In-vitro induction method of stomach organoid and application of in-vitro induction method

Abstract

The invention relates to an in-vitro induction method of stomach organoids and application of the in-vitro induction method. The invention provides a culture medium for stomach organoids and an in-vitro induction method and application of the culture medium.

Description

Technical Field

[0001] The present disclosure relates to the field of cell differentiation and regenerative medicine, and in particular to the in vitro culture of organoids. Background Art

[0002] The stomach is an important digestive organ that connects the esophagus and duodenum and is one of the important barriers for the interaction between the internal and external environments. Helicobacter pylori infection, poor diet, alcohol intake and smoking can damage the gastric epithelium, thereby inducing spasmolytic peptide expressing metaplasia (SPEM) or intestinal metaplasia (IM), which then develops into chronic atrophic gastritis (CAG). The occurrence of intestinal metaplasia or atrophic gastritis is closely related to the changes in gastric epithelial cell types, which are accompanied by the loss of parietal cells and chief cells, as well as the excessive proliferation of surface mucous cells (Pit cells) and the appearance of TFF2-positive SPEM cells and intestinal epithelial cells such as goblet cells and absorptive cells (Enterocyte). Studies have shown that Helicobacter pylori induces the loss of acid secretion function of parietal cells, or even apoptosis of parietal cells, which causes abnormal gastric pH and is accompanied by excessive proliferation of surface mucous cells, leading to gastritis and even gastric cancer.

[0003] In response to the above problems or clinical explorations, traditional research models are two-dimensional cell lines or animal models. However, it is difficult for cell lines to truly reflect the cell composition or three-dimensional structure of the human stomach, and the research conclusions are quite different from the actual situation in vivo. Animal models and humans have species differences, and the cost is high, which is not suitable for large-scale promotion. Organoids are three-dimensional cell clusters that are similar to the structure and function of in vivo tissues and organs after in vitro culture and expansion. Therefore, they have a variety of cell compositions, can partially perform tissue and organ functions, and participate in physiological homeostasis and the occurrence and development of diseases, and are therefore widely used. Gastric organoids have been widely used as an important technical means to study epithelial cell renewal and damage repair, as well as to simulate infections by exogenous pathogens such as Helicobacter pylori.

[0004] In the prior art, three-dimensional (3D) human gastric organoids are formed by in vitro culture using human adult stem cells, human embryonic stem cells or pluripotent stem cells, or human gastric epithelial cells are established by two-dimensional culture methods using human adult stem cells in gas-liquid exchange. However, these methods for establishing human gastric organoids all have defects.

[0005] Based on the in vitro culture of human adult stem cells into three-dimensional human gastric organoids, an induced differentiation system for surface mucus cells was established. The composition of the culture medium contained in this technology is complex, and it is difficult to determine the induction efficiency or maturity of surface mucus cells, and there is no process for wall-free cell induction.

[0006] The gastric fundus organoid system induced by human embryonic stem cells and pluripotent stem cells requires stringent conditions for biological samples, and the operation is complex and time-consuming, making it difficult to determine the induction efficiency or maturity of surface mucus cells and parietal cells.

[0007] The human gastric epithelial cell system based on gas-liquid exchange culture is more complicated to operate, and it is difficult for ordinary technical levels to meet the requirements of this system, and it is also time-consuming. In addition, the culture medium components of the surface mucous cells and parietal cells induced by this system are complex and costly.

[0008] Since the existing gastric epithelial organoid culture model is difficult to simulate the cell composition in the body, and cannot restore the cell damage repair or inflammation process caused by pathogen infection in the body, adjusting the gastric organoid culture system, promoting the differentiation of gastric surface mucous cells and parietal cells, and improving the maturity of gastric organoids are key issues facing the gastric organoid field.

[0009] Therefore, it is necessary to provide a simple and easy-to-operate differentiation culture method to effectively induce surface mucous cells and parietal cells. Summary of the invention

[0010] In order to solve one of the above-mentioned technical problems existing in the prior art, the present invention discloses an improved three-dimensional culture method of gastric organoids based on in vivo and in vitro gastric epithelial cell differentiation regulation models, which is helpful for the subsequent culture of complete regional gastric organoids and related research applications.

[0011] According to one aspect of the present disclosure, a culture medium for promoting cell differentiation is provided, wherein the culture medium comprises 10 to 30 ng / ml of BMP4.

[0012] In some embodiments, the culture medium is a culture medium for promoting surface mucous cell differentiation, which includes: BMP4; and a member of the epidermal growth factor (EGF) family.

[0013] In some embodiments, the EGF family member can be selected from one or more of EGF, transforming growth factor α (TGFα), amphiregulin (AREG), β-acetylglobulin (BTC), heparin-binding epidermal growth factor-like growth factor (HB-EGF), epiregulin (EPR), epigenetic protein (EPG), neuregulin (NRG), and neuregulin-2s (NRG2s). In a specific embodiment, the culture medium for promoting surface mucous cell differentiation includes EGF.

[0014] In some embodiments, in the culture medium for promoting surface mucous cell differentiation, the concentration of the epidermal growth factor EGF family member is 20-100 ng / ml, preferably 30-80 ng / ml, and most preferably 50 ng / ml.

[0015] In some embodiments, the culture medium for promoting surface mucous cell differentiation may include 10-30 ng / ml of BMP4. In some embodiments, the second culture medium may include 15-25 ng / mL of BMP4, for example, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, 20 ng / ml, 21 ng / ml, 22 ng / ml, 23 ng / ml, 24 ng / ml or 25 ng / ml of BMP4. In some embodiments, the culture medium for promoting surface mucous cell differentiation may include 20 ng / ml of BMP4.

[0016] The present disclosure found that the simultaneous addition of EGF family members (such as EGF) and BMP4 to the culture medium can significantly increase the proportion of surface mucous cells in gastric organoids, even as high as more than 90%.

[0017] The culture medium for promoting surface mucous cell differentiation disclosed herein does not contain a BMP signaling pathway inhibitor (eg, Noggin), a Wnt signaling pathway agonist (eg, R-spondin1), and / or a GSK3β inhibitor (eg, CHIR-99021).

[0018] In some embodiments, the culture medium for promoting surface mucous cell differentiation may also include, but is not limited to, one or more of the following substances: serum-free additives, such as N2 and B27; glutamine or its analogs, such as L-glutamine or GlutaMAX; antioxidants, such as N-acetylcysteine.

[0019] In some embodiments, in the culture medium for promoting surface mucous cell differentiation, the serum-free additive may include N2 and B27.

[0020] In some embodiments, the culture medium for promoting surface mucous cell differentiation may include 0.5-5mM GlutaMAX. In some embodiments, the culture medium for promoting surface mucous cell differentiation may include 1.0mM-4mM GlutaMAX, for example, including 1.0mM, 1.5mM, 2.0mM, 2.5mM, 3.0mM, 3.5mM or 4.0mM, GlutaMAX. In some embodiments, the culture medium for promoting surface mucous cell differentiation may include 2mM GlutaMAX.

[0021] In some embodiments, the antioxidant may be N-acetylcysteine. In some embodiments, the culture medium for promoting surface mucous cell differentiation may include 0.1 to 2 mM N-acetylcysteine, for example, 0.1 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, mM, 1.8 mM or 2.0 mM N-acetylcysteine. In some embodiments, the culture medium for promoting surface mucous cell differentiation may include 1 mM N-acetylcysteine.

[0022] In some embodiments, the culture medium is a culture medium that promotes parietal cell differentiation, which includes: BMP4; and a neural stem cell differentiation inducer.

[0023] In some embodiments, the neural stem cell differentiation inducer may be Isx-9. In some embodiments, the culture medium for promoting parietal cell differentiation may include 10 to 30 μM Isx-9. In some embodiments, the culture medium for promoting parietal cell differentiation may include 15 to 25 μM Isx-9, for example, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM or 25 μM Isx-9. In some embodiments, the culture medium for promoting parietal cell differentiation may include 20 μM Isx-9.

[0024] In some embodiments, the culture medium for promoting parietal cell differentiation may include 10-30 ng / ml of BMP4. In some embodiments, the culture medium for promoting parietal cell differentiation may include 15-25 ng / mL of BMP4, for example, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, 20 ng / ml, 21 ng / ml, 22 ng / ml, 23 ng / ml, 24 ng / ml or 25 ng / ml of BMP4. In some embodiments, the culture medium for promoting parietal cell differentiation may include 20 ng / ml of BMP4.

[0025] The present disclosure surprisingly found that neural stem cell differentiation inducers known to induce neuronal cell differentiation and endocrine cell differentiation in small intestinal organoids, such as Isx-9, improve parietal cell differentiation, and its combination with BMP4 can further significantly promote parietal cell differentiation and increase the expression of parietal cell markers.

[0026] In some embodiments, the culture medium that promotes parietal cell differentiation may further include metformin. In some embodiments, the culture medium that promotes parietal cell differentiation may include 50 to 150 μM of metformin. In some embodiments, the culture medium that promotes parietal cell differentiation may include 80 to 120 μM of metformin, for example, including 80 μM, 85 μM, 90 μM, 95 μM, 100 μM, 105 μM, 110 μM, 115 μM or 120 μM of metformin. In some embodiments, the culture medium that promotes parietal cell differentiation may include 100 μM of metformin. The addition of metformin can further promote the differentiation of parietal cells and increase the expression of parietal cell markers (such as ATP4B).

[0027] In some embodiments, the culture medium for promoting parietal cell differentiation may further include an ERR agonist, such as DY131, SLU-PP-332. In some embodiments, the ERR agonist may be DY131. In some embodiments, the culture medium for promoting parietal cell differentiation may contain 1 to 10 μM DY131. In some embodiments, the culture medium for promoting parietal cell differentiation may contain 3 to 8 μM DY131, for example, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM or 8 μM DY131. In some embodiments, the culture medium for promoting parietal cell differentiation may contain 5 μM DY131. The addition of an ERR agonist (such as DY131) can further promote the differentiation of parietal cells and increase the expression of parietal cell markers (such as ATP4B).

[0028] In some embodiments, the culture medium for promoting parietal cell differentiation may also contain a MEK pathway inhibitor, such as PD0325901, CI-1040, Trametinib, Cobimetinib, Binimetinib, Selumetinib, Refametinib, Pimasertib, RO4987655, AZD8330. In some embodiments, the MEK pathway inhibitor may be PD0325901. In some embodiments, the culture medium for promoting parietal cell differentiation may contain 0.5 to 5 μM PD0325901. In some embodiments, the culture medium for promoting parietal cell differentiation may contain 1.0 to 4.0 μM PD0325901, for example, including 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM or 4.0 μM PD0325901. In some embodiments, the culture medium for promoting parietal cell differentiation may contain 2 μM PD0325901.

[0029] In some embodiments, the culture medium for promoting parietal cell differentiation further comprises 10-30 μM Isx-9, 50-150 μM metformin, 1-10 μM DY131 and 0.5-5 μM PD0325901.

[0030] In some embodiments, the culture medium may include: serum-free additives. In some embodiments, the culture medium may include: N2 and / or B27. In some embodiments, the culture medium may include: 1×N2 and / or 1×B27.

[0031] In some embodiments, the culture medium further comprises: glutamine or its analogs. In some embodiments, the culture medium may comprise L-glutamine and / or GlutaMAX. In some embodiments, the culture medium may comprise 0.5 to 5 mM GlutaMAX. In some embodiments, the culture medium may comprise 1.0 mM to 4 mM GlutaMAX, for example, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM or 4.0 mM GlutaMAX. In some embodiments, the culture medium may comprise 2 mM GlutaMAX.

[0032] In some embodiments, the culture medium further comprises: an antioxidant. In some embodiments, the culture medium may include N-acetylcysteine. In some embodiments, the culture medium may include 0.1 to 2 mM N-acetylcysteine. In some embodiments, the culture medium may include 0.1 to 2 mM N-acetylcysteine, for example, including 0.1 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, mM, 1.8 mM or 2.0 mM N-acetylcysteine. In some embodiments, the culture medium may include 1.0 mM N-acetylcysteine.

[0033] In some embodiments, the culture medium further comprises: a basal culture medium. In some embodiments, the basal culture medium is selected from MEM, DMEM, IMDM, HAM F12, PRMI1640 and DMEM / F12 culture medium.

[0034] According to another aspect of the present disclosure, a method for inducing gastric organoids is provided, the method comprising the following steps: 1) obtaining gastric glands; 2) culturing using a first culture medium so that the obtained gastric glands grow into first gastric organoids; and 3) replacing the first culture medium with the above-mentioned cell differentiation promoting culture medium provided in the present disclosure for culturing.

[0035] In some embodiments, the first gastric organoid mainly includes proliferative cells and neck mucous cells. In some embodiments, the first gastric organoid includes more than 70% proliferative cells and neck mucous cells. In some embodiments, the first gastric organoid includes more than 60% neck mucous cells. In some embodiments, the first gastric organoid includes about 60% to 70% neck mucous cells, for example, including about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70% neck mucous cells. In some embodiments, the first gastric organoid includes about 10% to 30% proliferative cells, for example, including about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% proliferative cells.

[0036] In some embodiments, the first gastric organoid lacks cells that function as gastric glands, such as surface mucous cells or parietal cells.

[0037] In some embodiments, the first culture medium may include, but is not limited to, one or more of the following substances: serum-free additives, such as N2 and B27; glutamine or its analogs, such as L-glutamine or GlutaMAX; antioxidants, such as N-acetylcysteine; cell growth factors, such as epidermal growth factor (EGF), fibroblast growth factor (such as FGF1-FGF23); BMP signaling pathway inhibitors, such as Noggin, LDN-193189, Dorsomorphin, DMH1; Wnt signaling pathway agonists, such as R-spondin 1; GSK-3β inhibitors, such as CHIR-99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, Tideglusib, AR-A014418, LY2090314, SB216763, AZD1080; TGF-β receptor ALK-5 inhibitors, such as A-83-01; ROCK1 inhibitors, such as Y27632, Y-32885, Y-399 83. fasudil; hormones, such as gastrin, ghrelin, leptin, somatostatin, secretin, gastric inhibitory polypeptide (GIP), cholecystokinin (CCK), motilin, peptide YY (PYY), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), and oxyntomodulin (OXM).

[0038] In some embodiments, in the first culture medium, the serum-free additive may include N2 and B27, and the added amount may be the amount conventionally added in the art. In some embodiments, the first culture medium may include 1×N2. In some embodiments, the first culture medium may include 1×B27.

[0039] In some embodiments, the first culture medium may include 0.5-5 mM GlutaMAX. In some embodiments, the first culture medium may include 1.0 mM-4 mM GlutaMAX, for example, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM or 4.0 mM GlutaMAX. In some embodiments, the first culture medium may include 2 mM GlutaMAX.

[0040] In some embodiments, the BMP signaling pathway inhibitor may be Noggin. In some embodiments, the first culture medium may include 60 to 150 ng / ml of Noggin. In some embodiments, the first culture medium may include 80 to 120 ng / mL of Noggin, for example, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 110 ng / mL or 120 ng / mL of Noggin. In a specific embodiment, the first culture medium may include 100 ng / mL of Noggin.

[0041] In some embodiments, the Wnt signaling pathway agonist may be R-spondin 1. In some embodiments, the first culture medium may include 300-700 ng / mL of R-spondin 1. In some embodiments, the first culture medium may include 400-600 ng / mL of R-spondin 1, for example, 400 ng / mL, 450 ng / mL, 500 ng / mL, 550 ng / mL or 600 ng / mL of R-spondin 1. In a specific embodiment, the first culture medium may include 500 ng / mL of R-spondin 1.

[0042] In some embodiments, the GSK-3β inhibitor may be CHIR-99021. In some embodiments, the first culture medium may include 1 to 10 μM CHIR-99021. In some embodiments, the first culture medium may include 3 to 8 μM CHIR-99021, for example, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM or 8 μM CHIR-99021. In some embodiments, the first culture medium may include 5 μM CHIR-99021.

[0043] In some embodiments, the TGF-β receptor ALK-5 inhibitor can be A-83-01. In some embodiments, the first culture medium can include 1 to 10 nM A-83-01. In some embodiments, the first culture medium can include 3 to 8 nM A-83-01, for example, 3 nM, 3.5 nM, 4 nM, 4.5 nM, 5.0 nM, 5.5 nM, 6 nM, 6.5 nM, 7 nM, 7.5 nM or 8 nM A-83-01. In some embodiments, the first culture medium can include 5 nM A-83-01.

[0044] In some embodiments, the ROCK1 inhibitor may be Y27632. In some embodiments, the first culture medium may include 5 to 15 μM Y27632. In some embodiments, the first culture medium may include 8 to 12 μM Y27632, for example, 8 μM, 8.5 μM, 9 μM, 9.5 μM, 10 μM, 10.5 μM, 11 μM, 11.5 μM or 12 μM Y27632. In some embodiments, the first culture medium may include 10 μM Y27632.

[0045] In some embodiments, the hormone may include gastrin. In some embodiments, the first culture medium may include 5 to 15 nM of Gastrin. In some embodiments, the first culture medium may include 8 to 12 nM of Gastrin, for example, 8 nM, 8.5 nM, 9 nM, 9.5 nM, 10 nM, 10.5 nM, 11 nM, 11.5 nM or 12 nM of Gastrin. In some embodiments, the first culture medium may include 10 nM of Gastrin.

[0046] In some embodiments, the antioxidant can be N-acetylcysteine. In some embodiments, the first culture medium can include 0.1 to 2 mM N-acetylcysteine, for example, including 0.1 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, mM, 1.8 mM or 2.0 mM N-acetylcysteine. In some embodiments, the first culture medium can include 1.0 mM N-acetylcysteine.

[0047] In some embodiments, the first culture medium may include epidermal growth factor and / or FGF. In some embodiments, the FGF may be selected from one or more of FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22 and FGF23. In a specific embodiment, the FGF may be FGF10.

[0048] In some embodiments, the first culture medium may include epidermal growth factor (EGF) and / or FGF10.

[0049] In some embodiments, the first culture medium may include 20-70 ng / mL of EGF. In some embodiments, the first culture medium may include 30-60 ng / mL of EGF, for example, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL or 70 ng / mL of EGF. In some embodiments, the first culture medium may include 50 ng / mL of EGF.

[0050] In some embodiments, the first culture medium may include 60-150 ng / ml of FGF10. In some embodiments, the first culture medium may include 80-120 ng / mL of FGF10, for example, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 105 ng / mL, 110 ng / mL, 115 ng / mL or 120 ng / mL of FGF10. In some embodiments, the first culture medium may include 100 ng / mL of FGF10.

[0051] In some embodiments, in step 2), the gastric glands are mixed with matrix gel and then cultured with the first culture medium. In some embodiments, the matrix gel can be Matrigel matrix gel.

[0052] In some embodiments, the first gastric organoid can maintain growth of the gastric organoid in the first culture medium for a long period of time.

[0053] In some embodiments, the first gastric organoid may have high expression of, for example, one or more of AAGNT, MUC6, CD44, OLFM4, TNFRSF19, AQP5, and Mki67. In some embodiments, the markers of proliferating cells may include those known in the art, for example, may include, but are not limited to, Mki67, PCNA, TOP2A, BIRC5, and STMN1.

[0054] In some embodiments, the gastric glands may be derived from mammals. For example, the gastric glands may be derived from humans, non-human primates, mice, rats, sheep, cattle, cats, dogs, pigs or rabbits.

[0055] In some embodiments, in step 3), the culture medium that promotes cell differentiation is a second culture medium, and the second culture medium enables the first gastric organoid to differentiate into a second gastric organoid rich in surface mucous cells, wherein the second culture medium may include 10 to 30 ng / ml of BMP4.

[0056] In some embodiments, the second gastric organoid enriched in surface mucus cells may include more than about 90% surface mucus cells, for example, may include more than about 90%, more than 91%, more than 92%, more than 93%, more than 94% or more than 95% surface mucus cells.

[0057] In some embodiments, the second culture medium may include bone morphogenetic protein 4 (BMP4) and an epidermal growth factor (EGF) family member.

[0058] In some embodiments, the second culture medium may include 10-30 ng / ml of BMP4. In some embodiments, the second culture medium may include 15-25 ng / mL of BMP4, for example, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, 20 ng / ml, 21 ng / ml, 22 ng / ml, 23 ng / ml, 24 ng / ml or 25 ng / ml of BMP4. In some embodiments, the second culture medium may include 20 ng / ml of BMP4.

[0059] In some embodiments, the second culture medium may further include an epidermal growth factor EGF family member. In some embodiments, the EGF family member may be selected from one or more of EGF, transforming growth factor alpha (TGFα), amphiregulin (AREG), β-acetylglobulin (BTC), heparin-binding epidermal growth factor-like growth factor (HB-EGF), epiregulin (EPR), epigenetic protein (EPG), neuregulin (NRG), and neuregulin-2s (NRG2s). In a specific embodiment, the culture medium for promoting surface mucous cell differentiation includes EGF.

[0060] In some embodiments, in the second culture medium, the concentration of the EGF family member may be 20 to 100 ng / ml. In some embodiments, the concentration of the EGF family member may be 30 to 80 ng / ml, such as 30 ng / ml, 35 ng / ml, 40 ng / ml, 45 ng / ml, 50 ng / ml, 55 ng / ml, 60 ng / ml, 65 ng / ml, 70 ng / ml, 75 ng / ml or 80 ng / ml. In a specific embodiment, the concentration of the EGF family member may be 50 ng / ml. In the second culture medium of the present disclosure, no other growth factors are included, such as fibroblast growth factor.

[0061] In some embodiments, the second culture medium may further include, but is not limited to, one or more of the following substances: serum-free supplements, such as N2 and B27; glutamine or its analogs, such as L-glutamine or GlutaMAX; antioxidants, such as N-acetylcysteine.

[0062] In some embodiments, in the second culture medium, the serum-free supplement may include N2 and B27.

[0063] In some embodiments, the second culture medium may include 0.5-5 mM GlutaMAX. In some embodiments, the second culture medium may include 1.0 mM-4 mM GlutaMAX, for example, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM or 4.0 mM GlutaMAX. In some embodiments, the second culture medium may include 2 mM GlutaMAX.

[0064] In some embodiments, the antioxidant can be N-acetylcysteine. In some embodiments, the second culture medium can include 0.1 to 2 mM N-acetylcysteine, for example, including 0.1 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, mM, 1.8 mM or 2.0 mM N-acetylcysteine. In some embodiments, the second culture medium can include 1 mM N-acetylcysteine.

[0065] In some embodiments, the second gastric organoid rich in surface mucus cells may include highly expressed one or more of UEAI, MUCL3, TFF1, GKN2, GKN1, MUC5AC, and FOXQ1.

[0066] In some embodiments, the surface mucus cell markers may be known in the art, such as, but not limited to, one or more of UEAI, MUCL3, TFF1, GKN2, GKN1, MUC5AC, and FOXQ1.

[0067] In some embodiments, the diameter of the second gastric organoid enriched for surface mucous cells is significantly increased compared to the first gastric organoid.

[0068] In some embodiments, the second gastric organoid can survive in the second culture medium for about 4-5 days before significant cell death occurs.

[0069] In some embodiments, the differentiation of surface mucus cells can be induced by culturing for 2 to 3 days after the first culture medium is replaced with the second culture medium.

[0070] In some embodiments, in step 3), the culture medium that promotes cell differentiation is a third culture medium for culturing, so that the first gastric organoids differentiate into third gastric organoids rich in parietal cells, and the third culture medium may include 10 to 30 ng / ml of BMP4.

[0071] In some embodiments, the third parietal cell-enriched gastric organoid may contain more than 90% of parietal cell precursor cells, for example, may include about more than 90%, more than 91%, more than 92%, more than 93%, more than 94% or more than 95% of parietal cell precursor cells.

[0072] In some embodiments, the third culture medium includes 10-30 ng / ml of BMP4 and a neural stem cell differentiation inducer.

[0073] In some embodiments, the third culture medium may include: BMP4; and a neural stem cell differentiation inducer.

[0074] In some embodiments, the neural stem cell differentiation inducer may be Isx-9. In some embodiments, the third culture medium may include 10 to 30 μM Isx-9. In some embodiments, the third culture medium may include 15 to 25 μM Isx-9, for example, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM or 25 μM Isx-9. In some embodiments, the third culture medium may include 20 μM Isx-9.

[0075] In some embodiments, the third culture medium may further include metformin. In some embodiments, the third culture medium may include 50 to 150 μM of metformin. In some embodiments, the third culture medium may include 80 to 120 μM of metformin, for example, 80 μM, 85 μM, 90 μM, 95 μM, 100 μM, 105 μM, 110 μM, 115 μM or 120 μM of metformin. In some embodiments, the third culture medium may include 100 μM of metformin.

[0076] In some embodiments, the third culture medium may include 10-30 ng / ml of BMP4. In some embodiments, the second culture medium may include 15-25 ng / mL of BMP4, for example, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, 20 ng / ml, 21 ng / ml, 22 ng / ml, 23 ng / ml, 24 ng / ml or 25 ng / ml of BMP4. In some embodiments, the third culture medium may include 20 ng / ml of BMP4.

[0077] In some embodiments, the third culture medium may further include an ERR agonist, such as DY131, SLU-PP-332. In some embodiments, the ERR agonist may be DY131. In some embodiments, the third culture medium may include 1 to 10 μM DY131. In some embodiments, the third culture medium may include 3 to 8 μM DY131, for example, including 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM or 8 μM DY131. In some embodiments, the third culture medium may include 5 μM DY131.

[0078] In some embodiments, the third culture medium may also contain a MEK pathway inhibitor, such as PD0325901, CI-1040, Trametinib, Cobimetinib, Binimetinib, Selumetinib, Refametinib, Pimasertib, RO4987655, AZD8330. In some embodiments, the MEK pathway inhibitor may be PD0325901. In some embodiments, the third culture medium may contain 0.5 to 5 μM PD0325901. In some embodiments, the third culture medium may contain 1.0 to 4.0 μM PD0325901, for example, including 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM or 4.0 μM PD0325901. In some embodiments, the third culture medium may contain 2 μM PD0325901.

[0079] In some embodiments, the third gastric organoid enriched in parietal cells may include one or more of the highly expressed proteins, such as EZRIN, GHRL, CHGB, CKB, ATP4A, EGR1, and PLA2G1B.

[0080] In some embodiments, the markers of the parietal cells may be those known in the art, such as, but not limited to, EZRIN, CKB, ATP4A, and ATP4B.

[0081] In some embodiments, the diameter of the third parietal cell enriched gastric organoid is significantly decreased compared to the first gastric organoid.

[0082] In some embodiments, the third gastric organoid can survive in the third culture medium for about 4-5 days before significant cell death occurs.

[0083] In some embodiments, the differentiation of parietal cells can be induced by culturing for 2 to 3 days after the first culture medium is replaced with the third culture medium.

[0084] In some embodiments, the second gastric organoid and the second gastric organoid have reduced expression of a proliferation marker, such as Mki67, compared to the first gastric organoid.

[0085] In some embodiments, the first culture medium, the second culture medium and / or the third culture medium may include antibiotics, for example, may include penicillin and streptomycin.

[0086] In some embodiments, the basal medium of the first culture medium, the second culture medium and / or the third culture medium can use the basal medium conventionally used in the art. In some embodiments, the basal medium of the first culture medium, the second culture medium and / or the third culture medium can be selected from, but not limited to, MEM, DMEM, IMDM, HAMF12, PRMI1640, DMEM / F12 culture medium, etc.

[0087] According to another aspect, a gastric organoid obtained by the above method is provided, wherein the first gastric organoid mainly comprises proliferative cells and neck mucous cells. In some embodiments, the first gastric organoid comprises more than 70% proliferative cells and neck mucous cells. In some embodiments, the first gastric organoid comprises more than 60% neck mucous cells. In some embodiments, the first gastric organoid comprises about 60% to 70% neck mucous cells, for example, about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70% neck mucous cells. In some embodiments, the first gastric organoid comprises about 10% to 30% proliferative cells, for example, comprises about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% proliferative cells.

[0088] In some embodiments, the first gastric organoid lacks cells that function as gastric glands, such as surface mucous cells or parietal cells.

[0089] In some embodiments, the first gastric organoid may have high expression of, for example, one or more of AAGNT, MUC6, CD44, OLFM4, TNFRSF19, AQP5, and Mki67. In some embodiments, the markers of proliferating cells may include those known in the art, for example, may include, but are not limited to, Mki67, PCNA, TOP2A, BIRC5, and STMN1.

[0090] According to another aspect, a gastric organoid rich in surface mucus cells obtained by the above method or the above medium for promoting surface mucus cell differentiation is provided. In some embodiments, the second gastric organoid rich in surface mucus cells may include more than about 90% of surface mucus cells, for example, may include more than about 90%, more than 91%, more than 92%, more than 93%, more than 94% or more than 95% of surface mucus cells.

[0091] In some embodiments, the second gastric organoid rich in surface mucus cells may include highly expressed one or more of UEAI, MUCL3, TFF1, GKN2, GKN1, MUC5AC, and FOXQ1.

[0092] In some embodiments, the surface mucus cell markers may be known in the art, such as, but not limited to, one or more of UEAI, MUC5AC, GKN1, GKN2, TFF1, and MUCL3.

[0093] According to yet another aspect, provided is a gastric organoid enriched in parietal cells obtained by the above method or the above culture medium for promoting parietal cell differentiation.

[0094] In some embodiments, the third parietal cell-enriched gastric organoid may contain more than 90% of parietal cell precursor cells, for example, may include about more than 90%, more than 91%, more than 92%, more than 93%, more than 94% or more than 95% of parietal cell precursor cells.

[0095] In some embodiments, the third gastric organoid enriched in parietal cells may include one or more of the highly expressed proteins, such as EZRIN, GHRL, CHGB, CKB, ATP4A, EGR1, and PLA2G1B.

[0096] In some embodiments, the markers of the parietal cells may be those known in the art, such as, but not limited to, EZRIN, CKB, and ATP4A.

[0097] According to yet another aspect, a pharmaceutical preparation is provided, which includes the gastric organoid, the surface mucous cell-enriched gastric organoid, or the parietal cell-enriched gastric organoid of the present disclosure.

[0098] According to yet another aspect, provided is a use of the gastric organoid, the gastric organoid rich in surface mucous cells, or the gastric organoid rich in parietal cells of the present disclosure in the preparation of a medicament for preventing or treating a stomach-related disease.

[0099] According to yet another aspect, provided is a use of the gastric organoid, the gastric organoid enriched in surface mucous cells, or the gastric organoid enriched in parietal cells disclosed herein in preparing a gastric organoid model for the study of gastric-related diseases.

[0100] According to yet another aspect, provided is a use of the gastric organoid, the gastric organoid rich in surface mucous cells, or the gastric organoid rich in parietal cells of the present disclosure in screening drugs for preventing or treating stomach-related diseases.

[0101] According to yet another aspect, provided is a method for screening drugs for preventing or treating stomach-related diseases, the method comprising: contacting the gastric organoid, the gastric organoid rich in surface mucous cells, or the gastric organoid rich in parietal cells of the present disclosure with a candidate drug.

[0102] In some embodiments, the stomach-related disease may be caused by Helicobacter pylori infection. In some embodiments, the stomach-related disease may include gastric cancer, peptic ulcer disease, Ménetrier's disease, gastric injury, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 Shown are the proportions of various cell types and the expression profiles of different cell marker genes in epithelial cells of human gastric tissue and gastric organoids cultured under complete medium conditions. Figure 1 A shows the results of single-cell transcriptome sequencing of human gastric epithelial tissue (including gastric body and gastric antrum) and bioinformatics analysis. Figure 1 B shows the expression of surface mucous cell marker gene UEAI, epithelial cell marker gene E-CAD, parietal cell marker gene HK-Atpase, and proliferative cell marker genes MUC6 and MKI67 in gastric organoids cultured under complete medium conditions (left), as well as the cell proportion in gastric organoids (right). Figure 1 CD show the expression of surface mucus cell marker gene MUC5AC, neck mucus cell marker gene MUC6, parietal cell marker gene ATP4B, gastric mucosal chief cell marker gene PGC, proliferative cell marker gene MKI67, and antral stem cell marker gene LGR5 in gastric epithelial tissue and gastric body cells and antral cells of organoids under complete culture medium conditions, respectively.

[0104] Figure 2 The growth conditions of normal human gastric organoids in complete medium, differentiation medium-1 and differentiation medium-2 and the expression profiles of different cell marker genes are shown. Figure 2 A and Figure 2 B shows the diameter of gastric organoids in differentiation medium-1 and differentiation medium-2, respectively. Figure 2 C shows the relative expression levels of surface mucous cell marker genes MUC5AC, MUCL3, and TFF1 in gastric organoids cultured using complete medium, BMP4-component medium, and BMP4 plus EGF-component medium, respectively. Figure 2 D shows the expression of the parietal cell-related marker gene ESRRG in gastric organoids cultured using complete medium, Isx-9 component medium, Isx-9 plus BMP4 (BI) component medium, Isx-9 plus BMP4 and metformin (BIM) component medium, metformin and DY131 (BIMD) component medium, and Isx-9 plus BMP4, metformin, DY131 and PD0325901 (BIMDP) component medium. Figure 2 E shows the expression profiles of cell marker genes of gastric organoids cultured using complete medium, differentiation medium-1, and differentiation medium-2, respectively.

[0105] Figure 3 Shown are the differentiation results of surface mucous cells of normal human gastric organoids in complete medium and differentiation medium-1. Figure 3 A~ Figure 3 C shows the expression of surface mucous cell markers UEAI, MUC5AC, GKN2, proliferative cell marker Mki67, and epithelial cell marker E-CAD in organoids cultured in complete medium and differentiation medium-1, respectively.

[0106] Figure 4 The results of the differentiation of parietal cells of normal human gastric organoids in complete medium and differentiation medium-2 are shown. Figure 4 A to 4B respectively show the expression of the parietal cell-related marker gene ATP4B in gastric organoids cultured using complete medium, Isx-9 component medium, Isx-9 plus BMP4 (BI) component medium, Isx-9 plus BMP4 and metformin (BIM) component medium, metformin and DY131 (BIMD) component medium, and Isx-9 plus BMP4, metformin, DY131 and PD0325901 (BIMDP) component medium. Figure 4 C~ Figure 4E shows the expression of parietal cell markers HK-Atpase, EZRIN, and epithelial cell marker E-CAD, and proliferative cell marker Mki67 in organoids cultured in complete medium and differentiation medium-2, respectively.

[0107] Figure 5 The figure shows the proportion of each type of cell in normal human gastric organoids cultured in complete medium, differentiation medium-1 and differentiation medium-2 based on single-cell transcriptome sequencing analysis, as well as the similarity with the characteristics of similar cells in vivo. Figure 5 A shows the proportions of each type of cells when normal gastric organoids were cultured in complete medium, differentiation medium-1, and differentiation medium-2, respectively. Figure 5 B shows the results of correlation analysis between normal gastric organoids cultured in differentiation medium-1 and differentiation medium-2, and the differentiated surface mucous cells and parietal cells and the surface mucous cells and parietal cells of tissue origin, respectively. DETAILED DESCRIPTION

[0108] Existing human gastric organoids cultured based on human adult stem cells and complete culture medium are highly enriched in proliferating cells and lack differentiated functional cells, which leads to reduced maturity of human gastric organoids and significant differences in cell composition from the body, making it difficult to study the specific functions of various types of mature gastric epithelial cells.

[0109] The diversity of the cell composition and functional maturity of gastric organoids are important indicators for the application of gastric organoids, and are directly related to the application effects of gastric organoids in scientific research and clinical practice. Among them, the cell composition and functional maturity are affected by the in vitro culture system. By adjusting the culture medium formula, the cell composition and functional indicators of gastric organoids can be regulated. However, the gastric epithelial organoids of humans and mice cultured by the currently known culture systems are enriched with a large number of proliferating cells and contain fewer surface mucus cells. In addition, organoids based on three-dimensional culture of adult stem cells are difficult to induce parietal cells, resulting in the lack of cell composition, imbalanced cell proportions and incomplete functions of gastric organoids.

[0110] At the same time, the existing gastric epithelial organoid culture models are difficult to simulate the cell composition in the body, and are even more unable to restore the cell damage repair or inflammatory process caused by pathogen infection in the body. Therefore, adjusting the gastric organoid culture system, promoting the differentiation of gastric surface mucous cells and parietal cells, and improving the maturity of gastric organoids are key issues facing the gastric organoid field. However, the culture conditions for cell differentiation in organoids are currently unclear, and there is a lack of differentiation culture media for in vitro human gastric organoids, especially for the differentiation of surface mucous cells and parietal cells in organoids.

[0111] Based on the in vivo and in vitro gastric epithelial cell differentiation regulation model, the present invention establishes an improved differentiation culture medium and culture method for human gastric surface mucous cells and parietal cells, which can be used to construct a three-dimensional gastric organoid model, provide a basis for studying the specific functions of mature gastric epithelial cells and preventing the occurrence of gastric diseases, and facilitate the subsequent culture of gastric organoids and related research applications.

[0112] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0113] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0114] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0115] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0116] Gastric surface mucus cells, also known as apical cells, are the cell type in the gastric glands that is in most direct contact with food and the external environment. They have a fast renewal cycle and are the direct source of gastric mucus. They participate in the function of maintaining the homeostasis of the gastric microenvironment. At the same time, during the occurrence of gastritis, abnormal differentiation of proliferative cells will induce an increase in the number of surface mucus cells, resulting in an imbalance in the original cell ratio and aggravating the process of gastritis. Therefore, constructing human gastric organoids with differentiated and mature surface mucus cells is of great significance for studying the function and physiological and pathological balance of surface mucus cells. In some embodiments of the present disclosure, a culture medium for surface mucus cell differentiation is provided. The culture medium for surface mucus cell differentiation can promote the differentiation of surface mucus cells in human gastric organoids, contribute to the exploration of the regulation of gastric epithelial mucus secretion function, and is conducive to the development of drugs for targeted treatment of abnormal cell proliferation in the early stage of gastritis, which has positive significance for preventing gastritis from worsening and treating gastritis. In some embodiments, the gastric surface mucus cells of the present disclosure may be characterized by having significantly expressed UEAI. In some embodiments, compared with normal gastric organoids, the gastric surface mucus cells may be characterized by having an increased expression of UEAI.

[0117] Gastric parietal cells are the only cells in the gastric glands that can secrete gastric acid. They have a long renewal cycle and play a vital role in maintaining the homeostasis of gastric pH and digestive function. However, the culture conditions of gastric parietal cells are demanding. In the past, gastric organoids based on human adult stem cell culture usually lack such cells, which affects the research on the function of this type of cells. At the same time, gastritis is often accompanied by the death of parietal cells, which will further cause changes in the pH environment inside the stomach and inhibit the overall digestive ability of the stomach. Parietal cells are also particularly sensitive to Helicobacter pylori infection and are more susceptible to damage, thereby affecting the performance of gastric function. Therefore, constructing human gastric organoids with mature parietal cells is of great significance for studying the function and physiological and pathological balance of surface mucous cells. In some embodiments of the present disclosure, a culture medium for parietal cell differentiation is provided. The culture medium for parietal cell differentiation can promote the differentiation of parietal cells in human gastric organoids, help to better explore the regulatory mechanism of gastric acid secretion function, have direct application significance for the development of gastritis-related drugs and the therapeutic effect on parietal cells, and are conducive to the research and treatment of Helicobacter pylori infection that is widely present in China, and provide a more complete human gastric organoid model for gastric acid function research and related disease models. In some embodiments, the gastric parietal cells of the present disclosure may be characterized by having significantly expressed EZRIN. In some embodiments, the gastric parietal cells may be characterized by having elevated expression of EZRIN compared to normal gastric organoids.

[0118] The term "organoid" as used herein refers to a three-dimensional multicellular aggregate derived from stem cells that differentiate and self-organize to recapitulate the structural features and cell-cell interactions of mature tissues. The organoid is a three-dimensional aggregate of one or more cell types that mimics the surface appearance or true structure or function of a tissue or organ.

[0119] As used herein, the term "induce" is related to a process or action that causes a specific effect on the phenotype of a cell. Such an effect can be in the form of causing a phenotypic change, for example, differentiation to another cell phenotype; or it can be to maintain the cell in a specific cell form, for example, preventing dedifferentiation or promoting cell survival.

[0120] N2 and B27 are both serum-free additives, which are suitable for most neural cell cultures and can improve the cell culture effect.

[0121] GlutaMAX is an alternative to L-glutamine that has better stability and can improve cellular status.

[0122] N-acetylcysteine ​​is an antioxidant that has the functions of interfering with the generation of free radicals, scavenging generated free radicals, and regulating the metabolic activity of cells.

[0123] Epidermal Growth Factor (EGF) is a cell growth factor that stimulates cell growth.

[0124] Noggin is an important molecule that inhibits the BMP signaling pathway and belongs to a group of diffusible proteins.

[0125] R-spondin 1 is a secreted protein in the R-Spondin family of Wnt / β-catenin signaling regulatory proteins, which promotes cell proliferation by activating Wnt signaling.

[0126] CHIR-99021 is a GSK-3β inhibitor that can significantly activate the Wnt / β-catenin signaling pathway.

[0127] A-83-01 is an inhibitor of TGF-β (Transforming growth factor beta) type I receptor ALK-5, which can promote cell growth.

[0128] Y27632 is a ROCK1 inhibitor that can significantly inhibit the anoikis phenomenon of organoids and promote survival in cultured organoids.

[0129] FGF10 is a heparin-binding protein that promotes epithelial cell growth, proliferation, and differentiation.

[0130] Gastrin is a hormone that promotes acid secretion from gastric parietal cells and regulates gastric motility.

[0131] BMP4 is bone morphogenetic protein 4, which belongs to the TGFβ superfamily and is involved in the regulation of stem cell differentiation and proliferation in the gastrointestinal tract.

[0132] Isx-9 (Isoxazole 9) is a neural stem cell differentiation inducer that can activate EMT induced by TGFβ and non-canonical Wnt signaling pathways.

[0133] Metformin is a drug used to treat type 2 diabetes.

[0134] DY131 is a selective ERRγ and ERRβ agonist.

[0135] PD0325901 is a selective and non-ATP-competitive MEK pathway inhibitor.

[0136] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0137] It should be noted that, if the specific conditions are not specified in the examples, the experimental conditions are carried out according to conventional conditions, manufacturer recommendations or publicly reported experimental conditions. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially. If the manufacturer of the reagents used is specified, similar products from other manufacturers are substitutable.

[0138] Example

[0139] Example 1. Cultivation of human gastric organoids based on a 3D culture system

[0140] The specific method for culturing human gastric organoids based on a 3D culture system is as follows:

[0141] Obtain the gastric body tissue isolated during sleeve gastrectomy and wash it three times with pre-cooled PBS buffer with a pH of 7.4 to remove the chyme. Incubate the remaining tissue on ice for 30 minutes with PBS buffer containing 10mM EDTA. Replace the PBS buffer containing EDTA with fresh pre-cooled PBS buffer, and then use a scalpel to scrape vigorously to obtain the gastric glands. Transfer the obtained gland suspension to a 15mL centrifuge tube, let it stand for 1 minute, and take the supernatant to remove the precipitated tissue blocks. Centrifuge at 600rpm for 3 minutes to obtain the precipitate and remove the unprecipitated single cells. The precipitate after centrifugation is a relatively pure gastric body gland, which can be used for in vitro culture.

[0142] Every 200 separated gastric glands were mixed with Matrigel (BD Biosciences), added to the cell culture plate, placed in a 37° C., 5% carbon dioxide cell culture incubator for 15-20 minutes to wait for the Matrigel to solidify, and then added with complete culture medium for culture. The complete culture medium contained basal medium Advanced DMEM / F12 (ThermoFisher), serum replacements 1×N2 (ThermoFisher) and 1×B27 (ThermoFisher), 100U / ml penicillin-streptomycin (ThermoFisher), 2mM GlutaMAX (ThermoFisher), 1mM N-acetylcysteine ​​(Selleck), 50ng / mL epidermal growth factor (EGF, nearshore protein), 100ng / mL Noggin (nearshore protein), 500ng / mL R-spondin 1 (nearshore protein), 5μM CHIR-99021 (Selleck), 2μM A-83-01 (Selleck), 100ng / ml FGF10 (nearshore protein), 10nM Gastrin (TOCRIS) and 10μM Y27632 (Selleck).

[0143] Under this culture condition, the cells were observed under an optical microscope after 2 to 3 days of culture. Figure 2 A. Human gastric organoids can be grown for long periods of time in complete culture medium.

[0144] By performing single-cell transcriptome sequencing of human gastric epithelial tissue (including gastric body and gastric antrum) and performing bioinformatics analysis, it can be seen that a high proportion of surface mucous cells and parietal cells (including parietal cell precursor cells and parietal cells) are enriched in the epithelial cells of human gastric tissue ( Figure 1 A). However, gastric organoids cultured in complete medium, such as Figure 1 As shown in B, the main enrichment is in the neck mucous cells and proliferative cells (up to 40-60%), while the proportion of surface mucous cells and parietal cells is very low (less than 10%). RNA extraction was performed according to the instructions of the Cell / Tissue Total RNA Extraction Kit V2 (Vazyme / Novozyme), and RNA was extracted from gastric epithelial tissue (distinguishing between the gastric body and gastric antrum) and organoids cultured in complete medium, and real-time quantitative PCR was performed to detect the expression of various epithelial cell marker genes. Figure 1 C and Figure 1As shown in D, compared with gastric epithelial tissue, in organoids grown under complete culture medium conditions, the surface mucous cell and parietal cell marker genes MUC5AC and ATP4B were significantly lower than the tissue level expression, but expressed a certain level of neck mucous cell and proliferative cell marker genes MUC6 and MKI67, and highly expressed the gastric antral stem cell marker gene LGR5.

[0145] Example 2. Differentiation and culture of surface mucous cells in human gastric organoids

[0146] In this embodiment, the culture medium is adjusted to induce the differentiation culture of surface mucus cells. The complete culture medium in Example 1 is replaced with the replacement differentiation medium-1, and placed in a 37°C, 5% carbon dioxide cell culture incubator for culture. Among them, the differentiation medium-1 is based on Advanced DMEM / F12 (ThermoFisher) as the basic culture medium, and also contains: 1×N2 (ThermoFisher), 1×B27 (ThermoFisher), 100U / ml penicillin-streptomycin (ThermoFisher), 2mMGlutaMAX (ThermoFisher), 1mM N-acetylcysteine ​​(Selleck), 50ng / ml epidermal growth factor (nearshore protein) and 20ng / ml BMP4 (R&D).

[0147] After culturing in differentiation medium-1 for 2 to 3 days, the cells were observed under an optical microscope. Figure 2 As shown in A.

[0148] Example 3. Differentiation and culture of parietal cells in human gastric organoids

[0149] In this example, the culture medium was adjusted to induce differentiation of parietal cells.

[0150] The complete medium in Example 1 was replaced with differentiation medium-2, and the cells were cultured in a cell culture incubator at 37°C and 5% carbon dioxide. Differentiation medium-2 can induce parietal cell differentiation, and is based on Advanced DMEM / F12 and contains: 1×N2 (ThermoFisher), 1×B27 (ThermoFisher), 5U / ml penicillin-streptomycin (ThermoFisher), 2mM GlutaMAX (ThermoFisher), 1mM N-acetylcysteine ​​(Selleck), 20ng / mlBMP4 (R&D), 20μM Isoxazole 9 (Isx-9, Selleck), 100μM metformin (Selleck), 5μM DY131 (Selleck), and 2μM PD0325901 (Selleck).

[0151] After culturing in differentiation medium-2 for 2 to 3 days, the cells were observed under an optical microscope. Figure 2 As shown in A.

[0152] Example 4. Size statistics and transcriptome analysis of gastric organoids cultured in different culture media.

[0153] For the organoids obtained in complete medium, differentiation medium-1 and differentiation medium-2 obtained in Examples 1 to 3, 10 to 15 photos were collected under an optical microscope, and the diameters of the organoids in each medium were counted. Figure 2 As shown in B. Figure 2 As can be seen from the results of B, the diameter of the gastric organoids formed in differentiation medium-1 increased, while the diameter of the gastric organoids in differentiation medium-2 decreased significantly.

[0154] To investigate the effects of different differentiation inducers or their combinations on the differentiation of surface mucous cells and parietal cells, the culture medium (Advanced DMEM / F12 (ThermoFisher), 1×N2 (ThermoFisher), 1×B27 (ThermoFisher), 100U / ml penicillin-streptomycin (ThermoFisher), 2mM GlutaMAX (ThermoFisher), 1mM N-acetylcysteine ​​(Selleck)) was supplemented with 20ng / ml BMP4 (R&D), 50ng / ml EGF (Jinbin), 20μM Isoxazole 9 (Isx-9, Selleck), 100μM Metformin (Selleck), 5μM DY131 (Selleck) and 2μM PD0325901 (Selleck) or their combinations (as shown in Table 1 below) to detect the expression of markers of surface mucous cells and parietal cells.

[0155] Table 1 Composition of different culture media

[0156] Group BMP4 EGF Isoxazole9 Metformin DY131 PD0325901 BMP4(B) √ / / / / / BMP4+EGF √ √ / / / / ISX-9(I) / / √ / / / BI √ / √ / / / BIM √ / √ √ / / BIMD √ / √ √ √ / BIMDP √ / √ √ √ √

[0157] Complete medium was used as control (Ctrl).

[0158] The organoids were cultured for two days using complete medium and the differentiation medium of the different groups shown in the table above. RNA was extracted using the Cell / Tissue Total RNA Extraction Kit V2 (Vazyme / Novozyme) according to the instructions. Real-time quantitative qPCR was then performed to detect the surface mucus cell-related marker genes MUC5AC, MUCL3, TFF1 and GKN2, and the parietal cell-related marker genes ATP4B and ESRRG. The results are shown in Figure 2 C. Figure 2 D. Figure 3 A. Figure 4 A and Figure 4 As shown in B.

[0159] like Figure 2 C and Figure 3 As shown in A, BMP4 treatment can induce the upregulation of the expression of these marker genes, and the addition of EGF can enhance the differentiation-inducing effect of BMP4.

[0160] like Figure 2 D. Figure 4 A and Figure 4 As shown in B, Isx-9 treatment can induce upregulation of the marker gene ESRRG expression, and the addition of BMP4 can enhance the differentiation-inducing effect of Isx-9. Metformin has been reported to promote the differentiation and maturation of parietal cells in mice. DY131, as an agonist of ESRRG, has the effect of promoting the differentiation and maturation of parietal cells, and PD0325901 has a certain promoting effect on the induction of parietal cell differentiation by embryonic stem cells. Therefore, these three small molecules were added together with BMP4 and Isx-9 as differentiation medium-2 to jointly induce the differentiation and maturation of parietal cells.

[0161] In addition, RNA was extracted from organoids obtained in complete medium, differentiation medium-1, and differentiation medium-2 using the Cell / Tissue Total RNA Extraction Kit V2 (Vazyme / Novozyme) according to the instructions. The extracted RNA was sequenced and subjected to bioinformatics statistical analysis. Figure 2 E shows a heat map of differentially expressed genes under three culture conditions, where the differentially expressed genes selected were 3 to 5 marker genes of different types of gastric epithelial cells.

[0162] from Figure 2 E It can be seen that gastric organoids cultured in complete medium are mainly composed of proliferating cells and stem cells. Compared with complete medium, the expression of surface mucus cell marker genes MUC5AC, GKN1, GKN2, FOXQ1 and TFF1 increased in gastric organoids cultured in differentiation medium-1. Similarly, in gastric organoids in differentiation medium-2, the expression of parietal cell marker genes ATP4A, ESRRG and CKB increased, accompanied by the increase in the expression of endocrine cell marker genes CHGB, GHRL, GAST and HDC.

[0163] Example 5. Immunofluorescence staining analysis of gastric organoids cultured in different culture media.

[0164] In this example, immunofluorescence staining was performed on organoids cultured under complete medium, differentiation culture-1 and differentiation culture-2 conditions, respectively.

[0165] Simply put, the organoids are resuspended by blowing with 1ml of pre-cooled PBS, centrifuged at 1000rpm for 1 minute, the supernatant is discarded, the organoid pellet is taken, and 1ml of 4% paraformaldehyde is added to fix it directly overnight. The next day, the tissue or organoid samples are transferred to water and incubated at room temperature for 30 minutes. Then, they are incubated in 70%, 80%, 90%, 100%, and 100% anhydrous ethanol respectively, and finally, after two incubations in xylene for 30 minutes each, the tissue or organoid samples are immersed in paraffin and incubated for at least 1 hour. The embedded tissue or organoid samples are paraffin sectioned to obtain paraffin sections containing organoids.

[0166] The procedure for immunofluorescence staining of paraffin sections is as follows:

[0167] 1) Soak the paraffin section sample in xylene solution twice, 10 minutes each time, to fully dewax;

[0168] 2) Next, the paraffin sections were placed in a gradient of alcohol for dehydration, immersing in 100%, 100%, 90%, 80%, and 70% anhydrous ethanol for 5 minutes each;

[0169] 3) Rinse the paraffin sections twice in fresh PBS for 3 minutes each;

[0170] 4) Prepare sodium citrate antigen repair solution (pH=6.0), fully immerse the paraffin sections in the antigen repair solution, and heat them in a microwave oven at medium-high heat for 15 minutes; after heating, let them stand at room temperature for 2 hours and cool to room temperature;

[0171] 5) Transfer the paraffin sections to PBS and wash twice, 3 minutes each time;

[0172] 6) Use an immunohistochemical pen to circle the target area, add 0.1% Triton X-100, and permeabilize at 4 degrees for 15 minutes;

[0173] 7) Prepare 3% BSA + 0.01% Triton X-100 blocking solution, add to the target area, and block at room temperature for 1 hour;

[0174] 8) Dissolve the corresponding primary antibody in the above blocking solution according to a certain ratio, add it dropwise to the target area, and incubate at 4 degrees overnight;

[0175] 9) On the next day, recover the primary antibody solution and place the paraffin sections in new PBS and wash three times, 10 minutes each time;

[0176] 10) Dissolve the secondary antibody and DAPI in a blocking solution according to a certain ratio, add drops to the target area, and incubate at room temperature for 1-2 hours away from light; 11) Transfer the paraffin sections to new PBS and wash 3 times, 10 minutes each time;

[0177] 12) Add fluorescence anti-quenching agent dropwise onto the paraffin sections, cover with coverslips, apply nail polish around the sections, and leave at room temperature for half an hour to complete the sealing;

[0178] 13) Place the sealed paraffin sections on a fluorescence confocal microscope Olympus (FV3000) to collect and photograph the fluorescence signal.

[0179] Anti-UEAI antibody (Thermofisher) was used to mark surface mucous cells; anti-E-CAD antibody (BD) was used to mark epithelial cells; anti-EZRIN protein antibody (Santa) was used to mark parietal cells; anti-MKI67 antibody was used to mark proliferating cells; DAPI (Thermofisher) was used as a nuclear dye.

[0180] The results are as follows Figure 3-4 As shown. Figure 3 As can be seen in B-3C, compared with organoids cultured in complete medium, the proportion of surface mucus cells marked by UEAI in organoids cultured in differentiation medium-1 increased significantly (greater than 80%), while the proportion of proliferating cells marked by MKI67 decreased significantly. Figure 4 As can be seen from C-4E, compared with organoids cultured in complete medium, the proportion of parietal cells labeled by HK-Atpase and EZRIN in organoids cultured in differentiation medium-2 was significantly increased, while the proportion of proliferating cells labeled by MKI67 was significantly decreased.

[0181] Example 6. Single-cell RNA sequencing (scRNA-seq) analysis of gastric organoids cultured in different culture media.

[0182] In this example, scRNA-seq was used to analyze the cell types and their proportions in organoids cultured under complete medium, differentiation medium-1, and differentiation medium-2.

[0183] First, samples were prepared for scRNA-seq. Organoids were digested using TryplE (Gibco). After digestion, cells were filtered through a 40 μm sieve to obtain a single-cell suspension. Propidium iodide (PI; 5 μg / mL) staining was then performed to identify and exclude dead cells. PI-negative cells were then sorted by fluorescence-activated cell sorting (FACS) using a Beckman instrument. The sorted single cells were loaded onto a single-cell chip using the 10X Genomics Chromium Single Cell 3' kit. A cDNA library was constructed and sequenced using an Illumina Novasek 6000 sequencer (Illumina).

[0184] Using Rstudio software and bioinformatics statistical analysis, cells were grouped based on the expression of marker genes of each cell type, and the cell proportions of different cell groups were counted. The results are shown in Figure 5 A. From Figure 5 A It can be seen that under complete culture conditions, gastric organoids mainly contain neck mucous cells and proliferative cells; under the conditions of differentiation medium-1, gastric organoids are highly enriched in surface mucous cells (surface mucous cells-1 and surface mucous cells-2 can be understood as different subpopulations of surface mucous cells); under parietal cell differentiation culture conditions, gastric organoids are mainly composed of parietal cell precursor cells and parietal cells.

[0185] The Spearman correlation of specific cell types between the organoids rich in surface mucus cells obtained in Example 2 and the organoids rich in parietal cells obtained in Example 3 and tissues was further analyzed based on bioinformatics. The results are shown in Figure 5 B. From Figure 5 B It can be seen that by comparing with the single-cell transcriptome data of human gastric epithelial cells, the surface mucus cells differentiated in the surface mucus cell-rich organoids obtained in Example 2 and the surface mucus cells of tissue origin are highly correlated in the correlation analysis of characteristic genes (Rs=0.9); the parietal cells differentiated in the parietal cell-rich organoids obtained in Example 3 and the parietal cells of tissue origin are also highly correlated in the correlation analysis of characteristic genes (Rs=0.72).

[0186] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A culture medium for promoting cell differentiation, comprising 10 to 30 ng / ml of BMP4.

2. The culture medium according to claim 1, characterized in that The culture medium is used to promote the differentiation of surface mucous cells. Preferably, the culture medium further comprises a member of the epidermal growth factor (EGF) family, More preferably, the EGF family member is selected from one or more of EGF, transforming growth factor α, amphiregulin, β-acetylglobulin, heparin-binding epidermal growth factor-like growth factor, epiregulin, epigenetic protein, neuregulin, and neuregulin-2s; Further preferably, in the culture medium, the concentration of the epidermal growth factor EGF family member is 20-100 ng / ml, preferably 30-80 ng / ml, most preferably 50 ng / ml.

3. The culture medium according to claim 1, characterized in that The culture medium is used to promote the differentiation of parietal cells. Preferably, the culture medium further comprises a neural stem cell differentiation inducer, more preferably, the neural stem cell differentiation inducer is Isx-9, and further preferably, the culture medium comprises 10 to 30 μM Isx-9; Preferably, the culture medium further comprises metformin, more preferably 50 to 150 μM, preferably 80 to 120 μM, most preferably 100 μM metformin; Preferably, the culture medium further comprises an ERR agonist, more preferably DY131 and / or SLU-PP-332; Preferably, the culture medium further comprises a MEK pathway inhibitor, more preferably comprising one or more of PD0325901, CI-1040, Trametinib, Cobimetinib, Binimetinib, Selumetinib, Refametinib, Pimasertib, RO4987655, and AZD8330; more preferably, the MEK pathway inhibitor is PD0325901, Preferably, the culture medium further comprises 10-30 μM Isx-9, 50-150 μM metformin, 1-10 μM DY131 and 0.5-5 μM PD0325901.

4. The culture medium according to claim 1, characterized in that The culture medium further comprises: a serum-free additive, preferably N2 and / or B27; more preferably 1×N2 and / or 1×B27; Glutamine or its analogs, preferably L-glutamine and / or GlutaMAX; more preferably 0.5-5 mM GlutaMAX; An antioxidant, preferably N-acetylcysteine, more preferably 0.1 to 2 mM N-acetylcysteine; The basic culture medium is preferably MEM, DMEM, IMDM, HAM F12, PRMI1640 and DMEM / F12 culture medium.

5. A method for inducing gastric organoids, characterized in that: The method comprises the following steps: 1) Obtain gastric glands; 2) culturing using a first culture medium so that the obtained gastric glands grow into first gastric organoids; and 3) replacing the first culture medium with the culture medium for promoting cell differentiation according to any one of claims 1 to 4 for culturing.

6. The method according to claim 5, characterized in that The first gastric organoid comprises more than 70% proliferating cells and neck mucous cells, Preferably, the first gastric organoid comprises more than 60% of neck mucous cells, Preferably, the first gastric organoid comprises 10% to 30% proliferative cells.

7. The method according to claim 5, characterized in that The first culture medium comprises one or more of the following substances: Serum-free supplements, preferably N2 and B27; Glutamine or its analogs, preferably L-glutamine and / or GlutaMAX; more preferably GlutaMAX; more preferably, the concentration of GlutaMAX is 0.5 to 5 mM, most preferably 2 mM, An antioxidant, preferably N-acetylcysteine; more preferably, the concentration of N-acetylcysteine ​​is 0.1 to 2 mM, most preferably 1 mM, Cell growth factor, preferably an epidermal growth factor EGF family member and / or a fibroblast growth factor FGF; more preferably, the EGF family member is selected from EGF, transforming growth factor α (TGFα), amphiregulin (AREG), β-acetylglobulin (BTC), heparin-binding epidermal growth factor-like growth factor (HB-EGF), epiregulin (EPR), epigenetic protein (EPG), neuregulin (NRG), neuregulin-2s (NRG2s), preferably EGF; the concentration of the epidermal growth factor EGF family member is 20-70 ng / mL, preferably 30-60 ng / mL, and most preferably 50 ng / ml; more preferably, FGF is FGF10, and the concentration is more preferably 60-150 ng / ml, preferably 80-120 ng / mL, and most preferably 100 ng / ml, The BMP signaling pathway inhibitor is preferably selected from one or more of Noggin, LDN-193189, Dorsomorphin and DMH1, more preferably Noggin; more preferably, the concentration of Noggin is 60 to 150 ng / ml, preferably 80 to 120 ng / mL, and most preferably 100 ng / mL Wnt signaling pathway agonist, preferably R-spondin 1, more preferably at a concentration of 300 to 700 ng / mL, preferably 400 to 600 ng / mL, most preferably 500 ng / mL; GSK-3β inhibitor, preferably one or more selected from CHIR-99021, BIO, IM-12, TWS119, 1-Azakenpaullone, CHIR98014, Tideglusib, AR-A014418, LY2090314, SB216763, AZD1080, more preferably CHIR-99021, the concentration is more preferably 1-10 μM, preferably 3-8 μM, most preferably 5 μM; TGF-β receptor ALK-5 inhibitor, preferably A-83-01, more preferably at a concentration of 1 to 10 nM, preferably 3 to 8 nM, most preferably 5 nM; ROCK1 inhibitor, preferably one or more selected from Y27632, Y-32885, Y-39983, fasudil, more preferably Y27632, more preferably at a concentration of 5 to 15 μM, preferably 8 to 12 μM, most preferably 10 μM; The hormone is preferably one or more of gastrin, ghrelin, leptin, somatostatin, secretin, gastric inhibitory peptide, cholecystokinin, motilin, peptide YY, glucagon-like peptide-1, glucagon-like peptide-2, and oxyntomodulin, more preferably gastrin, and the concentration is more preferably 5-15nM, preferably 8-12nM, and most preferably 10nM.

8. The method according to claim 5, characterized in that In step 2), the gastric glands are mixed with the matrix gel and then cultured with the first culture medium. Preferably, the matrix glue is Matrigel matrix glue.

9. The method according to claim 5, characterized in that The gastric glands are derived from mammals, Preferably, the gastric glands are derived from humans, non-human primates, mice, rats, sheep, cows, cats, dogs, pigs or rabbits.

10. The method according to claim 5, characterized in that In step 3), the medium for promoting cell differentiation is a second medium, and the second medium allows the first gastric organoid to differentiate into a second gastric organoid rich in surface mucous cells, wherein the second medium comprises 10 to 30 ng / ml of bone morphogenetic protein 4 (BMP4), more preferably 15 to 25 ng / ml, and most preferably 20 ng / ml of BMP4; Preferably, the second culture medium further comprises an epidermal growth factor EGF family member; more preferably, the EGF family member is selected from EGF, transforming growth factor α (TGFα), amphiregulin (AREG), β-acetylglobulin (BTC), heparin-binding epidermal growth factor-like growth factor (HB-EGF), epiregulin (EPR), epigenetic protein (EPG), neuregulin (NRG), neuregulin-2s (NRG2s), preferably EGF; More preferably, the concentration of the epidermal growth factor EGF family member is 20-100 ng / ml, preferably 30-80 ng / ml, and most preferably 50 ng / ml.

11. The method according to claim 5, characterized in that In step 3), the culture medium for promoting cell differentiation is a third culture medium, and the third culture medium allows the first gastric organoid to differentiate into a third gastric organoid rich in parietal cells, wherein the third culture medium comprises 10 to 30 ng / ml of BMP4 and a neural stem cell differentiation inducer; Preferably, the third culture medium comprises 15-25 ng / ml BMP4, most preferably 20 ng / ml BMP4; Preferably, the third culture medium comprises metformin, more preferably comprises 50 to 150 μM, preferably 80 to 120 μM, most preferably 100 μM metformin; Preferably, the neural stem cell differentiation inducer comprises Isx-9, more preferably comprises 10 to 30 μM, preferably 15 to 25 μM, most preferably 20 μM Isx-9; Preferably, the third culture medium further comprises an ERR agonist, more preferably DY131 and / or SLU-PP-332; Preferably, the third culture medium further comprises a MEK pathway inhibitor, more preferably one or more of PD0325901, CI-1040, Trametinib, Cobimetinib, Binimetinib, Selumetinib, Refametinib, Pimasertib, RO4987655, and AZD8330; more preferably, the MEK pathway inhibitor is PD0325901.

12. The method according to any one of claims 5 to 11, characterized in that The basic culture medium is selected from MEM, DMEM, IMDM, HAM F12, PRMI1640 and DMEM / F12 culture medium.

13. A gastric organoid, a gastric organoid enriched in surface mucous cells, or a gastric organoid enriched in parietal cells obtained using the culture medium of any one of claims 1 to 4 or the method of any one of claims 5 to 12, Preferably, the gastric organoids include more than 70% proliferating cells and neck mucous mucous cells; Preferably, the gastric organoids rich in surface mucus cells comprise more than 90% of surface mucus cells. Preferably, the parietal cell-enriched gastric organoid comprises more than 90% of parietal cell precursor cells. 14 . A pharmaceutical preparation comprising the gastric organoid, the gastric organoid enriched in surface mucous cells, or the gastric organoid enriched in parietal cells of claim 13 .

15. Use of the gastric organoid, the gastric organoid rich in surface mucous cells or the gastric organoid rich in parietal cells according to claim 13 in the preparation of a medicament for preventing or treating a gastric-related disease, Preferably, the stomach-related diseases include gastric cancer, peptic ulcer disease, Ménétrier's disease or gastric injury. Preferably, the stomach-related disease is caused by Helicobacter pylori infection.

16. Use of the gastric organoid, the gastric organoid rich in surface mucous cells or the gastric organoid rich in parietal cells according to claim 13 in preparing a gastric organoid model for the study of gastric-related diseases, Preferably, the stomach-related diseases include gastric cancer, peptic ulcer disease, Ménétrier's disease or gastric injury. Preferably, the stomach-related disease is caused by Helicobacter pylori infection.

17. Use of the gastric organoid, the gastric organoid rich in surface mucous cells or the gastric organoid rich in parietal cells according to claim 13 in screening drugs for preventing or treating gastric-related diseases, Preferably, the stomach-related diseases include gastric cancer, peptic ulcer disease, Ménétrier's disease or gastric injury. Preferably, the stomach-related disease is caused by Helicobacter pylori infection.

18. A method for screening a drug for preventing or treating a stomach-related disease, the method comprising: contacting the gastric organoid, the gastric organoid rich in surface mucous cells, or the gastric organoid rich in parietal cells of claim 13 with a candidate drug, Preferably, the stomach-related diseases include gastric cancer, peptic ulcer disease, Ménétrier's disease or gastric injury. Preferably, the stomach-related disease is caused by Helicobacter pylori infection.

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