Stomach body three-dimensional organoid culture medium as well as preparation method and culture method thereof

Abstract

The invention discloses a stomach body three-dimensional organoid culture medium as well as a preparation method and a culture method thereof, and belongs to the technical field of organoid culture. The stomach body three-dimensional organoid culture medium comprises a basic culture medium and a stomach body organoid growth supplement, the stomach body organoid growth supplement comprises a Wnt signaling pathway agonist, an FGF (Fibroblast Growth Factor) family member, a TGF-beta signaling pathway inhibitor, hyaluronic acid and retinoic acid. The invention further discloses a preparation method of the gastric body three-dimensional organoid culture medium and a gastric body three-dimensional organoid culture method. The method can solve the problems that the existing organoid culture technology is difficult to effectively maintain and amplify the stomach organoid with the functional oxyntocytes, and the existing organoid culture technology has unstable pedigree and incomplete structure.

Description

Technical Field

[0001] This invention belongs to the field of organoid culture technology, specifically relating to a three-dimensional organoid culture medium for the stomach body and its preparation and culture methods. Background Technology

[0002] Organoid technology, as a rapidly developing three-dimensional in vitro culture system in recent years, can simulate organ development, homeostasis maintenance and disease occurrence in matrix gel, and has been widely used in developmental biology, disease mechanism analysis and drug screening.

[0003] The gastric body, as the main functional area of ​​the stomach, is responsible for the secretion of gastric acid, pepsin, and mucus by its epithelial cells, playing a central role in digestion. Dysfunction of the gastric body is closely related to diseases such as gastritis, peptic ulcers, and even gastric cancer. Therefore, constructing stable and reliable gastric body organoid models is of great significance for a deeper understanding of the regulatory network of gastric epithelial stem cells, exploring the pathogenesis of gastric diseases, and evaluating the effects of drugs and nutrients. Currently, the culture systems for mouse and human gastric body organoids are relatively mature, enabling long-term expansion of gastric body stem cells and differentiation into functional cells such as parietal cells, chief cells, and endocrine cells, thus promoting a paradigm shift in gastric physiology and pathology research. However, while mouse and human models have provided a wealth of knowledge for gastric biology, research on gastric body organoid culture systems for other species, especially animals with important agricultural and economic value and biomedical model status, such as pigs, dogs, cattle, sheep, and even non-human primates, lags significantly behind. Existing gastric body research in these animals still mainly relies on two traditional methods: primary cell culture and in vivo experiments. Primary gastric epithelial cells rapidly lose polarity, cell connectivity, and expression of specific biomarkers under two-dimensional culture conditions, making it difficult to reproduce their in vivo differentiation state. Furthermore, their passage capacity is extremely weak, failing to meet the needs of long-term experiments and mechanistic exploration. While in vivo experiments can reflect the overall physiological environment, they are costly, time-consuming, and have low throughput. They are also limited by ethical and individual variability constraints, hindering high-throughput screening and precise molecular mechanism analysis. More critically, the microenvironment of gastric epithelial stem cells differs significantly among different species; for example, Wnt signaling dependence, growth factor requirements, and extracellular matrix composition all exhibit species specificity. Directly applying established human or mouse culture systems to large animals like pigs often results in low organoid formation efficiency, growth arrest, or even rapid apoptosis, failing to achieve long-term stable expansion and functional maintenance. This situation severely restricts progress in cross-species comparative biology research, animal disease model construction, and the elucidation of zoonotic disease mechanisms.

[0004] Therefore, there is an urgent need to develop a three-dimensional gastric organoid culture medium with clearly defined components, standardization, and adaptability to species-specific needs, and to establish corresponding preparation and culture methods. This would fill the gap in gastric organoid research in pigs and other economic animals and biomedical models, and provide an efficient and reliable technical platform for animal nutrition regulation, drug target validation, regenerative medicine, and precision animal husbandry. Summary of the Invention

[0005] The problem to be solved by the present invention is to provide a three-dimensional gastric organoid culture medium and its preparation and culture methods, so as to solve the problems that existing organoid culture technologies are difficult to effectively maintain and expand gastric organoids with functional acid-secreting cells, and have unstable lineages and incomplete structures.

[0006] The technical solution adopted to solve its technical problems is a three-dimensional organoid culture medium for the stomach body, including a basic culture medium and a stomach body organoid growth supplement; Gastric organoid growth supplements include Wnt signaling pathway agonists, FGF growth factor family members, TGF-β signaling pathway inhibitors, hyaluronic acid, and retinoic acid.

[0007] The beneficial effects of the above-mentioned technical solution in this invention are as follows: By simultaneously introducing a Wnt signaling pathway agonist and a TGF-β signaling pathway inhibitor into the culture medium, this invention effectively simulates the balance between proliferation and differentiation inhibition in the gastric body stem cell niche. The Wnt agonist activates the β-catenin signaling pathway, which is essential for stem cell maintenance, while the TGF-β inhibitor blocks the premature initiation of mesenchymal transition and differentiation. The synergistic effect of these two agents significantly improves the survival rate, colony formation efficiency, and long-term passage capacity of gastric body epithelial stem cells in non-model animals such as pigs, dogs, and cattle, solving the problem of organoids failing to form or rapidly undergoing apoptosis when directly applying human or mouse culture systems. Simultaneously, hyaluronic acid, as an important glycosaminoglycan component of the extracellular matrix, not only provides a hydration microenvironment mimicking the in vivo matrix for gastric epithelial stem cells, promoting cell adhesion and migration, but also regulates stem cell self-renewal through the CD44 receptor, complementing the structural support provided by the matrix gel. This significantly improves the three-dimensional morphological integrity of organoids, reduces central necrosis during culture, and increases the recovery rate of organoids after passage. FGF growth factor family members can specifically promote the proliferation of gastric epithelial progenitor cells and the directed differentiation of parietal cell lineages; while retinoic acid can maintain the regional characteristics of gastric glands and inhibit glandular metaplasia into the gastric antrum or intestine. It can not only expand functional parietal cells, chief cells and endocrine cells, but also reproduce the unique glandular structure and polar arrangement of the gastric body in vitro, making the organoids more functionally similar to the gastric body tissue in vivo.

[0008] Preferably, the basal culture medium is L Wnt-3A cell-specific culture medium.

[0009] Preferably, the Wnt signaling pathway agonist is CHIR99021, with a working concentration of 10-20 μM.

[0010] More preferably, the Wnt signaling pathway agonist is CHIR99021, with a working concentration of 15 μM.

[0011] Preferably, the FGF growth factor family member is FGF10, with a working concentration of 50~500 ng / mL.

[0012] More preferably, the FGF growth factor family member is FGF10, with a working concentration of 250 ng / mL.

[0013] Preferably, the TGF-β signaling pathway inhibitor is A83-01, with a working concentration of 0.5~5 μM.

[0014] More preferably, the TGF-β signaling pathway inhibitor is A83-01, with a working concentration of 2.5 μM.

[0015] Preferably, the working concentration of hyaluronic acid is 1~2 mg / mL; and the working concentration of retinoic acid is 0.1~1 μM.

[0016] More preferably, the working concentration of hyaluronic acid is 1 mg / mL; and the working concentration of retinoic acid is 0.5 μM.

[0017] This invention also provides a method for preparing the above-mentioned three-dimensional organoid culture medium for the gastric body, comprising the following steps: A gastric organoid growth supplement was prepared by mixing Wnt signaling pathway agonists, FGF growth factor family members, TGF-β signaling pathway inhibitors, hyaluronic acid, and retinoic acid, and then added to the basal culture medium. The mixture was then vortexed and thoroughly mixed.

[0018] This invention also provides a method for culturing three-dimensional organoids of the gastric body, comprising the following steps: Gastric stem cells and matrix gel were seeded onto a cell culture plate and solidified to form three-dimensional droplets. Then, preheated gastric three-dimensional organoid culture medium was added for in vitro three-dimensional culture to form gastric three-dimensional organoids.

[0019] Preferably, the volume ratio of gastric stem cells to matrix gel is 1:(1~2); the curing temperature of the three-dimensional gel droplets is 35~38℃ and the curing time is 25~35min.

[0020] More preferably, the volume ratio of gastric stem cells to matrix gel is 1:1.2; the curing temperature of the three-dimensional gel droplets is 37°C and the curing time is 30 min.

[0021] Preferably, the in vitro three-dimensional culture is carried out at 35~38℃ and 4~6% CO2, and the gastric body three-dimensional organoid culture medium is replaced every 2~3 days during the in vitro three-dimensional culture process.

[0022] More preferably, the in vitro three-dimensional culture is carried out at 37°C and 5% CO2, and the gastric three-dimensional organoid culture medium is replaced every 2-3 days during the in vitro three-dimensional culture process.

[0023] Preferably, the three-dimensional organoid culture medium and the three-dimensional organoid culture method for the stomach body are applicable to mammals.

[0024] More preferably, the three-dimensional organoid culture medium and method for culturing three-dimensional organoids of the stomach body are applicable to pigs.

[0025] The present invention has the following beneficial effects: The gastric body three-dimensional organoid culture medium of the present invention can successfully construct and maintain gastric body three-dimensional organoids with typical three-dimensional cavity structure and clear polarity in vitro for a long time, such as porcine gastric body three-dimensional organoids. These organoids can stably express the gastric body-specific marker GIF and contain various gastric body epithelial cell types, such as SOX9-positive stem cells. This system provides a highly biomimetic, stable and reliable standardized in vitro platform for studying the physiological and pathological mechanisms of gastric acid secretion in economic animals and humans, establishing gastric injury models such as gastric ulcers, and evaluating the digestive properties of nutrients. It has broad application prospects in the fields of drug development and animal husbandry. Attached Figure Description

[0026] Figure 1 The image shows a GIF of a three-dimensional organoid of the porcine stomach body obtained in Example 1 and an immunofluorescence staining image of SOX9 protein. Figure 2 The images show the three-dimensional organoid characteristics of the stomach body obtained in Comparative Example 1 and Example 1; where (a) is a white light image of the stomach body organoids cultured for 5 days in Comparative Example 1 and Example 1; (b) is a statistical chart of the diameter of the formed organoids; and (c) is a statistical chart of the organoid formation efficiency. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.

[0028] Example 1 A three-dimensional organoid culture medium for gastric body, comprising L Wnt-3A cell-specific culture medium and gastric body organoid growth supplement; The gastric organoid growth supplement includes CHIR99021, a Wnt signaling pathway agonist at a concentration of 15 μM; FGF10, a member of the FGF growth factor family, at a concentration of 250 ng / mL; A83-01, a TGF-β signaling pathway inhibitor, at a concentration of 2.5 μM; hyaluronic acid at a concentration of 1 mg / mL; and retinoic acid at a concentration of 0.5 μM.

[0029] This embodiment also provides a method for preparing the above-mentioned three-dimensional organoid culture medium for the gastric body, including the following steps: A gastric organoid growth supplement was prepared by mixing the Wnt signaling pathway agonist CHIR99021, FGF growth factor family member FGF10, TGF-β signaling pathway inhibitor A83-01, hyaluronic acid, and retinoic acid. This supplement was then added to L Wnt-3A cell culture medium and vortexed to mix thoroughly.

[0030] This embodiment also provides a method for culturing three-dimensional organoids of the gastric body, including the following steps: Porcine gastric body stem cells and matrix gel were mixed on ice at a volume ratio of 1:1.2 and 25 μL was added dropwise to a preheated 48-well cell culture plate. The plate was then incubated at 37°C for 30 min to form three-dimensional gel droplets. 250 μL of gastric body three-dimensional organoid culture medium was added to each well to cover the droplets. The gastric body three-dimensional organoid culture medium was changed every 2–3 days. After 3–5 days of culture, obvious cystic organoid formation was observed. When the organoid density exceeded 80%, the plate was passaged. The droplets were washed with 4°C PBS buffer to digest the organoids into small cell clusters. These clusters were then re-embedded in fresh matrix gel following the same steps to form gastric body three-dimensional organoids.

[0031] Example 2 A three-dimensional organoid culture medium for gastric body, comprising L Wnt-3A cell-specific culture medium and gastric body organoid growth supplement; The gastric organoid growth supplement includes CHIR99021, a Wnt signaling pathway agonist at a concentration of 10 μM; FGF10, a member of the FGF growth factor family, at a concentration of 50 ng / mL; A83-01, a TGF-β signaling pathway inhibitor, at a concentration of 0.5 μM; hyaluronic acid at a concentration of 1 mg / mL; and retinoic acid at a concentration of 0.1 μM.

[0032] The preparation method of the gastric body three-dimensional organoid culture medium and the gastric body three-dimensional organoid culture method in this embodiment are the same as those in Example 1.

[0033] Example 3 A three-dimensional organoid culture medium for gastric body, comprising L Wnt-3A cell-specific culture medium and gastric body organoid growth supplement; The gastric organoid growth supplement includes CHIR99021, a Wnt signaling pathway agonist at a concentration of 20 μM; FGF10, a member of the FGF growth factor family, at a concentration of 500 ng / mL; A83-01, a TGF-β signaling pathway inhibitor, at a concentration of 5 μM; hyaluronic acid at a concentration of 2 mg / mL; and retinoic acid at a concentration of 1 μM.

[0034] The preparation method of the gastric body three-dimensional organoid culture medium and the gastric body three-dimensional organoid culture method in this embodiment are the same as those in Example 1.

[0035] Comparative Example 1 A three-dimensional organoid culture medium for gastric body, comprising L Wnt-3A cell-specific culture medium and gastric body organoid growth supplement; The gastric organoid growth supplement includes CHIR99021, a Wnt signaling pathway agonist at a concentration of 15.2 μM, FGF10, a member of the FGF growth factor family at a concentration of 250.5 ng / mL, and A83-01, a TGF-β signaling pathway inhibitor at a concentration of 2.8 μM.

[0036] The preparation method of the gastric body three-dimensional organoid culture medium and the gastric body three-dimensional organoid culture method in this comparative example are the same as those in Example 1.

[0037] Experimental Example 1: Phenotypic Identification of Three-Dimensional Organoids of the Porcine Stomach Body Three-dimensional organoids of the gastric body cultured to day 5 were subjected to immunofluorescence identification. The specific steps were as follows: 4% paraformaldehyde was added, and the organoids were fixed at room temperature for 10 min. After fixation, the organoids were washed three times with PBS for 5 min each time. 0.1%–0.3% Triton X-100 was added, and the organoids were incubated at room temperature for 10 min. The organoids were then washed three times with PBS for 5 min each time. Blocking buffer (5% BSA) was added, and the organoids were incubated at room temperature for 2 h. The blocking buffer was removed, and the organoids were incubated overnight at 4°C with primary antibody dilution buffer. The organoids were washed three times with PBS for 5 min each time. Secondary fluorescent antibody was added, and the organoids were incubated at room temperature in the dark for 2 h. The organoids were then washed three times with PBS for 5 min each time. Finally, DAPI was added to the gastric body organoids, and the organoids were incubated for 10 min. After washing three times with PBS, the organoids were mounted and observed and photographed under a fluorescence microscope. The results are as follows: Figure 1 As shown, the scale bars in the figure are all 50 μm.

[0038] from Figure 1 As can be seen from the data, the porcine gastric body three-dimensional organoids cultured in this invention contain gastric body stem cell marker SOX9 and gastric body parietal cell marker GIF, confirming their gastric body origin and differentiation potential.

[0039] Experiment Example 2: Verification of the Role of Key Components Equal amounts of the gastric body three-dimensional organoid culture medium from Example 1 and Comparative Example 1 were inoculated with stem cells isolated from gastric body tissue. After 5 days of culture, the number of organoids formed (spheres with a diameter >50 μm) was counted. The results are as follows: Figure 2 As shown, Figure 2 The scale bars in Figure (a) are all 100 μm.

[0040] from Figure 2 The results show that the obtained three-dimensional organoids of the pig stomach body are sac-like or glandular structures; there is no significant difference in the diameter of the three-dimensional organoids of the pig stomach body cultured in the three-dimensional organoid culture medium of Example 1 and Comparative Example 1; however, the formation efficiency of the three-dimensional organoids of the pig stomach body cultured in the three-dimensional organoid culture medium of Example 1 is higher than that of Comparative Example 1, which proves that hyaluronic acid and retinoic acid in the three-dimensional organoid culture medium of the stomach body can play a certain synergistic role in promoting the formation of three-dimensional organoids of the stomach body.

[0041] The present invention has been described according to the above embodiments. It should be understood that the above embodiments do not limit the present invention in any way. All technical solutions obtained by equivalent substitution or equivalent transformation fall within the scope of the present invention.

Claims

1. A three-dimensional organoid culture medium for the stomach body, characterized in that, Including basal culture medium and gastric organoid growth supplements; The gastric organoid growth supplement includes Wnt signaling pathway agonists, FGF growth factor family members, TGF-β signaling pathway inhibitors, hyaluronic acid, and retinoic acid.

2. The three-dimensional organoid culture medium for the stomach body as described in claim 1, characterized in that, The basal culture medium is L Wnt-3A cell-specific culture medium.

3. The three-dimensional organoid culture medium for the stomach body as described in claim 1, characterized in that, The Wnt signaling pathway agonist is CHIR99021, with a working concentration of 10–20 μM.

4. The three-dimensional organoid culture medium for the stomach body as described in claim 1, characterized in that, The FGF growth factor family member is FGF10, with a working concentration of 50~500 ng / mL.

5. The three-dimensional organoid culture medium for the stomach body as described in claim 1, characterized in that, The TGF-β signaling pathway inhibitor is A83-01, with a working concentration of 0.5~5 μM.

6. The three-dimensional organoid culture medium for the stomach body as described in claim 1, characterized in that, The working concentration of the hyaluronic acid is 1~2 mg / mL; the working concentration of the retinoic acid is 0.1~1 μM.

7. The method for preparing the three-dimensional organoid culture medium for the gastric body according to any one of claims 1 to 6, characterized in that, Includes the following steps: A gastric organoid growth supplement was prepared by mixing Wnt signaling pathway agonists, FGF growth factor family members, TGF-β signaling pathway inhibitors, hyaluronic acid, and retinoic acid, and then added to the basal culture medium. The mixture was then vortexed and thoroughly mixed.

8. A method for culturing three-dimensional organoids of the stomach body, characterized in that, Includes the following steps: Gastric stem cells and matrix gel are seeded onto a cell culture plate and solidified to form three-dimensional droplets. Then, preheated gastric three-dimensional organoid culture medium as described in any one of claims 1 to 6 is added for in vitro three-dimensional culture to form gastric three-dimensional organoids.

9. The method for culturing three-dimensional organoids of the stomach body as described in claim 8, characterized in that, The volume ratio of gastric stem cells to matrix gel is 1:(1~2); the curing temperature of the three-dimensional gel droplets is 35~38℃, and the curing time is 25~35 min.

10. The method for culturing three-dimensional organoids of the stomach body as described in claim 8, wherein the in vitro three-dimensional culture is carried out at 35~38℃ and 4~6% CO2, and the culture medium for the three-dimensional organoids of the stomach body is replaced every 2~3 days during the in vitro three-dimensional culture process.

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