CN114561341A - Three-dimensional islet organoid based on iPSC and construction method thereof - Google Patents

Three-dimensional islet organoid based on iPSC and construction method thereof Download PDF

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CN114561341A
CN114561341A CN202210351863.8A CN202210351863A CN114561341A CN 114561341 A CN114561341 A CN 114561341A CN 202210351863 A CN202210351863 A CN 202210351863A CN 114561341 A CN114561341 A CN 114561341A
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林昊
孙博
叶正
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Southeast University
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Abstract

The invention relates to a method for constructing three-dimensional islet organoids based on pluripotent stem cells (iPSCs), which belongs to the technical field of biomedicine, and the method does not use matrigel, but adopts agarose to combine with gellan gum to realize three-dimensional conglobation of cells, and finally induces the three-dimensional islet organoids containing all 4 islet cells by adding compounds with definite components in different induction periods.

Description

Three-dimensional islet organoid based on iPSC and construction method thereof
Technical Field
The disclosure belongs to the technical field of biomedicine, and particularly relates to a three-dimensional pancreatic islet organoid construction technology based on pluripotent stem cells (iPSCs).
Background
Pancreatic islet function is lost in type 1 diabetic patients, and the only way to completely cure type 1 diabetes is islet transplantation. The three-dimensional islet organoids are constructed by utilizing pluripotent stem cells (iPSCs), so that a wide islet source is provided for islet transplantation. The existing method for constructing the islet organoid is long in time and multiple in steps, and needs to use patents of foreign companies: and (3) matrigel. The matrigel is used for supporting the islets to form a three-dimensional structure, but the matrigel is expensive and complex to operate, the islets need to be slowly melted on ice for more than one hour in advance each time, and the temperature is higher than 10 ℃ and the islets are coagulated. Most importantly, matrigel has different components from batch to batch due to the limitation of the production process. Therefore, the three-dimensional islet organoid construction technology using matrigel has fatal problems in industrial large-scale mass production in the future, and the uniformity of the produced islet organoids in clinical application cannot be guaranteed. Furthermore, the natural islets contain 4 cells in total, whereas the islet organoids obtained by the existing construction methods usually contain only 2-3 cells.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a three-dimensional pancreatic islet organoid construction technology based on pluripotent stem cells (iPSCs), and solves the problem that the construction of the pancreatic islet organoids is difficult in the prior art.
The purpose of the disclosure can be realized by the following technical scheme:
a pluripotent stem cell (iPSC) -based three-dimensional islet organoid construction technique, comprising the steps of;
preparation of S1 three-dimensional gellan gum culture medium
(1) Gellan gum powder (Sigma) was formulated with ultrapure water into a 0.3% (w/v) aqueous solution and autoclaved.
(2) Then, a three-dimensional gellan gum medium working solution was prepared by adding the following components using mTeSR1 solution (StemCell corporation) as a solvent: gellan gum (final concentration 0.015-0.02%), methylcellulose (final concentration 0.2-0.3%), ROCK inhibitor (final concentration 8-15 μ M), and streptomycin (final concentration 1%).
S2: 0.8-1% agarose is prepared with ultrapure water, autoclaved, and poured onto a flat plate while hot. Add 1ml of three-dimensional gellan gum medium to each dish and equilibrate for at least 20 minutes.
S3: induced pluripotent stem cells (ipscs) of human origin were digested with 500ul accutase at 37 ℃ for 3 to 5 minutes, and the cells were resuspended in three-dimensional gellan gum medium to prepare a single cell suspension. The cell suspension was plated on an agarose plate and cultured for 2-5 days, to visualize the iPSC cell pellet.
S4: the following 5 kinds of culture solutions were prepared
Solution A comprises MCDB131500ml, 600 mu l of 50% glucose 400-;
the solution B comprises MCDB131500ml, 600 mul of 50 percent glucose 400-acetic acid, 30.6-0.7g of NaHCO, 10g of BSA, 10 mul of ITS-X, 5ml of GlutaMax, 20-30mg of vitamin C and 5ml of streptomycin qinghao;
solution C comprises MCDB131500ml, 600 mul of 50% glucose 400-;
solution D comprises MCDB131500ml, 600 mul of 50 percent glucose 400-;
the E solution comprises MCDB131500ml, 3000 mu.l of 50% glucose 2500-.
S5: the culture medium was changed to solution A and gellan gum (0.015-0.02%), methylcellulose (0.2-0.3%), activin A (100ng/ml), CHIR99021(2-4uM) were added and cultured for 1 day.
S6: the medium was changed to solution A and gellan gum (0.015-0.02%), methylcellulose (0.2-0.3%), activin A (100ng/ml) were added and cultured for 1 day.
S7: the medium was changed to solution B and FGF7(30-60ng/ml) was added and the cells were cultured for 2 days.
S8: the medium was changed to liquid C and FGF7(30-60ng/ml), SANT-1 (0.2-0.3. mu.M), retinoic acid (0.8-1. mu.M), LDN193189(100-150nM), Repsox (5-10. mu.M), TPB (200-250nM) were added thereto and cultured for 3 days.
S9: the medium was changed to liquid C and FGF7(30-60ng/ml), SANT-1 (0.2-0.3. mu.M), retinoic acid (0.8-1. mu.M), LDN193189(100-150nM), Repsox (5-10. mu.M), TPB (100-120nM) were added thereto and cultured for 3 days.
S10: the medium was changed to D medium and added with SANT-1 (0.2-0.3. mu.M), retinoic acid (40-80nM), LDN193189(100-150nM), Repsox (5-10. mu.M), T3 (1-1.2. mu.M), and cultured for 3 days
S11: replacing the culture medium with E solution, adding LDN193189(100-150nM), GSiXX (80-100nM), Repsox (5-10. mu.M), T3 (1-1.2. mu.M), and culturing for 7 days;
s12: changing the culture medium to E solution, adding Trolox (10-15 μ M), R428(2-2.5 μ M), N-acetylcysteine (1-1.2mM), Repsox (5-10 μ M), T3(1-1.2 μ M), and culturing for 14 days;
s13: the medium was changed to E medium and added with Trolox (10-15. mu.M), R428 (2-2.5. mu.M), N-acetylcysteine (1-1.2mM), Repsox (5-10. mu.M), T3 (1-1.2. mu.M), WNT4(100-200ng/ml), and cultured for 7 days;
an iPSC-based three-dimensional islet organoid constructed from agarose in combination with gellan gum.
An iPSC-based three-dimensional islet organoid cultured by the culture solution of claim 3.
An iPSC-based three-dimensional islet organoid constructed by one of the methods of claims 1-6.
A diabetes treatment drug product comprising insulin synthesized by one of the three-dimensional islet organoids of claims 7-9.
Has the advantages that:
1. the invention adopts agarose combined gellan gum to replace matrigel, thereby realizing great reduction of cost.
2. The operation is simple, and the process repeatability is high; the invention adopts agarose combined gellan gum to replace matrigel, and has simple operation and high process repeatability. The matrigel needs to be stored at the temperature of-20 ℃, needs to be melted on ice for at least 1 hour when being taken and used every time, and is condensed when the temperature is higher than 10 ℃, so that the operation is inconvenient. The agarose and the gellan gum can be prepared at normal temperature, can be stored for a long time at normal temperature after being sterilized at high pressure, and are convenient to use.
3. The three-dimensional islet organoid has good homogeneity; the invention adopts agarose combined with gellan gum to replace matrigel, and the three-dimensional islet organoid has good homogeneity. Matrigel is an extracellular basement membrane matrix extracted from mouse tumors. Due to individual differences of mice, the components of different batches of matrigel are different, which may result in poor uniformity of three-dimensional islet organoids. The agarose is combined with the gellan gum, the components are determined, the three-dimensional islet organoid is easy to produce in large scale in industry, and the obtained three-dimensional islet organoid has good uniformity
4. The three-dimensional islet organoid obtained by inducing the A-E liquid is closer to the cell composition of natural islets; the three-dimensional islet organoid obtained by the present invention contains all 4 known islet cells, i.e. alpha cells, beta cells, delta cells, PP cells, which are closer to the cell composition of natural islets than other methods. While other inventions contain only 2-3 of the above cells, the absence of 1-2 cells will result in pancreatic islet insufficiency.
Drawings
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present disclosure, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
Fig. 1 is an effect diagram of embodiment 1 of the present disclosure;
fig. 2 is an effect diagram of embodiment 2 of the present disclosure;
fig. 3 is an effect diagram of embodiment 3 of the present disclosure.
Detailed Description
The technical solutions in the embodiments of the present disclosure will be described clearly and completely with reference to the drawings in the embodiments of the present disclosure, and it is obvious that the embodiments described are only some embodiments of the present disclosure, rather than all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments disclosed herein without inventive step, are intended to be within the scope of the present disclosure.
Example 1: a pluripotent stem cell (iPSC) -based three-dimensional islet organoid construction technique, comprising the steps of;
a pluripotent stem cell (iPSC) -based three-dimensional islet organoid construction technique, comprising the steps of;
preparation of S1 three-dimensional gellan gum culture medium
(1) Gellan gum powder (Sigma) was formulated with ultrapure water into a 0.3% (w/v) aqueous solution and autoclaved.
(2) Then, a three-dimensional gellan gum culture medium working solution was prepared by adding the following components using mTeSR1 solution (StemCell corporation) as a solvent: gellan gum (final concentration 0.015%), methylcellulose (final concentration 0.2%), ROCK inhibitor (final concentration 8 μ M), penicillin streptomycin (final concentration 1%);
s2: 0.8% agarose was prepared with ultrapure water, autoclaved, and poured onto a flat plate while hot. Add 1ml of three-dimensional gellan gum medium to each dish and equilibrate for at least 20 minutes.
S3: induced pluripotent stem cells (ipscs) of human origin were digested with 500ul accutase at 37 ℃ for 3 minutes, and the cells were resuspended in three-dimensional gellan gum medium to prepare a single cell suspension. The cell suspension was plated on an agarose plate and cultured for 2 days, to visualize the iPSC cell pellet.
S4: the following 5 kinds of culture solutions were prepared
Solution A comprises MCDB131500ml, 400 μ l of 50% glucose, NaHCO31.2 g, 10g of BSA, 10 μ l of ITS-X, 5ml of GlutaMax, 20mg of vitamin C and 5ml of streptomycin qinghaosu;
solution B including MCDB131500ml, 50% glucose 400. mu.l, NaHCO30.6g, BSA10g, ITS-X10. mu.l, GlutaMax 5ml, vitamin C20 mg, and streptomycin 5 ml;
solution C including MCDB131500ml, 50% glucose 400 μ l, NaHCO30.6g, BSA10g, ITS-X2.5 ml, GlutaMax 5ml, vitamin C20 mg, and streptomycin 5 ml;
solution D comprises MCDB131500ml, 400 μ l of 50% glucose, NaHCO30.6g, BSA10g, ITS-X2.5 ml, GlutaMax 5ml, vitamin C20 mg, streptomycin 5ml, heparin 5mg, and zinc sulfate 1.2 mg;
solution E comprises MCDB131500ml, 2500 mul of 50% glucose, 30.7g of NaHCO, 10g of BSA, 2.5ml of ITS-X, 5ml of GlutaMax, 20mg of vitamin C, 5ml of streptomycin, 5mg of heparin and 1.2mg of zinc sulfate.
S5: the medium was changed to solution A and gellan gum (0.015%), methylcellulose (0.2%), activin A (100ng/ml), CHIR99021(2uM) were added thereto and cultured for 1 day.
S6: the medium was changed to solution A and gellan gum (0.015%), methylcellulose (0.2%), activin A (100ng/ml) were added thereto and cultured for 1 day.
S7: the medium was changed to solution B and FGF7(30ng/ml) was added and the cells were cultured for 2 days.
S8: the medium was changed to solution C and FGF7(30ng/ml), SANT-1 (0.2. mu.M), retinoic acid (0.8. mu.M), LDN193189(100nM), Repsox (5. mu.M), TPB (200nM) were added thereto and cultured for 3 days.
S9: the medium was changed to solution C and FGF7(30ng/ml), SANT-1 (0.2. mu.M), retinoic acid (0.8. mu.M), LDN193189(100nM), Repsox (5. mu.M), TPB (100nM) were added thereto and cultured for 3 days.
S10: replacing the medium with D solution, adding SANT-1(0.2 μ M), retinoic acid (40nM), LDN193189(100nM), Repsox (5 μ M), T3(1 μ M), and culturing for 3 days;
s11: the medium was changed to E medium and LDN193189(100nM), GSiXX (80nM), RepSox (5. mu.M), T3(1. mu.M) were added thereto and cultured for 7 days;
s12: the medium was changed to E medium and Trolox (10. mu.M), R428(2. mu.M), N-acetylcysteine (1mM), Repsox (5. mu.M), T3(1. mu.M) were added thereto, followed by culture for 14 days;
s13: the medium was changed to E medium, and Trolox (10. mu.M), R428(2. mu.M), N-acetylcysteine (1mM), Repsox (5. mu.M), T3(1. mu.M), WNT4(100ng/ml) were added thereto and cultured for 7 days.
Example 2
A pluripotent stem cell (iPSC) -based three-dimensional islet organoid construction technique, comprising the steps of;
preparation of S1 three-dimensional gellan gum culture medium
(1) Gellan gum powder (Sigma) was formulated with ultrapure water into a 0.3% (w/v) aqueous solution and autoclaved.
(2) Then, a three-dimensional gellan gum medium working solution was prepared by adding the following components using mTeSR1 solution (StemCell corporation) as a solvent: gellan gum (final concentration 0.02%), methylcellulose (final concentration 0.3%), ROCK inhibitor (final concentration 15 μ M), streptomycin (final concentration 1%);
s2: 1% agarose was prepared using ultrapure water, autoclaved, and poured onto plates while hot. 1ml of three-dimensional gellan gum medium was added to each dish and equilibrated for at least 20 minutes.
S3: induced pluripotent stem cells (ipscs) of human origin were digested with 500ul accutase at 37 ℃ for 5 minutes, and the cells were resuspended in three-dimensional gellan gum medium to prepare a single cell suspension. The cell suspension was plated on an agarose plate and cultured for 5 days, and the iPSC cell pellet was observed.
S4: the following 5 kinds of culture solutions were prepared
Solution A comprises MCDB131500ml, 600 μ l of 50% glucose, NaHCO31.2-1.5g, BSA10g, ITS-X10 μ l, GlutaMax 5ml, vitamin C30 mg, and streptomycin 5 ml;
solution B including MCDB131500ml, 50% glucose 600 μ l, NaHCO30.7g, BSA10g, ITS-X10 μ l, GlutaMax 5ml, vitamin C30 mg, and streptomycin 5 ml;
solution C comprises MCDB131500ml, 600 μ l of 50% glucose, NaHCO30.7g, 10g of BSA, 2.5ml of ITS-X, 5ml of GlutaMax, 30mg of vitamin C and 5ml of streptomycin qinghaosu;
solution D comprises MCDB131500ml, 600 mul of 50% glucose, NaHCO30.7g, 10g of BSA, 2.5ml of ITS-X, 5ml of GlutaMax, 30mg of vitamin C, 5ml of streptomycin, 5mg of heparin and 1.5mg of zinc sulfate;
solution E comprises MCDB131500ml, 3000 mu.l of 50% glucose, 30.9 g of NaHCO, 10g of BSA, 2.5ml of ITS-X, 5ml of GlutaMax, 30mg of vitamin C, 5ml of streptomycin, 5mg of heparin and 1.5mg of zinc sulfate.
S5: the medium was changed to solution A and gellan gum (0.02%), methylcellulose (0.3%), activin A (100ng/ml), CHIR99021(4uM) were added thereto and cultured for 1 day.
S6: the medium was changed to solution A and gellan gum (0.02%), methylcellulose (0.3%), and activin A (100ng/ml) were added thereto and cultured for 1 day.
S7: the medium was changed to solution B and FGF7(60ng/ml) was added and the cells were cultured for 2 days.
S8: the medium was changed to liquid C and FGF7(60ng/ml), SANT-1 (0.3. mu.M), retinoic acid (1. mu.M), LDN193189(150nM), Repsox (10. mu.M), TPB (250nM) were added thereto and cultured for 3 days.
S9: the medium was changed to liquid C and FGF7(60ng/ml), SANT-1 (0.3. mu.M), retinoic acid (1. mu.M), LDN193189(150nM), Repsox (10. mu.M), TPB (120nM) were added and cultured for 3 days;
s10: replacing the medium with D solution, adding SANT-1(0.3 μ M), retinoic acid (80nM), LDN193189(150nM), Repsox (10 μ M), T3(1.2 μ M), and culturing for 3 days;
s11: the medium was changed to E medium and LDN193189(150nM), GSiXX (100nM), RepSox (10. mu.M), T3 (1.2. mu.M) were added thereto and cultured for 7 days;
s12: the medium was changed to E medium, and Trolox (15. mu.M), R428 (2.5. mu.M), N-acetylcysteine (1.2mM), Repsox (10. mu.M), T3 (1.2. mu.M) were added thereto and cultured for 14 days;
s13: the medium was changed to E medium, and Trolox (15. mu.M), R428 (2.5. mu.M), N-acetylcysteine (1.2mM), Repsox (10. mu.M), T3 (1.2. mu.M), WNT4(200ng/ml) were added thereto and cultured for 7 days.
Example 3
Preparation of S1 three-dimensional gellan gum culture medium
(1) Gellan gum powder (Sigma) was formulated with ultrapure water into a 0.3% (w/v) aqueous solution and autoclaved.
(2) Then, a three-dimensional gellan gum medium working solution was prepared by adding the following components using mTeSR1 solution (StemCell corporation) as a solvent: gellan gum (final concentration 0.015%), methylcellulose (final concentration 0.25%), ROCK inhibitor (final concentration 10 μ M), streptomycin (final concentration 1%).
S2: 1% agarose was prepared using ultrapure water, autoclaved, and poured onto plates while hot. 1ml of three-dimensional gellan gum medium was added to each dish and equilibrated for at least 20 minutes.
S3: induced pluripotent stem cells (ipscs) of human origin were digested with 500ul accutase at 37 ℃ for 4 minutes, and the cells were resuspended in three-dimensional gellan gum medium to prepare a single cell suspension. The cell suspension was plated on an agarose plate and cultured for 3 days, and the iPSC cell pellet was observed.
S4: the following 5 kinds of culture solutions were prepared
Solution A comprises MCDB131500ml, 500 μ l of 50% glucose, NaHCO31.25 g, 10g of BSA, 10 μ l of ITS-X, 5ml of GlutaMax, 24mg of vitamin C and 5ml of streptomycin qinghaosu;
solution B comprises MCDB131500ml, 500 mul of 50 percent glucose, NaHCO30.625g, 10g of BSA, 10 mul of ITS-X, 5ml of GlutaMax, 24mg of vitamin C and 5ml of streptomycin qinghao;
solution C comprises MCDB131500ml, 500 μ l of 50% glucose, NaHCO30.625g, BSA10g, ITS-X2.5 ml, GlutaMax 5ml, vitamin C24 mg, and streptomycin 5 ml;
solution D comprises MCDB131500ml, 500 mul of 50% glucose, NaHCO30.625g, 10g of BSA, 2.5ml of ITS-X, 5ml of GlutaMax, 24mg of vitamin C, 5ml of streptomycin, 5mg of heparin and 1.45mg of zinc sulfate;
solution E comprises MCDB131500ml, 50% glucose 2700 μ l, NaHCO30.89 g, BSA10g, ITS-X2.5 ml, GlutaMax 5ml, vitamin C24 mg, streptomycin 5ml, heparin 5mg, and zinc sulfate 1.45 mg.
S5: the medium was changed to solution A and gellan gum (0.015%), methylcellulose (0.25%), activin A (100ng/ml), CHIR99021 (3. mu.M) were added and cultured for 1 day.
S6: the medium was changed to solution A and gellan gum (0.015%), methylcellulose (0.25%), and activin A (100ng/ml) were added thereto and cultured for 1 day.
S7: the medium was changed to solution B and FGF7(50ng/ml) was added and the cells were cultured for 2 days.
S8: the medium was changed to liquid C, and FGF7(50ng/ml), SANT-1 (0.25. mu.M), retinoic acid (1. mu.M), LDN193189(120nM), Repsox (10. mu.M), TPB (200nM) were added thereto and cultured for 3 days.
S9: the medium was changed to liquid C, and FGF7(50ng/ml), SANT-1 (0.25. mu.M), retinoic acid (1. mu.M), LDN193189(120nM), Repsox (10. mu.M), TPB (100nM) were added thereto and cultured for 3 days.
S10: replacing the medium with D solution, adding SANT-1(0.25 μ M), retinoic acid (50nM), LDN193189(120nM), Repsox (10 μ M), T3(1 μ M), and culturing for 3 days;
s11: the medium was changed to E medium and LDN193189(120nM), GSiXX (100nM), RepSox (10. mu.M), T3(1. mu.M) were added thereto and cultured for 7 days;
s12: the medium was changed to E medium and Trolox (10. mu.M), R428 (2.5. mu.M), N-acetylcysteine (1mM), Repsox (10. mu.M), T3(1. mu.M) were added thereto, followed by culture for 14 days;
s13: the medium was changed to E medium, and Trolox (10. mu.M), R428 (2.5. mu.M), N-acetylcysteine (1mM), RepSox (10. mu.M), T3(1. mu.M), WNT4(150ng/ml) were added thereto and cultured for 7 days.
The invention aims to establish a novel construction method of the three-dimensional islet organoid based on the iPSC, the method does not use matrigel, but uses agarose to combine with gellan gum to realize cell conglobation, and finally achieves the aims of greatly reducing the technical cost, being simple to operate, being repeatable in process flow and being good in uniformity of the produced three-dimensional islet organoid.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, and such changes and modifications are within the scope of the invention as claimed.

Claims (10)

1. The three-dimensional islet organoid construction method based on the iPSC is characterized in that the three-dimensional islet organoid realizes three-dimensional clustering of cells into balls through agarose and gellan gum.
2. The method for constructing the three-dimensional islet organoid based on the iPSC according to claim 1, comprising the preparation of a three-dimensional gellan gum culture medium, wherein the preparation of the three-dimensional gellan gum culture medium comprises the following steps;
(1) gellan Gum powder (sigma), prepared into 0.3% (w/v) water solution with ultrapure water, and autoclaved;
(2) then, a three-dimensional gellan gum medium working solution was prepared by adding the following components using mTeSR1 solution (StemCell corporation) as a solvent: gellan gum (final concentration 0.015-0.02%), methylcellulose (final concentration 0.2-0.3%), ROCK inhibitor (final concentration 8-15 μ M), and streptomycin (final concentration 1%).
3. The method for constructing the three-dimensional islet organoid based on the ipscs according to claim 1, wherein the culture solution of the three-dimensional islet organoid comprises one or more of the following free combinations;
solution A comprises MCDB131500ml, 600 mu l of 50% glucose 400-;
the solution B comprises MCDB131500ml, 600 mul of 50 percent glucose 400-acetic acid, 30.6-0.7g of NaHCO, 10g of BSA, 10 mul of ITS-X, 5ml of GlutaMax, 20-30mg of vitamin C and 5ml of streptomycin qinghao;
solution C comprises MCDB131500ml, 600 mul of 50% glucose 400-;
solution D comprises MCDB131500ml, 600 mul of 50 percent glucose 400-;
the E solution comprises MCDB131500ml, 3000 mu.l of 50% glucose 2500-.
4. The method for constructing the three-dimensional islet organoid based on the ipscs according to claim 3, wherein the method for culturing the three-dimensional islet organoid comprises the following steps:
(1) the culture medium was changed to solution A and gellan gum (0.015-0.02%), methylcellulose (0.2-0.3%), activin A (100ng/ml), CHIR99021(2-4uM) were added and cultured for 1 day.
(2) The medium was changed to solution A and gellan gum (0.015-0.02%), methylcellulose (0.2-0.3%), activin A (100ng/ml) were added and cultured for 1 day.
(3) The medium was changed to solution B and FGF7(30-60ng/ml) was added thereto for 2 days.
(4) The medium was changed to liquid C and FGF7(30-60ng/ml), SANT-1 (0.2-0.3. mu.M), retinoic acid (0.8-1. mu.M), LDN193189(100-150nM), Repsox (5-10. mu.M), TPB (200-250nM) were added thereto and cultured for 3 days.
(5) The medium was changed to liquid C and FGF7(30-60ng/ml), SANT-1 (0.2-0.3. mu.M), retinoic acid (0.8-1. mu.M), LDN193189(100-150nM), Repsox (5-10. mu.M), TPB (100-120nM) were added thereto and cultured for 3 days.
(6) The medium was changed to D medium and added with SANT-1 (0.2-0.3. mu.M), retinoic acid (40-80nM), LDN193189(100-150nM), Repsox (5-10. mu.M), T3 (1-1.2. mu.M), cultured for 3 days S11: the medium was changed to E solution, and LDN193189(100-150nM), GSiXX (80-100nM), RepSox (5-10. mu.M), T3 (1-1.2. mu.M) were added thereto and cultured for 7 days.
5. The method for constructing the three-dimensional islet organoid based on the ipscs according to claim 4, wherein the method for culturing the three-dimensional islet organoid comprises the following steps: the medium was changed to E solution, and Trolox (10-15. mu.M), R428 (2-2.5. mu.M), N-acetylcysteine (1-1.2mM), Repsox (5-10. mu.M), T3 (1-1.2. mu.M) were added thereto and cultured for 14 days.
6. The method for constructing the three-dimensional islet organoid based on the ipscs according to claim 5, wherein the method for culturing the three-dimensional islet organoid comprises the following steps: the medium was changed to E medium and added with Trolox (10-15. mu.M), R428 (2-2.5. mu.M), N-acetylcysteine (1-1.2mM), Repsox (5-10. mu.M), T3 (1-1.2. mu.M), WNT4(100-200ng/ml), and cultured for 7 days.
7. A three-dimensional islet organoid based on iPSC, characterized in that the three-dimensional islet organoid is constructed by agarose in combination with gellan gum.
8. An iPSC-based three-dimensional islet organoid, wherein said three-dimensional islet organoid is cultured from the culture solution of claim 3.
9. An iPSC-based three-dimensional pancreatic islet organoid constructed by one of the methods of claims 1-6.
10. A diabetes treatment product comprising insulin, wherein said insulin is synthesized by one of the three-dimensional islet organoids of claims 7-9.
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