3d intestinal organoid differentiation method based on human pluripotent stem cells, and induced culture medium and application thereof

Abstract

The application discloses a 3D intestinal organoid differentiation method based on human pluripotent stem cells, an induced culture medium thereof and application, relates to the technical field of stem cells, and the culture medium formula combination of the application can make the intestinal organoid differentiate into various cell types such as epithelial cells, neuroendocrine cells and endothelial cells, and is a key sign for successful differentiation and mature function of the intestinal organoid. The 3D intestinal organoid differentiation method of the application generates the intestinal organoid differentiated into histological structures such as intestinal crypts and can make the intestinal organoid appear peristalsis in the maintenance stage, so that the intestinal organoid is from structural bionics to functional simulation, and the significance is far more than simple morphological simulation. Through systematic culture medium formula, clear operation process and multi-stage induction strategy, the 3D intestinal organoid with structural integrity, cell diversity and functional activity is successfully constructed, and the system provides an efficient, reliable and scalable in-vitro model platform for intestinal biology research and related application.

Description

Technical Field

[0001] This invention relates to the field of stem cell technology, specifically to a 3D intestinal organoid differentiation method based on human pluripotent stem cells, its induction culture medium, and its applications. Background Technology

[0002] The gut is a crucial organ for digestion, nutrient absorption, and maintaining immune homeostasis in the human body. Its complex structure, composed of crypts and villi, contains various functionally diverse cell types, such as intestinal epithelial cells, goblet cells, Paneth cells, and enteroendocrine cells. Traditional gut research models, such as two-dimensional (2D) cell line cultures and animal models, have significant limitations in simulating human intestinal physiology and pathology. 2D cell lines cannot reproduce the three-dimensional structure and cellular diversity of the gut, while animal models face bottlenecks such as species differences, low throughput, high cost, and ethical concerns.

[0003] In recent years, the emergence of three-dimensional (3D) intestinal organoid technology has brought revolutionary breakthroughs to intestinal research. Organoids are three-dimensional micro-organoids that are self-assembled and differentiated in vitro from adult stem cells or pluripotent stem cells, capable of highly mimicking the complex structure, cell type, and specific functions of the source tissue. In 2009, Clevers' team successfully cultured mouse intestinal Lgr5+ adult stem cells into intestinal organoids with crypt-village-like structures in a Matrigel matrix containing specific growth factors (such as EGF, Noggin, and R-spondin1). Since then, human intestinal organoid technology has also developed rapidly, becoming a powerful tool for studying intestinal development, disease mechanisms, host-microbe interactions, and evaluating drug toxicity and efficacy.

[0004] Despite the significant success of existing technologies, current mainstream 3D intestinal organoid culture systems still face numerous unresolved technical challenges, severely limiting their standardization and large-scale application. Existing methods (such as Canadian patent CA3062600A1) disclose a method for preparing intestinal organoids from pluripotent stem cells, including the following steps: differentiating pluripotent stem cells into endoderm-like cells; differentiating the obtained endoderm into intestinal stem cells; culturing the obtained intestinal stem cells to form spheroids; and forming the differentiated spheroids into intestinal organoids. These steps include culturing in the presence of MEK1 / 2 inhibitors, DNA methylation inhibitors, TGF-β receptor inhibitors, and γ-secretase inhibitors, in addition to epidermal growth factor, BMP inhibitors, and Wnt signaling activators. However, the organoids generated by this invention often exhibit significant heterogeneity in size, morphology, and cellular composition, lacking a unified standardized quality control system. Furthermore, conventional organoid models typically lack key microenvironmental components such as immune cells, vascular systems, neural innervation, and microbiota, making it impossible to fully simulate the complex physiological and pathological states in vivo. Due to the factors mentioned above, the translation of existing intestinal organoid technologies from laboratory research to clinical and industrial fields such as precision medicine and new drug development faces enormous challenges. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, a method for differentiating 3D intestinal organoids based on human pluripotent stem cells and its induction culture medium are provided to solve the problem that the intestinal organoids cultured by existing 3D intestinal organoid culture systems are only at the level of structural biomimicry.

[0006] To achieve the above objectives, a first aspect of the present invention provides a culture medium composition for inducing human pluripotent stem cells to differentiate into intestinal organoids, the composition comprising:

[0007] The first induction medium consists of RPMI 1640 medium, B27 additive, Activin A, FGF4 and Y27632;

[0008] The second induction medium consists of RPMI 1640 medium, non-essential amino acids, GlutaMax, B27 additive, FGF4, BMP4 and Y27632.

[0009] The third induction medium includes RPMI 1640 medium, non-essential amino acids, GlutaMax, B27 additive, FGF4 and Wnt signaling pathway activator;

[0010] The fourth induction medium includes Advanced DMEM / F-12 medium, N2 additive, B27 additive, GlutaMax, non-essential amino acids, EGF, R-spondin-1, Noggin and Y-27632;

[0011] The fifth induction medium contains Advanced DMEM / F-12 medium, N2 additive, B27 additive, GlutaMax, non-essential amino acids, EGF, R-spondin-1, and Noggin;

[0012] The maintenance culture medium, comprising Advanced DMEM / F-12 medium, GlutaMax, all-trans retinoic acid, and Forskolin, is provided in a second aspect of the present invention as a method for 3D intestinal organoid differentiation based on human pluripotent stem cells, comprising the following steps:

[0013] Human pluripotent stem cells were subjected to primary differentiation in a first induction medium to obtain primary differentiation products. The first induction medium included RPMI 1640 medium, B27 additive, Activin A, FGF4 and Y27632.

[0014] The primary differentiation product is transferred to a second induction medium for secondary differentiation to obtain a secondary differentiation product. The second induction medium includes RPMI 1640 medium, non-essential amino acids, GlutaMax, B27 additive, FGF4, BMP4 and Y27632.

[0015] The secondary differentiation product was transferred to a third induction medium for tertiary differentiation to obtain a tertiary differentiation product. The third induction medium included RPMI 1640 medium, non-essential amino acids, GlutaMax, B27 additive, FGF4, and Wnt signaling pathway activator.

[0016] The tertiary differentiation product was transferred to a fourth induction medium for quaternary differentiation to obtain a quaternary differentiation product. The fourth induction medium included Advanced DMEM / F-12, N2 additive, B27 additive, GlutaMax, non-essential amino acids, EGF, R-spondin-1, Noggin and Y-27632.

[0017] The fourth-grade differentiation product was transferred to the fifth induction medium for fifth-grade differentiation to obtain the fifth-grade differentiation product. The fifth induction medium included Advanced DMEM / F-12 medium, N2 additive, B27 additive, GlutaMax, non-essential amino acids, EGF, R-spondin-1 and Noggin.

[0018] The fifth-grade differentiation products were dynamically cultured in a maintenance medium to obtain mature intestinal organoids. The maintenance medium included Advanced DMEM / F-12 medium, GlutaMax, all-trans retinoic acid, and Forskolin.

[0019] Furthermore, the first induction medium comprises RPMI 1640 medium, 1X of B27 additive, 50-200 ng / ml of Activin A, 1-10 ng / ml of FGF4, and 1-10 μM of Y27632.

[0020] Furthermore, the second induction medium comprises RPMI 1640 medium, 1X of non-essential amino acids, 1X of GlutaMax, 0.5-1X of B27, 10-50 ng / ml of FGF4, 10-50 ng / ml of BMP4, and 1-10 μM of Y27632.

[0021] Furthermore, the third induction medium is RPMI 1640 medium, 1X of non-essential amino acids, 1X of GlutaMax, 0.5~1X of B27 additive, 80~200ng / ml of FGF4, and 200~500ng / ml of Wnt3a.

[0022] Furthermore, the fourth induction medium includes Advanced DMEM / F-12 medium, 0.5X N2, 0.5X B27, 1X GlutaMax, 1X non-essential amino acids, 20~100 ng / ml EGF, 50~300 ng / ml R-spondin-1, 10~200 ng / ml Noggin, and 1~20 μM Y-27632.

[0023] Furthermore, the fifth induction medium includes Advanced DMEM / F-12 medium, 0.5X N2 additive, 0.5X B27 additive, 1X GlutaMax, 1X non-essential amino acids, 20~100 ng / ml EGF, 50~300 ng / ml R-spondin-1, and 10~200 ng / ml Noggin.

[0024] Furthermore, the maintenance culture medium includes Advanced DMEM / F-12 medium, 1X GlutaMax, 0.1~1μM all-trans retinoic acid and 1~10μM Forskolin.

[0025] A third aspect of the present invention provides an intestinal organoid, which is differentiated and generated using the 3D intestinal organoid differentiation method described in any of the above.

[0026] A fourth aspect of the present invention provides a method for evaluating the pharmacokinetics or toxicity of a test substance using intestinal organoids as described above.

[0027] The fifth aspect of the present invention provides the use of the intestinal organoids described above in the preparation of medicaments or transplant formulations for treating intestinal injury, intestinal dysfunction or inflammatory bowel disease.

[0028] The beneficial effects of this invention are as follows: the culture medium used in the 3D intestinal organoid differentiation method based on human pluripotent stem cells is simple in composition, relatively easy to prepare, and the required raw materials are readily available. While ensuring normal function, it saves on culture costs, and the organoids can be stably preserved for a long time, maintaining their efficacy even when kept at 4 degrees Celsius after preparation. This invention provides specific components of the induction culture medium for each stage, greatly reducing the uncertainty of the formulation and the reliance on operator experience. The raw materials for the induction culture medium are highly commercialized; all culture medium components can be purchased from common suppliers (such as Thermo Fisher, Nearshore Protein, MCE, etc.), ensuring a stable supply chain and facilitating rapid technology promotion and large-scale production. The preparation methods for the culture medium at each stage are clearly defined, which is beneficial for experimental reproducibility and batch consistency, suitable for high-throughput screening and standardized production. The induction culture medium used in this invention has a simplified composition, avoiding redundancy. Compared with existing complex culture medium systems, this invention, through optimized ratios, reduces the amount of expensive growth factors used while ensuring differentiation efficiency.

[0029] The culture medium formulation provided by the 3D intestinal organoid differentiation method based on human pluripotent stem cells of this invention can differentiate intestinal organs into various cell types such as epithelial cells, neuroendocrine cells, and endothelial cells, which is a key indicator of successful differentiation and functional maturity of intestinal organs.

[0030] The present invention utilizes a 3D intestinal organoid differentiation method based on human pluripotent stem cells to generate intestinal organoids that exhibit histological structures such as intestinal crypts and demonstrate peristalsis during the maintenance phase. This allows the intestinal organoids to move beyond structural biomimicry to functional simulation, with significance far exceeding mere morphological imitation. The intestinal organoids generated by the differentiation method of this invention possess the expression capabilities of multiple drug-metabolizing enzymes, which can be used for in vitro evaluation of drug absorption, metabolism, toxicity, and drug-drug interactions. The intestinal organoids generated by the differentiation method of this invention can be used for pathological model construction, mechanistic studies, drug screening, and personalized medicine. Furthermore, the intestinal organoids generated by the differentiation method of this invention can serve as transplantation materials for intestinal tissue engineering and regenerative therapy, particularly suitable for research on the repair of refractory intestinal diseases such as inflammatory bowel disease.

[0031] This invention successfully constructed 3D intestinal organoids with structural integrity, cell diversity, and functional activity through a systematic culture medium formulation, a well-defined operational procedure, and a multi-stage induction strategy. Its technical solution combines standardization, economy, stability, and functional simulation, demonstrating strong translational potential not only for basic research but also for drug screening, toxicity evaluation, disease modeling, and regenerative medicine. This system provides an efficient, reliable, and scalable in vitro model platform for intestinal biology research and related applications. Attached Figure Description

[0032] Other features, objects, and advantages of this application will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0033] Figure 1 This is a photograph of the final differentiated intestinal organoid generated by the 3D intestinal organoid differentiation method based on human pluripotent stem cells according to an embodiment of the present invention.

[0034] Figure 2 This diagram illustrates the process of intestinal organoid differentiation induced by human iPSCs.

[0035] Figure 3 Optical photographs of different stages of intestinal organoid differentiation induced by human iPSCs.

[0036] Figure 4 This is an immunofluorescence image of the intestinal organoids differentiated in this embodiment.

[0037] Figure 5 HE-stained images of the intestinal organoids differentiated in this embodiment. Detailed Implementation

[0038] The present application will now be described in further detail 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 not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0039] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0040] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0041] The human iPSCs used were purchased from iXCells Biotechnologies USA, Inc., catalog number: 30HU-002, batch number: 400279-2-hPSC-A26F (https: / / ixcellsbiotech.com / ).

[0042] RPMI 1640 medium was purchased from Thermo-Gibco, catalog number C22400500BT;

[0043] B27 supplement (insulin-free) was purchased from Thermo-Gibco, product number A3695201;

[0044] Activin A was purchased from nearshore protein, catalog number GMP-C687;

[0045] FGF4 was purchased from nearshore protein, catalog number CR08;

[0046] Non-essential amino acids (NEAA) were purchased from Thermo-Gibco, catalog number 11140050;

[0047] GlutaMax was purchased from Thermo-Gibco, product number 35050-061;

[0048] B27 additive was purchased from Thermo-Gibco, product number 17504044;

[0049] BMP4 was purchased from nearshore protein, catalog number CR93;

[0050] Wnt3a was purchased from nearshore protein, catalog number C22R;

[0051] Advanced DMEM / F-12 medium was purchased from Thermo-Gibco, catalog number 12634-010;

[0052] EGF was purchased from Dongkang, product number Y00801;

[0053] R-spondin-1 was purchased from Dongkang, item number Y03501;

[0054] Noggin was purchased from nearshore protein sources, catalog number CB89;

[0055] Y-27632 was purchased from MCE, item number HY-10071;

[0056] N2 additive was purchased from Thermo-Gibco, product number A1370701;

[0057] All-trans retinoic acid was purchased from MCE, product number HY-14649;

[0058] Forskolin was purchased from MCE, product number HY-15371.

[0059] The mTesR Plus was purchased from Stemcell, item number 100-0276.

[0060] A first aspect of the present invention provides a culture medium composition for inducing human pluripotent stem cells to differentiate into intestinal organoids, the composition comprising:

[0061] The first induction medium consists of RPMI 1640 medium, B27 additive, Activin A, FGF4 and ROCK inhibitor Y27632;

[0062] The second induction medium consists of RPMI 1640 medium, non-essential amino acids, GlutaMax, B27 additive (insulin-free), FGF4, BMP4, and ROCK inhibitor Y27632.

[0063] The third induction medium includes RPMI 1640 medium, non-essential amino acids, GlutaMax, B27 additive, FGF4, and Wnt signaling pathway activator (which may be Wnt3a, CHIR99021, or other Wnt ligands).

[0064] The fourth induction medium includes Advanced DMEM / F-12 medium, N2 additive, B27 additive, GlutaMax, non-essential amino acids, EGF, R-spondin-1, Noggin and Y-27632;

[0065] The fifth induction medium contains Advanced DMEM / F-12 medium, N2 additive, B27 additive, GlutaMax, non-essential amino acids, EGF, R-spondin-1, and Noggin;

[0066] The maintenance medium contains Advanced DMEM / F-12 medium, GlutaMax, all-trans retinoic acid, and Forskolin.

[0067] In the first induction medium, RPMI 1640 medium serves as the basal medium, providing a nutrient and buffering system to maintain a stable cell growth environment. B27 supplement (insulin-free) is a supplement for neural cell culture, supporting early endoderm differentiation and avoiding insulin interference. Activin A, a member of the TGF-β superfamily, promotes endoderm differentiation and induces human pluripotent stem cells to differentiate into mature endoderm. FGF4, fibroblast growth factor 4, synergistically promotes endoderm differentiation and enhances cell survival in conjunction with Activin A. Specifically, the formulation of the first induction medium may include RPMI 1640 medium, 1X of B27 supplement (insulin-free), 50–200 ng / ml of Activin A, 1–10 ng / ml of FGF4, and 1–10 μM of the ROCK inhibitor Y27632.

[0068] In the second induction medium, RPMI 1640 medium serves as the basal medium, providing nutrients and a buffer system to maintain a stable cell growth environment. Non-essential amino acids are a mixture providing amino acids to support cell metabolism and protein synthesis. GlutaMax, a stable glutamine derivative, provides energy and nitrogen to maintain cell health. B27 supplement is a nerve culture supplement containing antioxidants, providing vitamins, hormones, and antioxidants to reduce oxidative damage. FGF4, fibroblast growth factor 4, synergistically promotes endoderm differentiation and enhances cell survival in conjunction with Activin A. BMP4, bone morphogenetic protein 4, promotes hindgut mesoderm differentiation and guides intestinal fate. Specifically, the formulation of the second induction medium may include RPMI 1640 medium, 1X of non-essential amino acids, 1X of GlutaMax, 0.5–1X of B27, 10–50 ng / ml of FGF4, 10–50 ng / ml of BMP4, and 1–10 μM of the ROCK inhibitor Y27632.

[0069] The second induction medium provides optimized and sufficient nutrients, hormones, and antioxidants to minimize cell apoptosis. The B27 additive in this medium is rich in antioxidants (such as vitamin E, vitamin C, glutathione, and superoxide dismutase). At a 1X concentration, these components neutralize excess reactive oxygen species produced by cellular metabolism, preventing oxidative damage.

[0070] In the third induction medium, RPMI 1640 medium serves as the basal medium, providing nutrients and a buffer system to maintain a stable cell growth environment. Non-essential amino acids are a mixture providing amino acids to support cell metabolism and protein synthesis. GlutaMax, a stable glutamine derivative, provides energy and nitrogen to maintain cell health. B27 supplement, an antioxidant-containing neurotrophic supplement, provides vitamins, hormones, and antioxidants to reduce oxidative damage. FGF4, fibroblast growth factor 4, synergistically promotes endoderm differentiation and enhances cell survival in conjunction with Activin A. Wnt3a, a ligand for the Wnt signaling pathway, activates the Wnt / β-catenin pathway, promoting intestinal progenitor cell proliferation and polarization. Specifically, the formulation of the third induction medium may include RPMI 1640 medium, 1X of non-essential amino acids, 1X of GlutaMax, 0.5-1X of B27 additive, 80-200 ng / ml of FGF4, and 200-500 ng / ml of Wnt3a. It is readily understood that the Wnt3a in the above formulation can be replaced by CHIR99021 or other Wnt ligands at the same or similar concentration range. Any Wnt signaling pathway activator capable of achieving the Wnt pathway activation effect of the embodiments of this invention should fall within the scope of protection of this invention. The third induction medium of this embodiment provides optimized and sufficient nutrients, hormones, and antioxidants to minimize cell apoptosis. The B27 additive contains abundant antioxidants (such as vitamin E, vitamin C, glutathione, superoxide dismutase, etc.). At a 1X concentration, these components can neutralize excess reactive oxygen species produced by cell metabolism, preventing oxidative damage.

[0071] The fourth induction medium comprises Advanced DMEM / F-12 medium, 0.5X N2 additive, 0.5X B27 additive, 1X GlutaMax, 1X non-essential amino acids, 20-100 ng / ml EGF, 50-300 ng / ml R-spondin-1, 10-200 ng / ml Noggin, and 1-20 μM Y-27632. Specifically, the formulation of the fourth induction medium may include Advanced DMEM / F-12 medium, 0.5X N2 additive, 0.5X B27 additive, 1X GlutaMax, 1X non-essential amino acids, 20-100 ng / ml EGF, 50-300 ng / ml R-spondin-1, 10-200 ng / ml Noggin, and 1-20 μM Y-27632.

[0072] In the fifth induction medium, Advanced DMEM / F-12 is an optimized basal medium, providing richer nutrients to support the formation of three-dimensional structures. N2 is a supplement for neural crest cell culture, supporting the differentiation of neuroepithelial cells and enteric nervous system precursors. B27 additive is a neural culture supplement containing antioxidants, providing vitamins, hormones, and antioxidants to reduce oxidative damage. GlutaMax is a stable glutamine derivative, providing energy and nitrogen sources to maintain cell health. Non-essential amino acids are a mixture of non-essential amino acids, providing amino acids to support cell metabolism and protein synthesis. EGF is epidermal growth factor, promoting epithelial cell proliferation and survival. R-spondin-1 is a Wnt signaling enhancer, enhancing Wnt signaling and promoting crypt formation and stem cell maintenance. Noggin is a BMP signaling inhibitor, inhibiting the BMP pathway and promoting epithelialization and villous formation. Specifically, the formulation of the fifth induction medium may include Advanced DMEM / F-12 medium, 0.5X N2 additive, 0.5X B27 additive, 1X GlutaMax, 1X non-essential amino acids, 20~100 ng / ml EGF, 50~300 ng / ml R-spondin-1, and 10~200 ng / ml Noggin.

[0073] In maintenance medium, Advanced F12 contains a richer variety of amino acids, vitamins, trace elements, and buffer systems than traditional DMEM / F-12. Its stable composition makes it suitable for dynamic shaker culture systems, promoting uniform oxygenation and nutrition for organoids. GlutaMax, a stable glutamine derivative, provides energy and nitrogen to maintain cell health. All-trans retinoic acid (a vitamin A derivative) promotes intestinal epithelial cell differentiation and barrier function maturation. Forskolin, an adenylate cyclase activator, increases cAMP levels, promoting intestinal endocrine cell differentiation and peristaltic function. Specifically, the maintenance medium formulation may include Advanced DMEM / F-12 medium, 1X GlutaMax, 0.1–1 μM all-trans retinoic acid, and 1–10 μM Forskolin.

[0074] A second aspect of the present invention provides a method for differentiating 3D intestinal organoids based on human pluripotent stem cells, which may include S1, a pluripotent stem cell expansion and culture stage, and S2, a multi-stage induction differentiation stage.

[0075] Specifically, the S1 pluripotent stem cell expansion and culture stage includes the following steps:

[0076] Pluripotent stem cells were seeded into stem cell maintenance medium for expansion culture. Theoretically, these pluripotent stem cells could be derived from iPSCs (Induced Pluripotent Stem Cells) or ESCs (Embryonic Stem Cells), both of which can differentiate; however, this embodiment uses iPSCs. The expansion culture phase can last 3-5 days. The stem cell maintenance medium can include mTesR Plus and Y-27632. Furthermore, the stem cells were statically cultured at 5-10% CO2, 30-37°C, and 100 rpm, with the medium changed daily.

[0077] S2, the multi-stage induced differentiation phase includes the following steps:

[0078] S21. The primary differentiation stage involves inducing primary differentiation of stem cells under dynamic conditions in the first induction medium. The primary differentiation stage lasts from day 3 to day 7. Stem cells are cultured at 5-10% CO2 and 30-37°C, with the medium changed every two days. The first induction medium includes RPMI 1640 medium, B27 supplement (without insulin), Activin A, FGF4, and the ROCK inhibitor Y27632. The ROCK inhibitor Y27632 can be added on the last day of the primary differentiation stage.

[0079] In the first induction medium, RPMI 1640 medium serves as the basal medium, providing a nutrient and buffering system to maintain a stable cell growth environment. B27 supplement (insulin-free) is a supplement for insulin-free neural cell culture, supporting early endoderm differentiation and avoiding insulin interference. Activin A, a member of the TGF-β superfamily, promotes endoderm differentiation and induces iPSC differentiation into mature endoderm. FGF4, fibroblast growth factor 4, synergistically promotes endoderm differentiation and enhances cell survival in conjunction with Activin A.

[0080] S22. The primary differentiation product is transferred to the second induction medium for secondary differentiation to obtain the secondary differentiation product. The second induction medium includes RPMI 1640 medium, non-essential amino acids, GlutaMax, B27 additive, FGF4, BMP4, and ROCK inhibitor Y27632. Y27632 can be added on the first day of culture for secondary differentiation.

[0081] In the second induction medium, RPMI 1640 medium serves as the basal medium, providing nutrients and a buffer system to maintain a stable cell growth environment. Non-essential amino acids are a mixture providing amino acids to support cell metabolism and protein synthesis. GlutaMax, a stable glutamine derivative, provides energy and nitrogen to maintain cell health. B27 supplement, an antioxidant-containing neurotrophic protein supplement, provides vitamins, hormones, and antioxidants to reduce oxidative damage. FGF4, fibroblast growth factor 4, synergistically promotes endoderm differentiation and enhances cell survival in conjunction with Activin A. BMP4, bone morphogenetic protein 4, promotes hindgut mesoderm differentiation and guides intestinal fate.

[0082] Specifically, the second induction medium includes RPMI 1640 medium, 1X of non-essential amino acids, 1X of GlutaMax, 0.5-1X of B27 additive, 10-50 ng / ml of FGF4 and 10-50 ng / ml of BMP4, and ROCK inhibitor Y27632.

[0083] In a preferred embodiment, the second induction medium comprises RPMI 1640 medium, 1X of non-essential amino acids, 1X of GlutaMax, 1X of B27, 10-50 ng / ml of FGF4, and 10-50 ng / ml of BMP4. In this embodiment, the second induction medium provides optimized and sufficient nutrients, hormones, and antioxidants to minimize apoptosis. The B27 in this medium is rich in antioxidants (such as vitamin E, vitamin C, glutathione, and superoxide dismutase). At a 1X concentration, these components neutralize excess reactive oxygen species produced by cellular metabolism, preventing oxidative damage.

[0084] The secondary differentiation stage involves transferring the primary differentiation products to a second induction medium for secondary differentiation under dynamic conditions, with the medium being replaced periodically.

[0085] The secondary differentiation stage lasts from day 8 to 12. Primary differentiation products are cultured at 5-10% CO2 and 30-37°C, with the medium changed every two days.

[0086] S23. The secondary differentiation product was transferred to the third induction medium for tertiary differentiation to obtain the tertiary differentiation product. The third induction medium was RPMI 1640 medium, non-essential amino acids, GlutaMax, B27 additive, FGF4, and Wnt3a.

[0087] In the third induction medium, RPMI 1640 medium serves as the basal medium, providing nutrients and a buffer system to maintain a stable cell growth environment. Non-essential amino acids are a mixture providing amino acids to support cell metabolism and protein synthesis. GlutaMax, a stable glutamine derivative, provides energy and nitrogen to maintain cell health. B27 supplement, an antioxidant-containing neurotrophic supplement, provides vitamins, hormones, and antioxidants to reduce oxidative damage. FGF4, fibroblast growth factor 4, synergistically promotes endoderm differentiation and enhances cell survival in conjunction with Activin A. Wnt3a, a ligand for the Wnt signaling pathway, activates the Wnt / β-catenin pathway, promoting intestinal progenitor cell proliferation and polarization.

[0088] Specifically, the third induction medium is RPMI 1640 medium, 1X of non-essential amino acids, 1X of GlutaMax, 0.5~1X of B27, 80~200 ng / ml of FGF4, and 200~500 ng / ml of Wnt3a.

[0089] In a preferred embodiment, the third induction medium comprises RPMI 1640 medium, 1X of non-essential amino acids, 1X of GlutaMax, 1X of B27 supplement, 80-200 ng / ml of FGF4, and 200-500 ng / ml of Wnt3a. In this embodiment, the third induction medium provides optimized and sufficient nutrients, hormones, and antioxidants to minimize apoptosis. The B27 supplement contains abundant antioxidants (such as vitamin E, vitamin C, glutathione, and superoxide dismutase). At a 1X concentration, these components neutralize excess reactive oxygen species produced by cellular metabolism, preventing oxidative damage.

[0090] The tertiary differentiation stage involves transferring the secondary differentiation products to a third induction medium for tertiary differentiation, with the medium being changed periodically.

[0091] The tertiary differentiation stage lasts from day 13 to 16. Secondary differentiation products are cultured at 5-10% CO2 and 30-37°C, with the medium changed every two days.

[0092] S24. The tertiary differentiation products are transferred to the fourth induction medium for quaternary differentiation to obtain quaternary differentiation products. The fourth induction medium includes Advanced DMEM / F-12 medium, N2 additive, B27 additive, GlutaMax, non-essential amino acids, EGF, R-spondin-1, Noggin, and Y-27632.

[0093] In the fourth induction medium, Advanced DMEM / F-12 is an optimized basal medium, providing richer nutrients to support the formation of three-dimensional structures. N2 is a supplement for neural crest cell culture, supporting the differentiation of neuroepithelial cells and enteric nervous system precursors. B27 is a neural culture supplement containing antioxidants, providing vitamins, hormones, and antioxidants to reduce oxidative damage. GlutaMax is a stable glutamine derivative, providing energy and nitrogen to maintain cell health. Non-essential amino acids are a mixture of non-essential amino acids, providing amino acids to support cell metabolism and protein synthesis. EGF is epidermal growth factor, promoting epithelial cell proliferation and survival. R-spondin-1 (R-spondin1) is a Wnt signaling enhancer, enhancing Wnt signaling and promoting crypt formation and stem cell maintenance. Noggin is a BMP signaling inhibitor, inhibiting the BMP pathway and promoting epithelialization and villous formation. Y-27632 is a ROCK inhibitor, reducing apoptosis and enhancing cell cluster survival and adhesion.

[0094] Specifically, the fourth induction medium includes Advanced DMEM / F-12 medium, 0.5X N2, 0.5X B27, 1X GlutaMax, 1X non-essential amino acids, 20-100 ng / ml EGF, 50-300 ng / ml R-spondin-1, 10-200 ng / ml Noggin, and 1-20 μM Y-27632.

[0095] The fourth-grade differentiation stage involves transferring the products from the third-grade differentiation stage to the fourth-grade induction medium for fourth-grade differentiation, and periodically changing the medium.

[0096] The fourth differentiation stage occurs from day 17 to 19. Tertiary differentiation products are cultured at 5-10% CO2 and 30-37°C, with the medium changed every two days.

[0097] S25. The quaternary differentiation product is transferred to the fifth induction medium for quinary differentiation to obtain the quinary differentiation product. The fifth induction medium includes Advanced DMEM / F-12 medium, N2, B27, GlutaMax, non-essential amino acids, EGF, R-spondin-1, and Noggin.

[0098] In the fifth induction medium, Advanced DMEM / F-12 is an optimized basal medium, providing richer nutrients to support the formation of three-dimensional structures. N2 is a supplement for neural crest cell culture, supporting the differentiation of neuroepithelial cells and enteric nervous system precursors. B27 is a neural culture supplement containing antioxidants, providing vitamins, hormones, and antioxidants to reduce oxidative damage. GlutaMax is a stable glutamine derivative, providing energy and nitrogen to maintain cell health. Non-essential amino acids are a mixture of non-essential amino acids, providing amino acids to support cell metabolism and protein synthesis. EGF is epidermal growth factor, promoting epithelial cell proliferation and survival. R-spondin-1 (R-spondin1) is a Wnt signaling enhancer, enhancing Wnt signaling and promoting crypt formation and stem cell maintenance. Noggin is a BMP signaling inhibitor, inhibiting the BMP pathway and promoting epithelialization and villous formation.

[0099] Specifically, the fifth induction medium includes Advanced DMEM / F-12 medium, 0.5X N2, 0.5X B27, 1X GlutaMax, 1X non-essential amino acids, 20-100 ng / ml EGF, 50-300 ng / ml R-spondin-1 and 10-200 ng / ml Noggin.

[0100] The fifth-grade differentiation stage involves transferring the products from the fourth-grade differentiation stage to the fifth-grade induction medium for fifth-grade differentiation, and the medium is changed periodically.

[0101] The fifth differentiation stage lasted from day 20 to 53. The fourth differentiation products were dynamically cultured under conditions of 5-10% CO2, 30-37℃, and 100 rpm, with the medium changed every two days.

[0102] S26. The products of the fifth differentiation stage were dynamically cultured in maintenance medium to obtain mature intestinal organoids.

[0103] Specifically, the maintenance culture medium includes AdvancedF12, 1X GlutaMax, 0.1~1μM all-trans retinoic acid and 1~10μM Forskolin.

[0104] In maintenance medium, Advanced DMEM / F-12 contains a richer variety of amino acids, vitamins, trace elements, and buffer systems than conventional DMEM / F-12. Its stable composition makes it suitable for dynamic shaker culture systems, promoting uniform oxygenation and nutrition across organoids. GlutaMax, a stable glutamine derivative, provides energy and nitrogen to maintain cell health. All-trans retinoic acid (a vitamin A derivative) promotes intestinal epithelial cell differentiation and barrier function maturation. Forskolin, an adenylate cyclase activator, increases cAMP levels, promoting intestinal endocrine cell differentiation and peristaltic function.

[0105] The maintenance and amplification phase involves dynamically culturing the quintuple differentiation products in a maintenance medium, with the medium being replaced periodically.

[0106] The amplification phase was maintained from day 54 to ∞. The quintile differentiation products were dynamically cultured at 5-10% CO2, 70 rpm, and 30-37℃, with the medium changed every two days.

[0107] This invention provides an intestinal organoid, which is generated by differentiation using the aforementioned 3D intestinal organoid differentiation method based on human pluripotent stem cells.

[0108] The culture medium used in the 3D intestinal organoid differentiation method based on human pluripotent stem cells of this invention has simple components, is relatively easy to prepare, and the required raw materials are readily available.

[0109] The culture medium formulation provided by the 3D intestinal organoid differentiation method based on human pluripotent stem cells of this invention can differentiate intestinal organs into various cell types such as epithelial cells, enteroendocrine cells, and endothelial cells, which is a key indicator of successful differentiation and functional maturity of intestinal organs.

[0110] The intestinal organoid differentiation method based on human pluripotent stem cells of this invention generates intestinal organoids that differentiate into histological structures such as intestinal crypts and can exhibit peristalsis during the maintenance phase, thus moving from structural biomimicry to functional simulation. Its significance far exceeds that of simple morphological simulation.

[0111] To further illustrate the present invention’s 3D intestinal organoid differentiation method based on human pluripotent stem cells, the following examples are provided. Example

[0112] This embodiment provides a method for 3D intestinal organoid differentiation based on human pluripotent stem cells, including the following steps:

[0113] Step 1: Stem cell expansion and culture.

[0114] Frozen healthy human blood-derived pluripotent stem cells (A26F) were removed from a -80°C freezer and rapidly thawed in a 37°C water bath. The cells were then gently mixed with 5 ml of DMEM / F12 medium, centrifuged at 4°C, 300 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in 1 ml of mTesR Plus and 10 μM Y-27632 solution and counted. Cells were then counted at 3 × 10⁶ cells per well in a 6-well plate. 5 Cells were seeded into plates. After 2 hours, the medium containing Y-27632 was removed, and 3 ml of fresh mTesR Plus was added. The medium was changed daily. When the cell confluence in the 6-well plate reached 80-90%, the next step of differentiation was performed.

[0115] Step 2: Multi-stage induction and differentiation of intestinal organoids.

[0116] 1. First stage of differentiation induction: Remove all the culture medium from the six-well plate in step one, add the first induction medium, and carry out the first differentiation induction culture. Culture for 4 days at 5-10% CO2 and 30-37℃. On the 4th day, add 5 μM Y-27632 to the first induction medium to obtain the primary differentiation product.

[0117] The names and amounts of the first induction culture medium are shown in Table 1 below:

[0118] Table 1. Composition of the first induction medium

[0119]

[0120] 2. Second Differentiation Induction Stage: Remove all culture medium from the six-well plate and add the second induction medium for secondary differentiation induction culture. On the first day of this stage, add 10 μM Y-27632 to the second induction medium. Incubate statically for 4 days at 5-10% CO2 and 30-37℃. The second induction medium needs to be changed every two days to obtain secondary differentiation products.

[0121] The names and amounts of the second induction medium are shown in Table 2 below:

[0122] Table 2. Composition of the second induction medium

[0123]

[0124] 3. Third stage of induction differentiation: Remove all the culture medium from the six-well plate, add the third induction medium for third differentiation induction culture, and incubate statically for 3 days at 5-10% CO2 and 30-37℃. Replace the third induction medium every two days to obtain the tertiary differentiation product.

[0125] The names and amounts of the third induction medium are shown in Table 3 below:

[0126] Table 3. Composition of the third induction medium

[0127]

[0128] 4. Fourth stage of differentiation induction: All culture medium was aspirated from the six-well plates. 5 ml of TryPLE digestion solution was added to each well, and the plates were incubated at 37°C for 10 minutes. After most cells became rounded under a microscope, they were gently pipetted to create a cell suspension. The cell suspension from each well of the six-well plate was transferred to a corresponding 15 ml centrifuge tube and centrifuged at 4°C, 300 rpm, for 5 minutes. The supernatant was then discarded, and the cells were resuspended in 9.6 ml of fourth-stage induction medium. Finally, 200 μL of cell suspension was transferred from each well to a 96-well low-adhesion U-shaped plate and incubated at 37°C for 3 days to obtain the fourth-stage differentiation products.

[0129] The names and amounts of the fourth induction culture medium are shown in Table 4 below:

[0130] Table 4. Composition of the fourth induction medium

[0131]

[0132] 5. Fifth stage of induction differentiation: 3D microspheres in 96-well low-adhesion U-shaped plates were transferred to 6-well low-adhesion plates, with 24 3D samples per well. 5 ml of fifth induction culture medium was added, and the plates were placed in a 37°C incubator on a horizontal shaker at 100 rpm for dynamic culture. Fresh culture medium was replaced every two days. The culture was carried out until day 50 to obtain the fifth-stage differentiation products.

[0133] The names and amounts of the fifth induction culture medium are shown in Table 5 below:

[0134] Table 5. Composition of the fifth induction medium

[0135]

[0136] 6. Maintenance phase: The products of the fifth differentiation stage are dynamically cultured in maintenance medium to obtain intestinal organoids.

[0137] The names and amounts of the maintenance culture media are shown in Table 6 below:

[0138] Table 6. Composition of the maintenance culture medium

[0139]

[0140] Combination Figure 1 , Figure 1 This is a photograph of the intestinal organoids that were finally differentiated in this embodiment. Figure 1 The spherical object indicated by the white arrow is an intestinal organoid cultured for 80 days.

[0141] Figure 2 This diagram illustrates the process of intestinal organoid differentiation induced by human iPSCs.

[0142] Figure 3 Optical photographs of different stages of intestinal organoid differentiation induced by human iPSCs. Figure 3 Optical photographs of the first induction differentiation stage show the presence of numerous dead cells and the beginning of cell aggregation. The purpose of this stage is to direct differentiation into well-defined endoderm cells. Optical photographs of the second induction differentiation stage show further cell aggregation into clusters. The purpose of this stage is to further specialize the well-defined endoderm cells differentiated in the first stage into more specific, regionally specific post-foregut progenitor cells. Optical photographs of the third induction differentiation stage show wrinkled structures in the cell clusters, with some areas exhibiting vesicular structures. The purpose of this stage is to further specialize and expand into specific, more mature intestinal progenitor cells. Optical photographs of the fourth induction differentiation stage show that after digestion into single cells, 3D solid spheres can be formed after standing in a 96-well plate for 3 days. Optical photographs of the fifth induction differentiation stage show that the intestinal organoids have large cavities and wrinkled structures similar to the human intestine. Optical photographs of the maintenance stage show peristalsis in the intestinal organoids, indicating that the intestinal organoid structure is more stable and functionally mature.

[0143] Figure 4 This is an immunofluorescence image of the intestinal organoids differentiated in this embodiment. EpCAM (green) is a marker of intestinal epithelial cells; CD31 (orange) is a marker of vascular endothelial cells; Chromogranin A (red) is a marker of neuroendocrine cells; and DAPI (blue) is the cell nucleus. Figure 4 This indicates that intestinal organoids contain multiple cell types, including epithelial cells, endocrine cells, and endothelial cells.

[0144] Figure 5 The image shows an HE-stained photograph of the intestinal organoids differentiated in this embodiment. The HE results indicate that the intestinal organoids have epithelial tissue, stroma, muscle tissue, and crypt structures similar to those of the human intestine.

[0145] In this embodiment, the electrophysiological signal detection results of the intestinal organoids generated on day 27 after differentiation are shown in Table 7 below.

[0146] Table 7. Electrophysiological signal detection results of intestinal organoids

[0147]

[0148] In Table 7, Firing Rate is the firing frequency, physiologically significant as it is the most basic indicator of neuronal activity. It represents the average number of action potentials (i.e., "fires" or "spikes") generated by a neuron per unit time (usually 1 second). Spike Amplitude is the action potential amplitude, physiologically significant as the voltage difference from baseline to peak of a recorded single action potential (spike), which can indicate the state of neurons in intestinal organoids: large, healthy neurons typically generate action potentials with larger amplitudes. Interspike Interval is the peak potential interval, physiologically significant as the time interval between two consecutive action potentials.

[0149] Since the core function of the intestine is peristalsis to propel its contents forward, the intestine possesses an independent enteric nervous system. Electrophysiological signals of intestinal organoids cultured for 27 days were detected using a commercially available high-density microelectrode array system (MaxWell Biosystem). The results showed that the intestinal organoids on day 27 exhibited partial electrical activity, with an average discharge rate of 0.46 Hz and an average discharge amplitude of 40.48 μV, indicating that they already possessed the electrophysiological basis for generating peristaltic-like movements.

[0150] This invention relates to the use of an intestinal organoid obtained by the differentiation method of this invention.

[0151] As a primary application, various analyses can be provided. The intestinal organoids of the present invention can be used in intestinal model systems and are useful for evaluating pharmacokinetics (absorption, metabolism, etc.) and toxicity in the intestine; that is, the intestinal organoids of the present invention can be utilized in the evaluation of the pharmacokinetics and toxicity of compounds.

[0152] Specifically, the intestinal organoids of the present invention are used to test the metabolism, absorption, membrane permeability, drug interactions, induction of drug-metabolizing enzymes, induction of drug transport proteins, and toxicity of test substances. That is, as one application of intestinal organoids, the present invention provides a method for evaluating the metabolism, absorption, membrane permeability, drug interactions, induction of drug-metabolizing enzymes, induction of drug transport proteins, and toxicity of test substances. This method includes the following steps:

[0153] Step I: The process of bringing the test substance into contact with the intestinal organoids obtained by the differentiation method of the present invention;

[0154] Step II: Determine / evaluate the metabolism, absorption, membrane permeability, drug interactions, induction of drug-metabolizing enzymes or drug transporters, or toxicity of the test substance.

[0155] The "contact" in step I is typically performed by adding the test substance to the culture medium. There is no particular limitation on the timing of the addition of the test substance. Therefore, it can be added at some point after culturing has begun in a medium that does not contain the test substance, or it can be started in a medium that already contains the test substance.

[0156] The analyte can be an organic or inorganic compound of various molecular sizes. Examples of organic compounds include nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (glycerophosphates, sphingolipids, glycosylglycerols, cerebrosides, etc.), prostaglandins, isoprene morphologies, terpenes, steroids, polyphenols, catechins, and vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.). Existing or candidate ingredients from pharmaceuticals, nutritional foods, food additives, pesticides, and cosmetics are also preferred analytes. Plant extracts, cell extracts, and culture supernatants can also be used as analytes. By simultaneously adding two or more analytes, interactions and synergistic effects between them can be investigated. The analyte can be of natural origin or synthesized. In the latter case, for example, combinatorial synthesis methods can be used to construct an effective analytical system.

[0157] The contact time of the substance being tested can be set arbitrarily. The contact time can be, for example, from 10 minutes to 3 days, preferably from 1 hour to 1 day. Contact can also be performed in multiple sessions.

[0158] Following step I, the metabolism, absorption, membrane permeability, drug interactions, induction by drug-metabolizing enzymes, induction by drug transporters, or toxicity of the analyte are measured / evaluated (step II). This measurement / evaluation can be performed immediately after step I, i.e., immediately after exposure to the analyte, without a substantial time interval, or it can be performed after a certain period (e.g., 10 minutes to 5 hours). Metabolism can be measured, for example, by detecting metabolites. In this case, the culture medium following step I is typically used as a sample for qualitative or quantitative determination of the desired metabolites. The appropriate measurement method can be selected based on the metabolites; for example, mass spectrometry, liquid chromatography, or immunological methods (e.g., fluorescence immunoassay (FIA) or enzyme immunoassay (EIA)) can be used.

[0159] When metabolites of the analyte are detected, it is determined or evaluated that "the analyte has been metabolized." Furthermore, the amount of metabolites can be used to evaluate the metabolic rate of the analyte. The metabolic efficiency of the analyte can be calculated based on the detection results of the metabolites and the amount of the analyte used (typically, the amount added to the culture medium).

[0160] The expression of drug-metabolizing enzymes (cytochrome P450 (especially human CYP3A4, cynomolgus monkey CYP3A8), uridine diphosphate-glucuronide transferases (especially UGT1A8, UGT1A10), sulfatases (especially SULT1A3, etc.)) in intestinal organoids can also be used as indicators to determine the metabolism of the test substance. The expression of drug-metabolizing enzymes can be evaluated at the mRNA or protein level. For example, an increase in the mRNA level of a drug-metabolizing enzyme can be considered an increase in gene expression. Similarly, an increase in the activity of a drug-metabolizing enzyme can be considered a metabolism of the test substance. Similar to using metabolites as indicators, quantitative determination / evaluation can be based on the expression level of drug-metabolizing enzymes.

[0161] To evaluate the absorption of a analyte, for example, by determining the residual amount of the analyte in the culture medium, the culture medium after step I is typically sampled to quantify the analyte. The assay method can be chosen appropriately based on the analyte. For example, mass spectrometry, liquid chromatography, and immunological methods (such as fluorescence immunoassay (FIA) and enzyme immunoassay (EIA)) can be used. Typically, when a decrease in the concentration of the analyte in the culture medium is confirmed, it is considered that "the analyte has been absorbed." Furthermore, the degree of reduction can be used to determine / evaluate the amount or efficiency of absorption of the analyte. It should be noted that absorption can also be evaluated by measuring the amount of the analyte ingested into the cells.

[0162] It should be noted that metabolic assays / evaluations and absorption assays / evaluations can be performed simultaneously or in parallel.

[0163] As a second use of the intestinal organoids obtained by the differentiation method of the present invention, transplant materials comprising intestinal organoids can be provided. The transplant materials of the present invention can be applied to the treatment of various intestinal diseases (e.g., refractory inflammatory bowel disease). In particular, they are envisioned for use as materials for the regeneration / reconstruction of damaged (including dysfunctional) intestinal tissue, i.e., they are expected to contribute to regenerative medicine. The transplant materials of the present invention can be used directly or after processing such as embedding in matrix gel or collagen gel as transplant materials. Furthermore, their use is also envisioned as screening candidate compounds for therapeutic drugs in various intestinal disease pathological models and elucidating pathological mechanisms.

[0164] The transplant material of this invention can also be used to construct in vivo experimental systems. For example, transplant material containing intestinal organoids made from human pluripotent stem cells can be transplanted into laboratory animals such as mice, rats, guinea pigs, hamsters, pigs, cynomolgus monkeys, macaques, and chimpanzees to create humanized animals (human intestinal models). Such humanized animals are particularly useful for pharmacokinetic and toxicity tests, and are expected to contribute to research on the effects of first-pass effects on oral medications, drug-induced enteritis, and other related topics.

[0165] Intestinal organoids made from iPSC cells from patients with intestinal diseases can be used not only as intestinal pathology models for drug evaluation systems, but also for various experiments in studies aimed at elucidating the mechanisms of pathogenesis, pathogenesis, and / or progression of intestinal diseases.

[0166] The above description is merely a preferred embodiment of this application and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in this application is not limited to technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the inventive concept. For example, technical solutions formed by substituting the above features with (but not limited to) technical features with similar functions disclosed in this application.

Claims

1. A culture medium composition for inducing human pluripotent stem cells to differentiate into intestinal organoids, characterized in that, This combination consists of the following culture media: The first induction medium consists of RPMI 1640 medium, 1X of B27 additive, 50-200 ng / ml of Activin A, 1-10 ng / ml of FGF4 and 1-10 μM of Y27632; The second induction medium consists of RPMI 1640 medium, 1X of non-essential amino acids, 1X of GlutaMax, 0.5-1X of B27 additive, 10-50 ng / ml of FGF4, 10-50 ng / ml of BMP4 and 1-10 μM of Y27632. The third induction medium consists of RPMI 1640 medium, 1X of non-essential amino acids, 1X of GlutaMax, 0.5-1X of B27 additive, 80-200 ng / ml of FGF4 and 200-500 ng / ml of Wnt signaling pathway activator, wherein the Wnt signaling pathway activator is Wnt3a. The fourth induction medium consists of Advanced DMEM / F-12 medium, 0.5X N2 additive, 0.5X B27 additive, 1X GlutaMax, 1X non-essential amino acids, 20-100 ng / ml EGF, 50-300 ng / ml R-spondin-1, 10-200 ng / ml Noggin, and 1-20 μM Y-27632; The fifth induction medium consists of Advanced DMEM / F-12 medium, 0.5X N2 additive, 0.5X B27 additive, 1X GlutaMax, 1X non-essential amino acids, 20~100 ng / ml EGF, 50~300 ng / ml R-spondin-1 and 10~200 ng / ml Noggin; The maintenance medium consisted of Advanced DMEM / F-12 medium, 1X GlutaMax, 0.1–1 μM all-trans retinoic acid, and 1–10 μM Forskolin.

2. A method for 3D intestinal organoid differentiation based on human pluripotent stem cells, characterized in that, Includes the following steps: Human pluripotent stem cells were subjected to primary differentiation in a first induction medium to obtain primary differentiation products. The first induction medium consisted of RPMI 1640 medium, 1X B27 additive, 50~200 ng / ml Activin A, 1~10 ng / ml FGF4, and 1~10 μM Y27632. The primary differentiation product was transferred to a second induction medium for secondary differentiation to obtain a secondary differentiation product. The second induction medium consisted of RPMI 1640 medium, 1X of non-essential amino acids, 1X of GlutaMax, 0.5-1X of B27 additive, 10-50 ng / ml of FGF4, 10-50 ng / ml of BMP4, and 1-10 μM of Y27632. The secondary differentiation product was transferred to a third induction medium for tertiary differentiation to obtain the tertiary differentiation product. The third induction medium consisted of RPMI 1640 medium, 1X of non-essential amino acids, 1X of GlutaMax, 0.5-1X of B27 additive, 80-200 ng / ml of FGF4, and 200-500 ng / ml of Wnt3a. The tertiary differentiation products were transferred to a fourth induction medium for quaternary differentiation to obtain quaternary differentiation products. The fourth induction medium consisted of Advanced DMEM / F-12 medium, 0.5X N2 additive, 0.5X B27 additive, 1X GlutaMax, 1X non-essential amino acids, 20~100 ng / ml EGF, 50~300 ng / ml R-spondin-1, 10~200 ng / ml Noggin, and 1~20 μM Y-27632. The quaternary differentiation products were transferred to a fifth induction medium for quinary differentiation to obtain quinary differentiation products. The fifth induction medium consisted of Advanced DMEM / F-12 medium, 0.5X N2 additive, 0.5X B27 additive, 1X GlutaMax, 1X non-essential amino acids, 20~100 ng / ml EGF, 50~300 ng / ml R-spondin-1, and 10~200 ng / ml Noggin. The fifth-grade differentiation products were dynamically cultured in a maintenance medium to obtain mature intestinal organoids. The maintenance medium consisted of Advanced DMEM / F-12 medium, 1X GlutaMax, 0.1~1μM all-trans retinoic acid, and 1~10μM Forskolin.

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