 0.4% (v / v), 0.6% (v / v), 0.8% (v / v), 1.0% (v / v), etc.), Trace Element A concentration is 0.5% (v / v) (options include, for example, 0.1% (v / v), 0.2% (v / v), 0.4% (v / v), 0.6% (v / v), 0.8% (v / v), 1.0% (v / v), etc.), and Element B concentration is 0.5% (v / v) (options include, for example, 0.1% (v / v), 0.2% (v / v), 0.4% (v / v), 0.6% (v / v), 0.The antibiotics used include: 8% (v / v), 1.0% (v / v), etc.; Trolox concentration of 15 μM (optionally, 5 μM, 10 μM, 20 μM, 25 μM, etc.); acetylcysteine ​​concentration of 1 mM (optionally, 0.1 mM, 0.5 mM, 1.5 mM, 2 mM, etc.); R-428 concentration of 5 μM (optionally, 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, etc.); and ZM-447439 concentration of 0.5 μM (optionally, 0.1 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1 μM, etc.); penicillin and / or streptomycin are used as the antibiotics.

[0119] Specifically, in step (8), the cell spheroid reaggregation specifically includes: taking 100-1500g of the culture medium after differentiation in step (7), centrifuging for 2-10min to collect cell spheroids at the bottom of the tube, discarding the supernatant after centrifugation, adding 100-1500g of 8-10mL of cell washing solution (using DPBS) to each group, centrifuging for 2-10min to wash once, discarding the supernatant, adding 8-12mL of LTrypLE (or Accutase, Trypsin) to each tube, digesting the cells at 37℃ for about 2-25min, taking them out and using a pipette to disperse the cells until a small number of cell spheroids are visible to the naked eye;

[0120] Add S6 medium to stop digestion and filter cells using a 20-150μm cell filter.

[0121] Take 100-3000g of filtered culture medium, centrifuge for 2-10 minutes, resuspend the cells in S6 medium and count them (note that the cell counting range is relatively accurate between 1E6 and 1E7, adjust the volume to this range for counting).

[0122] Dilute the cells to 1E4-5E7 / mL and seed them into 2-5 mL per well in a low-adsorption 6-well plate. Repolymerize the cell spheroids at 50-120 rpm.

[0123] In a specific embodiment,

[0124] The cell spheroid reaggregation specifically includes: taking 1000g of the culture medium after differentiation in step (7) (optional, such as 100g, 500g, 800g, 1200g, 1500g, etc.), centrifuging for 5min (optional, such as 2min, 4min, 6min, 10min, etc.) to collect cell spheroids at the bottom of the tube, discarding the supernatant after centrifugation, adding 9mL (optional, such as 8mL, 8.5mL, 10mL, etc.) of cell washing solution (using DPBS) 100-1500g to each group, centrifuging for 5min (optional, such as 2min, 4min, 6min, 10min, etc.) to wash once, discarding the supernatant, and adding 8-12mL to each tube. Digest cells with TrypLE (or Accutase, Trypsin) at 37°C for about 15 minutes (optional, such as 2 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, etc.), remove the cells and use a pipette to disperse them until a small number of cell spheroids are visible to the naked eye.

[0125] Add S6 medium to terminate digestion and filter cells using a 100 μm (optional, such as 20 μm, 60 μm, 80 μm, 150 μm, etc.) cell filter.

[0126] After centrifuging at 500g (optional, such as 100g, 500g, 800g, 1200g, 2000g, 3000g, etc.) for 2-10 minutes, resuspend the cells in S6 medium and count them (note that the most accurate cell counting range is between 1E6 and 1E7; adjust the volume to this range for counting).

[0127] Dilute the cells to 1E6 / mL (optional, such as 1E4 / mL, 1E5 / mL, 2E6 / mL, 3E7 / mL, 5E7 / mL, etc.), seed them into low-adsorption 6-well plates, 3 mL (optional, such as 2 mL, 4 mL, 5 mL, etc.) per well, and reassemble the cell spheroids at 80 rpm (optional, such as 50 rpm, 70 rpm, 90 rpm, 100 rpm, 120 rpm, etc.).

[0128] Example 1: A method for increasing the yield and improving the stability of pancreatic islet organoids differentiated from pluripotent stem cells, comprising:

[0129] Step (1): Select EB03 pluripotent stem cells with a confluence of 75%, digest them with Accutase for 5 min, terminate the digestion with basal medium, centrifuge and discard the supernatant, resuspend the cells in basal medium containing Y-27632, filter the cells through a 45 μm filter, and then count the cells.

[0130] Pluripotent stem cells were seeded into bioreactor flasks at a rate of 1E4 / mL, using mTeSR1 medium containing Y-27632 at a concentration of 25 μM. The total volume of the seeded pluripotent stem cell resuspension in the bioreactor flask plus the volume of mTeSR+Y-27632 medium was 100 mL. The culture was carried out at 45 rpm for 1 day.

[0131] In this embodiment, a total of 5 groups were inoculated, which were denoted as groups A, B, B2, C, and C2 respectively; in step (1), the operation on groups A, B, B2, C, and C2 was exactly the same.

[0132] Step (2):

[0133] Replace 90 mL of culture medium in each bioreactor bottle with mTeSR1 culture medium and continue culturing for 1 day.

[0134] In step (2), the operations for groups A, B, B2, C, and C2 are exactly the same.

[0135] Step (3.1): Wash the cells cultured in step (2) once with DPBS rinse solution, and then replace with S1D1 medium and culture for 1 day;

[0136] The S1D1 medium uses MCDB131 as the basal medium, and the basal medium is supplemented with sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin (BSA), human serum albumin (HSA), B-27, sodium bicarbonate (NaHCO3), GlutaMax, L-glutamine, glucose, vitamin C, Activin A, CHIR99021 and bFGF;

[0137] In the S1D1 medium, the concentrations were as follows: sodium pyruvate 1.5 mM, HEPES buffer 20 mM, antibiotic 30 g / L, bovine serum albumin 5 g / L, B-27 1% (v / v); human serum albumin 5 g / L, sodium bicarbonate 1 g / L, GlutaMax 3.5% (v / v); glutamine 200 mg / L; glucose 2 g / L, vitamin C 50 mg / L, Activin A 50 ng / mL, CHIR99021 3 μM, and bFGF 20 ng / mL; the antibiotics used were penicillin and / or streptomycin.

[0138] In step (3.1), the operations on groups A, B, B2, C, and C2 are exactly the same.

[0139] Step (3.2):

[0140] After changing to S1D2 medium, culture for 2-3 days;

[0141] The S1D2 medium uses MCDB131 as the basal medium, and sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, Activin A and bFGF are added to the basal medium.

[0142] In the S1D2 medium, the concentrations were as follows: sodium pyruvate 1.5 mM, HEPES buffer 20 mM, antibiotic 30 g / L, bovine serum albumin 50 g / L, B-27 1% (v / v); human serum albumin 50 g / L, sodium bicarbonate 1 g / L, GlutaMax 1% (v / v); glutamine 200 mg / L; glucose 2 g / L, vitamin C 50 mg / L, Activin A 50 ng / mL, and bFGF 20 ng / mL; penicillin was used as the antibiotic.

[0143] In step (3.2), all other culture conditions are the same, the only difference being that groups A, B, and B2 are cultured for 2 days, while groups C and C2 are cultured for 3 days; and in step (3.2), the medium is changed daily, half daily, or every other day; all groups use the same medium change method.

[0144] As shown in Table 1 and Figure 1 As shown, samples were taken from groups A, B, and B2 at S1D3 (i.e., at the end of differentiation in the S1 stage), and the positive rates of SOX17 / FOXA2 were detected. The data for groups A, B, and B2 were 76.3%, 76.3%, and 77.70%, respectively. Samples were taken from groups C and C2 at S1D4 (i.e., at the end of differentiation in the S1 stage), and the positive rates of SOX17 / FOXA2 were detected. The data for groups C and C2 were 83.50% and 81.50%, respectively. It can be seen that the DE double positivity rate in all five groups reached over 75%. Moreover, the DE differentiation efficiency in groups C and C-2 increased to over 80%. It is preliminarily judged that 4 days of differentiation in S1 is better than 3 days, that is, after changing the S1D2 medium in step (3.2) and culturing for 3 days, the corresponding DE double positivity is better.

[0145] In step (4), groups A, B, and B2 are cultured for 3 days; groups C and C2 are cultured for 2 days; and in step (4), the medium is changed daily, half daily, or every other day; each group uses the same medium change method.

[0146] S2 medium uses MCDB131 as the basal medium, and the basal medium is supplemented with sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin (BSA), human serum albumin (HSA), B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, insulin-transferrin-selenoethanol (ITS-X) and fibroblast growth factor 7 (FGF7).

[0147] In the S2 medium, the concentrations of sodium pyruvate (1.0 mM), HEPES buffer (25 mM), antibiotic (25 g / L), bovine serum albumin (50 g / L), B-27 (5% v / v), human serum albumin (50 g / L), sodium bicarbonate (1.0 g / L), GlutaMax (5% v / v), glutamine (200 mg / L), glucose (3 g / L), vitamin C (50 mg / L), insulin-transferrin-selenoethanolamine (1.0% v / v), and fibroblast growth factor 7 (50 ng / mL) were as follows:

[0148] The antibiotic used is penicillin and / or streptomycin;

[0149] Step (5):

[0150] To induce S3 stage differentiation: change the medium and culture in S3 medium for 1-2 days;

[0151] S3 culture uses MCDB131 or DMEM medium as the basal medium, and the basal medium is supplemented with sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, insulin-transferrin-selenoethanol, fibroblast growth factor 7, SANT1, retinoic acid, LDN-193189, TPPB, PdBu, Wnt-C59, Y-27632 and KAAD-Cyclopamine;

[0152] In S3 medium, the concentrations are as follows: sodium pyruvate 0-2.0 mM, HEPES buffer 0-50 mM, antibiotic 1-50 g / L, bovine serum albumin 0.5-100 g / L, B-27 0.1-10% (v / v), human serum albumin 0.5-100 g / L, sodium bicarbonate 0.5-2.0 g / L, GlutaMax 0.1-10% (v / v), glutamine 100-300 mg / L, glucose 0-4 g / L, and vitamin C 0-100 mg / L. The concentrations of L, insulin-transferrin-seleno-aminoethanol were 0-1.5% (v / v), fibroblast growth factor 7 was 20-200 ng / mL, SANT1 was 0.05-0.3 μM, retinoic acid was 1-5 μM, LDN-193189 was 50-250 nM, TPPB was 50-750 nM, PdBu was 0-0.75 μM, Wnt-C59 was 0-0.2 μM, Y-27632 was 0-25 μM, and KAAD-Cyclopamine was 0-0.45 μM.

[0153] In step (5) of this invention, groups A, B, and B2 are cultured for 1 day; groups C and C2 are cultured for 2 days; and in step (5), the medium is changed daily, half daily, or every other day; each group uses the same medium change method.

[0154] As shown in Table 1, Figure 2 As shown, samples were taken from groups A, B, and B2 at S3D1 (i.e., at the end of differentiation in stage S3), and the PDX1 positivity rates were measured. The data for groups A, B, and B2 were 78.4%, 82.2%, and 77.60%, respectively. Samples were taken from groups C and C2 at S3D2 (i.e., at the end of differentiation in stage S3), and the PDX1 positivity rates for groups C and C2 were 83.00% and 83.10%, respectively. It is evident that the PDX1 positivity rates for all five groups reached over 74%. Furthermore, the 2-day differentiation in stage S2 + 2-day differentiation in stage S3 approach resulted in a higher PDX1 positivity rate and better differentiation effect compared to the 3-day differentiation in stage S2 + 1-day differentiation in stage S3 approach.

[0155] Step (6):

[0156] S4 stage differentiation: Change the medium and culture in S4 medium for 4-7 days;

[0157] S4 medium uses MCDB131 or DMEM medium as the basal medium, and the basal medium is supplemented with sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, sodium heparin, insulin-transferrin-selenoethanolamine (ITS-X), fibroblast growth factor 7 (FGF7), epidermal growth factor (EGF), SANT1, retinoic acid, LDN-193189, TPPB, PdBu, nicotinamide, Activin A, Y-27632 and afedipine.

[0158] The concentrations of the following buffers are as follows: sodium pyruvate (0-2.0 mM), HEPES buffer (0-50 mM), antibiotic (1-50 g / L), bovine serum albumin (0.5-100 g / L), B-27 (0.1-10% v / v), human serum albumin (0.5-100 g / L), sodium bicarbonate (0.5-2.0 g / L), GlutaMax (0.1-10% v / v), glutamine (100-300 mg / L), glucose (0-4 g / L), vitamin C (0-100 mg / L), heparin sodium (0-50 μg / mL), and insulin-transferrin. The concentrations of seleno-aminoethanol were 0-1.5% (v / v), fibroblast growth factor 7 was 20-200 ng / mL, epidermal growth factor was 0-250 ng / mL, SANT1 was 0.05-0.3 μM, retinoic acid was 0.05-0.45 μM, LDN-193189 was 50-250 nM, TPPB was 50-750 nM, PdBu was 0-0.75 μM, nicotinamide was 0-20 mM, Activin A was 50-200 ng / mL, Y-27632 was 0-25 μM, and afedipine was 0-5 μM.

[0159] In step (6) of this invention, groups A, C, and C2 are cultured for 4 days; groups B and B2 are cultured for 5 days; and in step (6), the medium is changed daily, half daily, or every other day; each group uses the same medium change method.

[0160] As shown in Table 1, Figure 3As shown, samples were taken from groups A, C, and C2 on day 4 of stage S4, and the positive rates of PDX1 / NKX6.1 were detected. The data for groups A, C, and C2 were 43.3%, 58.3%, and 58.70%, respectively. Samples were taken from groups B and B2 on day 5 of stage S4, and the positive rates of PDX1 / NKX6.1 were detected. The data for groups B and B2 were 45.20% and 52.40%, respectively. The positive rates of PDX1 / NKX6.1 in all five groups reached over 43%; and the data for groups C and C2 were both higher than 58%, which was better than the data for groups B and B2. Therefore, in the S4 stage, 4 days of culture was more effective than 5 days. The reason why the data for group A was lower was that the culture time for group A in the S3 stage was 1 day, which affected the data here. Therefore, in summary, culture for 2 days in the S3 stage and 4 days in the S4 stage, and then cultured for 4 days after the end of the S4 stage, can achieve a positive rate of PDX1 / NKX6.1 higher than 58%.

[0161] Step (7):

[0162] To induce S5 stage differentiation: change the medium and culture in S5 medium for 3-10 days;

[0163] S5 medium uses MCDB131 or RPMI1640 as the basal medium, and the basal medium is supplemented with antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, sodium heparin, zinc sulfate, SANT1, retinoic acid, LDN-193189, Y-27632, Alk5 inhibitor II, T3 thyroxine, γ-secretase inhibitor (GSI-IX), Betacellulin, ISX9, aphidicolin, and Wnt-C59.

[0164] The concentrations of the antibiotics were 25 g / L, bovine serum albumin 50 g / L, B-27 5% (v / v), human serum albumin 50 g / L, sodium bicarbonate 1.0 g / L, GlutaMax 5% (v / v), glutamine 150 mg / L, glucose 2 g / L, vitamin C 50 mg / L, heparin sodium 25 μg / mL, zinc sulfate 50 μM, and SANT1 0. 15 μM, retinoic acid concentration of 0.5 μM, LDN-193189 concentration of 100 nM, Y-27632 concentration of 15 μM, Alk5 inhibitor II concentration of 10 μM, T3 thyroxine concentration of 1.0 μM, γ-secretase inhibitor XX concentration of 0.05 μM, Betacellulin concentration of 15 ng / mL, ISX9 concentration of 5 μM, afedipine concentration of 2.5 μM, and Wnt-C59 concentration of 0.1 μM;

[0165] In step (7), groups A, B, B2, C, and C2 are cultured in S5 medium for 3-10 days; and in step (7), the medium is changed daily, half daily, or every other day; each group uses the same medium change method.

[0166] In step (7) of this invention, on day 7 of S5 culture, the following cells were detected in groups A, B, B2, C, and C2: CHGA single positive, pancreatic α-cell GCG, early pancreatic α-cell C-Peptide / GCG, pancreatic β-cell C-Peptide / NKX6.1, pancreatic β-endocrine cells C-Peptide / CHGA, pancreatic δ-cell SST single positive, and enterochromaffin SLC18A1 single positive. Groups B and B2 were two parallel replicates with identical pretreatment; therefore, after 7 days of S5 culture, the cells were combined, mixed, and sampled for flow cytometry analysis. Groups C and C2 were also two parallel replicates with identical pretreatment; therefore, after 7 days of S5 culture, the cells were combined, mixed, and sampled for flow cytometry analysis.

[0167] like Figures 4-7 As shown, the positive rate of CHGA endocrine cells was between 63% and 69%, while the positive rates of SST pancreatic δ cells and SLC18A1 enterochromaffin cells were both below 5%, indicating that the differentiation method and formulation of S5 can ensure the differentiation of pancreatic progenitor cells into endocrine-related cells. Furthermore, some pancreatic β cells showed differentiation at the S5 stage, with C-Peptide / NKX6.1 showing a positive expression rate of 15-28%.

[0168] From a yield perspective, a cell count of 0.6-1.14E8 / vial is representative. However, the yields of groups A and C are higher than those of group B, indicating that optimizing the differentiation time in the first five stages can also lead to yield optimization, which is beneficial for increasing the yield of pancreatic islet organoids.

[0169] Step (8):

[0170] Reaggregation and further differentiation: First, cell spheroids are reaggregated and cultured in S6 medium for 3-21 days to obtain pancreatic islet organoids. In this example, the specific steps for cell spheroid reaggregation in groups A, B, B2, C, and C2 are as follows:

[0171] Reaggregation of Group A Cells: Take 1000g of the culture medium from Group A after differentiation in step (7), centrifuge for 6min to collect Group A cell spheroids at the bottom of the tube, discard the supernatant after centrifugation, add 9mL of cell washing buffer to each group, centrifuge for 8min to wash once, discard the supernatant, add 10mL TrypLE to each tube, digest the cells at 37℃ for about 15min, remove and pipette to disperse the cells until no cell spheroids are visible to the naked eye. Add 3 times the volume of S6 medium to stop digestion, and filter the cells using an 80μm cell filter. Take 1000g of the filtered culture medium, centrifuge for 8min, resuspend the cells in 5mL of S6 medium and count them (note that the cell counting range is relatively accurate between 1E6-1E7, adjust the volume to this range for counting). Then dilute the cells to 1E6 / mL, seed them in a low-adsorption 6-well plate, 3mL per well, and reaggregate the cell spheroids at 60rpm.

[0172] Group A counting results: cell viability 95.11%, total cell concentration 1.2E+07, viable cell concentration 1.14E+07, total cell count 6E+07, viable cell count 5.7E+07.

[0173] B and B2 group cell spheroid reaggregation: The treatment steps are the same as those for group A cell spheroid reaggregation, the only difference being that after washing the cells and discarding the supernatant, 10 mL of Gentle Cell Dissociation Reagent (GCDR) is added to each group and the cells are digested at 37°C for about 2-25 min.

[0174] The results of combining and counting B and B2 cells were as follows: cell viability 68.43%, total cell concentration 1.76E+07, viable cell concentration 1.21E+07, total cell count 1.76E+08, viable cell count 1.21E+08. After thorough mixing, the cells were transferred to a low-attachment 6-well plate (out of 12 wells).

[0175] Cell spheroid reaggregation in groups C and C2: The treatment steps are the same as those for group A.

[0176] The combined count results of C and C2 were as follows: cell viability 96.01%, total cell concentration 1.95E+07, viable cell concentration 1.87E+07, total cell count 1.95E+08, and viable cell count 1.87E+08. After thorough mixing, the cells were transferred to a low-attachment 6-well plate (out of 12 wells).

[0177] The experimental results show that TrypLE digestion resulted in better repolymerization than GCDR. In S6D1 (the first day of S6), group A, digested with TrypLE for 4 minutes, achieved a cell viability of 95%. Group B, digested with GCDR for 8 minutes, only achieved a cell viability of 68%. Group C, digested with TrypLE for 5 minutes with reduced pipetting, achieved a cell viability of 96%. Furthermore, regarding the repolymerization effect in S6, groups B and B2 showed worse repolymerization than groups C and C2, exhibiting cell spheroids of varying sizes (adhesion).

[0178] After cell spheroids in groups A, B, B2, C, and C2 were reassembled, they were cultured in S6 medium for 15 days. In step (8), the medium was changed daily, half daily, or every other day. All groups used the same medium change method to obtain pancreatic islet organoids. On day 9 and day 14 of the S6 stage, the flow cytometry results and GSIS results of C-Peptide / NKX6.1, C-Peptide / CHGA, CHGA single positive, C-Peptide / GCG were detected. Sampling was also taken to detect GSIS results, and sampling was taken to detect the immunofluorescence results of Merge / DAPI / NKX6.1 / C-Peptide, Merge / DAPI / CHGA / C-Peptide, and Merge / DAPI / GCG / C-Peptide.

[0179] According to Table 1, Figures 8-12 It can be seen that the positive ratio of CP / NKX6.1 pancreatic β cells in groups B and B2 is not as ideal as in groups A, C, and C2. Although GSIS shows a value greater than 1, the absolute value of insulin secreted per unit cell (uIU / 1000 cells) is lower than that in groups A, C, and C2. The main reason is that the previous differentiation stages resulted in a lower double positive ratio of PDX1 / NKX6.1 in pancreatic progenitor cells compared to other groups. This further illustrates that differentiation at day 4 is superior to differentiation at day 5, and that a high proportion of PDX1 / NKX6.1 is a prerequisite for promoting pancreatic β cell differentiation.

[0180] Furthermore, the overall positive rate of C-Peptide / NKX6.1 in the five groups was lower in S6D14 than in S6D9, indicating that the pancreatic β cells showed a decline or instability in vitro after differentiation and maturation. Although the proportion of CHGA single positivity increased, the C-Peptide / CHGA ratio decreased, indicating that the expression of endocrine-related factors in pancreatic organoids increased, but the proportion of pancreatic β cell subsets decreased. Moreover, the GSIS in groups A, C, and C2 was all less than 1, indicating a decline in the function of pancreatic β cells in sensing glucose stimulation and secreting insulin. The 9-day differentiation period in stage 6 (S6D9) yielded better results.

[0181] Table 1 Summary of flow cytometry detection at different differentiation stages under different conditions

[0182]

[0183]

[0184]

[0185] This is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for increasing the yield and improving the stability of pancreatic islet organoids differentiated from pluripotent stem cells, characterized in that, The method includes: (1) Seeding and culturing pluripotent stem cells: Seeding pluripotent stem cells and culturing them in a basic culture medium containing Y-27632; (2) Culture in a 3D environment: change the medium and continue culturing using basal culture medium; (3) Perform S1 stage differentiation: first culture in S1D1 medium for 1 day, then culture in S1D2 medium for 2-3 days; (4) Perform S2 stage differentiation: Change the medium and culture in S2 medium for 2-3 days; (5) Perform S3 stage differentiation: Change the medium and culture in S3 medium for 1-2 days; (6) Perform S4 stage differentiation: Change the medium and culture in S4 medium for 4-7 days; (7) Perform S5 stage differentiation: Change the medium and culture in S5 medium for 3-10 days; (8) Reaggregate and continue to differentiate: First, reaggregate the cell spheres and culture them in S6 medium for 3-21 days to obtain pancreatic islet organoids.

2. The method for increasing the yield and improving the stability of pancreatic islet organoids differentiated from pluripotent stem cells according to claim 1, characterized in that, Step (1) specifically involves: selecting pluripotent stem cells with a confluence of 60-95%, digesting them for 1-10 minutes, terminating the digestion with basal culture medium, centrifuging and discarding the supernatant, resuspending them in basal culture medium containing Y-27632, filtering the cells through a 20-100 μm filter, and then counting the cells. Pluripotent stem cells were seeded into 100 mL bioreactor flasks at a seeding rate of 1E3 / mL–1E7 / mL, using basal medium containing Y-27632, and cultured at a rotation speed of 30–100 rpm for 1 day. The basal culture medium includes any one of mTeSR1 medium, E8 medium, mTeSR Plus medium, mTeSRAOF medium, and NutriStem hPSC XF Medium. The concentration of Y-27632 in the basal culture medium containing Y-27632 is 2-50 μM.

3. The method for increasing the yield and improving the stability of pancreatic islet organoids differentiated from pluripotent stem cells according to claim 2, characterized in that, Step (2) specifically involves replacing 70-90 mL of culture medium in each bioreactor bottle with basic culture medium and continuing the culture for 0-2 days. The basal culture medium includes any one of mTeSR1 medium, E8 medium, mTeSR Plus medium, mTeSRAOF medium, and NutriStem hPSC XF Medium.

4. The method for increasing the yield and improving the stability of pancreatic islet organoids differentiated from pluripotent stem cells according to claim 1, characterized in that, Step (3) includes: (3.1) Wash the cells cultured in step (2) once with DPBS washing solution and replace with S1D1 medium for 1 day; The S1D1 medium uses MCDB131 or RPMI1640 as the basal medium, and the basal medium is supplemented with sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, Activin A, CHIR99021 and bFGF. In the S1D1 medium, the concentrations are as follows: sodium pyruvate 0-2.0 mM, HEPES buffer 0-50 mM, antibiotic 1-50 g / L, bovine serum albumin 0.5-100 g / L, B-27 0.1-10% (v / v); human serum albumin 0.5-100 g / L, sodium bicarbonate 0.5-2 g / L, GlutaMax 0.1-10% (v / v); glutamine 100-300 mg / L; glucose 0-4 g / L, vitamin C 0-100 mg / L, Activin A 50-200 ng / mL, CHIR99021 0-5 μM, and bFGF 0-50 ng / mL; wherein the antibiotics used are penicillin and / or streptomycin. (3.2) After changing to S1D2 medium, culture for 2-3 days; The S1D2 medium uses MCDB131 or RPMI1640 as the basal medium, and the basal medium is supplemented with sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, Activin A and bFGF. In the S1D2 medium, the concentrations of sodium pyruvate (0-2.0 mM), HEPES buffer (0-50 mM), antibiotic (1-50 g / L), bovine serum albumin (0.5-100 g / L), B-27 (0.1-10% v / v), human serum albumin (0.5-100 g / L), sodium bicarbonate (0.5-2 g / L), GlutaMax (0.1-10% v / v), glutamine (100-300 mg / L), glucose (0-4 g / L), vitamin C (0-100 mg / L), Activin A (50-200 ng / mL), and bFGF (0-50 ng / mL) are specified. The antibiotics used are penicillin and / or streptomycin.

5. The method for increasing the yield and improving the stability of pancreatic islet organoids differentiated from pluripotent stem cells according to claim 1, characterized in that, In step (4), the S2 medium uses MCDB131 or RPMI1640 as the basal medium, and sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, insulin-transferrin-selenoethanol and fibroblast growth factor 7 are added to the basal medium. In the S2 medium, the concentrations are as follows: sodium pyruvate 0-2.0 mM, HEPES buffer 0-50 mM, antibiotic 1-50 g / L, bovine serum albumin 0.5-100 g / L, B-27 0.1-10% (v / v), human serum albumin 0.5-100 g / L, sodium bicarbonate 0.5-2.0 g / L, GlutaMax 0.1-10% (v / v), glutamine 100-300 mg / L, glucose 0-4 g / L, vitamin C 0-100 mg / L, insulin-transferrin-selenoethanol 0-1.5% (v / v), and fibroblast growth factor 7 20-200 ng / mL; wherein the antibiotics are penicillin and / or streptomycin.

6. The method for increasing the yield and improving the stability of pancreatic islet organoids differentiated from pluripotent stem cells according to claim 1, characterized in that, In step (5), the S3 culture uses MCDB131 or DMEM medium as the basal medium, and sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, insulin-transferrin-selenoethanol, fibroblast growth factor 7, SANT1, retinoic acid, LDN-193189, TPPB, PdBu, Wnt-C59, Y-27632 and KAAD-Cyclopamine are added to the basal medium. In the S3 medium, the concentrations of sodium pyruvate (0-2.0 mM), HEPES buffer (0-50 mM), antibiotics (1-50 g / L), bovine serum albumin (0.5-100 g / L), B-27 (0.1-10% v / v), human serum albumin (0.5-100 g / L), sodium bicarbonate (0.5-2.0 g / L), GlutaMax (0.1-10% v / v), glutamine (100-300 mg / L), glucose (0-4 g / L), vitamin C (0-100 mg / L), and insulin-transferrin were present. The concentrations of protein-selenium-aminoethanol are 0-1.5% (v / v), fibroblast growth factor 7 is 20-200 ng / mL, SANT1 is 0.05-0.3 μM, retinoic acid is 1-5 μM, LDN-193189 is 50-250 nM, TPPB is 50-750 nM, PdBu is 0-0.75 μM, Wnt-C59 is 0-0.2 μM, Y-27632 is 0-25 μM, and KAAD-Cyclopamine is 0-0.45 μM; wherein, the antibiotics used are penicillin or streptomycin.

7. The method for increasing the yield and improving the stability of pancreatic islet organoids differentiated from pluripotent stem cells according to claim 1, characterized in that, In step (6), the S4 medium uses MCDB131 or DMEM medium as the basal medium, and sodium pyruvate, HEPES buffer, antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, heparin sodium, insulin-transferrin-selenoethanol, fibroblast growth factor 7, epidermal growth factor, SANT1, retinoic acid, LDN-193189, TPPB, PdBu, nicotinamide, Activin A, Y-27632 and afedipine are added to the basal medium. In the S4 medium, the concentrations of sodium pyruvate (0-2.0 mM), HEPES buffer (0-50 mM), antibiotics (1-50 g / L), bovine serum albumin (0.5-100 g / L), B-27 (0.1-10% v / v), human serum albumin (0.5-100 g / L), sodium bicarbonate (0.5-2.0 g / L), GlutaMax (0.1-10% v / v), glutamine (100-300 mg / L), glucose (0-4 g / L), vitamin C (0-100 mg / L), heparin sodium (0-50 μg / mL), and insulin-transferrin-seleno-amino acid... The concentrations of the following antibiotics are as follows: ethanol 0-1.5% (v / v), fibroblast growth factor 7 20-200 ng / mL, epidermal growth factor 0-250 ng / mL, SANT1 0.05-0.3 μM, retinoic acid 0.05-0.45 μM, LDN-193189 50-250 nM, TPPB 50-750 nM, PdBu 0-0.75 μM, nicotinamide 0-20 mM, Activin A 50-200 ng / mL, Y-27632 0-25 μM, and afedipine 0-5 μM; wherein, the antibiotics used are penicillin or streptomycin.

8. The method for increasing the yield and improving the stability of pancreatic islet organoids differentiated from pluripotent stem cells according to claim 1, characterized in that, In step (7), the S5 medium uses MCDB131 or RPMI1640 as the basal medium, and antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, sodium heparin, zinc sulfate, SANT1, retinoic acid, LDN-193189, Y-27632, Alk5 inhibitor II, T3 thyroxine, γ secretase inhibitor, Betacellulin, ISX9, afedipine and Wnt-C59 are added to the basal medium. In the S5 medium, the concentrations of antibiotics were 1-50 g / L, bovine serum albumin 0.5-100 g / L, B-27 0.1-10% (v / v), human serum albumin 0.5-100 g / L, sodium bicarbonate 0.5-2.0 g / L, GlutaMax 0.1-10% (v / v), glutamine 100-300 mg / L, glucose 0-4 g / L, vitamin C 0-100 mg / L, heparin sodium 0-50 μg / mL, zinc sulfate 0-100 μM, and SANT1. The concentrations of the antibiotics are 0-0.3 μM, 0.01-0.10 μM for retinoic acid, 50-250 nM for LDN-193189, 0-25 μM for Y-27632, 0-20 μM for Alk5 inhibitor II, 0.1-2 μM for T3 thyroxine, 0-0.1 μM for γ-secretase inhibitor, 0-25 ng / mL for Betacellulin, 0-10 μM for ISX9, 0-5 μM for afedipine, and 0-0.2 μM for Wnt-C59; wherein the antibiotics used are penicillin or streptomycin.

9. The method for increasing the yield and improving the stability of pancreatic islet organoids differentiated from pluripotent stem cells according to claim 1, characterized in that, In step (8), the cell spheroid reaggregation specifically includes: centrifuging and discarding the supernatant, collecting cell spheroids, adding cell washing solution and centrifuging to wash, discarding the supernatant and adding TrypLE, Accutase, Gentle Cell Dissociation Reagen or trypsin to digest the cells, using a pipette to disperse the cells, adding S6 medium to stop the digestion; filtering with a cell filter, centrifuging and resuspending the cells in S6 medium and counting; diluting the cells and seeding them in a cell culture plate to reaggregate the cell spheroids; The S6 medium uses MCDB131 or RPMI1640 as the basal medium, and antibiotics, bovine serum albumin, human serum albumin, B-27, sodium bicarbonate, GlutaMax, glutamine, glucose, vitamin C, sodium heparin, zinc sulfate, Y-27632, Alk5 inhibitor II, T3 thyroxine, non-essential amino acids, TraceA Element, Trace B Element, Trolox, acetylcysteine, R-428, and ZM-447439 are added to the basal medium. In the S6 medium, the concentrations of antibiotics were 1-50 g / L, bovine serum albumin 0.5-100 g / L, B-27 0.1-10% (v / v), human serum albumin 0.5-100 g / L, sodium bicarbonate 0.5-2.0 g / L, GlutaMax 0.1-10%, glutamine 100-300 mg / L, glucose 0-4 g / L, vitamin C 0-100 mg / L, heparin sodium 0-50 μg / mL, zinc sulfate 0-100 μM, Y-27632 0-25 μM, Alk5 inhibitor II 0-20 μM, T3 thyroxine 0.1-2 μM, non-essential amino acids 0-1.0% (v / v), and Trace A Element 0-1.0% (v / v). The concentrations of BElement (0-1.0% (v / v), Trolox (0-25 μM), acetylcysteine ​​(0-2 mM), R-428 (0-10 μM), and ZM-447439 (0-1 μM) are specified; wherein the antibiotics used are penicillin or streptomycin.

10. The method for increasing the yield and improving the stability of pancreatic islet organoids differentiated from pluripotent stem cells according to claim 9, characterized in that, During the cell spheroid reaggregation process, TrypLE was added to digest the cells.

11. The method for increasing the yield and improving the stability of pancreatic islet organoids differentiated from pluripotent stem cells according to claim 1, characterized in that, In step (8), the culture was carried out in S6 medium for 9 days.

12. The method for increasing the yield and improving the stability of pancreatic islet organoids differentiated from pluripotent stem cells according to claim 11, characterized in that, When cultured in S6 medium for 9 days, the GSIS value was >1, and the positive rate of CP / NKX6.1 pancreatic β cells reached 53%.

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