CN-121991884-A - Stomach three-dimensional organoid culture medium and preparation method and culture method thereof

Abstract

The invention discloses a gastric three-dimensional organoid culture medium, a preparation method and a culture method thereof, belonging to the technical field of organoid culture, wherein the gastric three-dimensional organoid culture medium comprises a basal culture medium and a gastric organoid growth supplement, and the gastric organoid growth supplement comprises Wnt signal path agonists, FGF growth factor family members, TGF-beta signal path inhibitors, hyaluronic acid and retinoic acid. The invention also discloses a preparation method of the gastric three-dimensional organoid culture medium and a gastric three-dimensional organoid culture method. The invention can solve the problems that the existing organoid culture technology is difficult to effectively maintain and expand the gastric organoid with functional acid secreting cells, and the pedigree is unstable and the structure is incomplete.

Inventors

• ZHOU JIAYI
• LIU DINGMING
• WANG ZIYANG
• YAN HUICHAO

Assignees

• 华南农业大学

Dates

Publication Date

20260508

Application Date

20260226

Claims (10)

1. 1. A gastric three-dimensional organoid medium comprising a basal medium and a gastric organoid growth supplement; the gastric organoid growth supplements include Wnt signaling pathway agonists, FGF growth factor family members, TGF-beta signaling pathway inhibitors, hyaluronic acid, and retinoic acid.
2. 2. The gastric three-dimensional organoid medium of claim 1, wherein the basal medium is L Wnt-3A cell-specific medium.
3. 3. The gastric three-dimensional organoid medium of claim 1, wherein the Wnt signaling pathway agonist is CHIR99021 at a working concentration of 10-20 μm.
4. 4. The gastric three-dimensional organoid medium of claim 1, wherein the FGF growth factor family member is FGF10 at a working concentration of 50-500 ng/mL.
5. 5. The gastric three-dimensional organoid medium of claim 1, wherein the TGF- β signaling pathway inhibitor is a83-01 at a working concentration of 0.5-5 μm.
6. 6. The gastric three-dimensional organoid medium of claim 1, wherein the working concentration of hyaluronic acid is 1-2 mg/mL and the working concentration of retinoic acid is 0.1-1 μm.
7. 7. The method for preparing the gastric three-dimensional organoid medium according to any one of claims 1 to 6, comprising the steps of: Mixing Wnt signal pathway agonist, FGF growth factor family member, TGF-beta signal pathway inhibitor, hyaluronic acid and retinoic acid to form stomach organoid growth supplement, adding into basal medium, and mixing.
8. 8. A method for three-dimensional organoid culture of a gastric body, comprising the steps of: inoculating the gastric stem cells and matrigel on a cell culture plate, solidifying to form three-dimensional gel drops, and adding the preheated gastric three-dimensional organoid culture medium according to any one of claims 1-6 for in-vitro three-dimensional culture to form the gastric three-dimensional organoid.
9. 9. The method for culturing the three-dimensional organoid of the stomach body according to claim 8, wherein the volume ratio of the stem cells of the stomach body to the matrigel is 1 (1-2), the curing temperature of the three-dimensional gel drop is 35-38 ℃ and the time is 25-35 min.
10. 10. The method for culturing a three-dimensional organoid of a stomach according to claim 8, wherein the in vitro three-dimensional culture is performed under the conditions of 35-38 ℃ and 4-6% CO 2 , and the culture medium of the three-dimensional organoid of the stomach is replaced every 2-3 d in the in vitro three-dimensional culture process.

Description

Stomach three-dimensional organoid culture medium and preparation method and culture method thereof Technical Field The invention belongs to the technical field of organoid culture, and particularly relates to a gastric three-dimensional organoid culture medium, a preparation method and a culture method thereof. Background The organoid technology is used as a three-dimensional in-vitro culture system which is rapidly developed in recent years, can simulate the development, steady state maintenance and disease occurrence process of organs in matrigel, and is widely applied to the fields of development biology, disease mechanism analysis, drug screening and the like. The stomach body is used as the main functional area of the stomach, the epithelial cells are responsible for secreting gastric acid, pepsin and mucus, and plays a core role in the digestion process, and the dysfunction of the stomach body is closely related to diseases such as gastritis, peptic ulcer and gastric cancer. Therefore, the construction of a stable and reliable gastric organoid model is of great significance for the deep understanding of the regulation network of gastric epithelial stem cells, the exploration of the pathogenesis of gastric diseases and the evaluation of the effects of drugs and nutrients. At present, the culture systems of the mouse and human gastric organoids are relatively mature, long-term expansion of gastric stem cells and differentiation of functional cells such as parietal cells, main cells and endocrine cells can be realized, and the paradigm shift of gastric physiology and pathology research is promoted. However, while mouse and human models provide a rich body of knowledge for gastric biology, research into other species, particularly animals with important agro-economic value and biomedical model status, such as pigs, dogs, cattle, sheep, and even non-human primates, has been severely delayed. The existing gastric body studies on these animals still rely mainly on two major traditional approaches, primary cell culture and in vivo experiments. The primary gastric epithelial cells can rapidly lose polarity, cell connection and specific marker expression under two-dimensional culture conditions, the in-vivo differentiation state is difficult to reproduce, the passage capacity is extremely weak, and the requirements of long-term experiments and mechanism exploration cannot be met, while in-vivo experiments can reflect the whole physiological environment, but have high cost, long period, low experimental flux, are limited by ethics and individual differences, and are difficult to carry out high-flux screening and accurate molecular mechanism analysis. More importantly, the micro-environments of the gastric epithelial stem cells among different species have obvious differences, such as Wnt signal dependence, growth factor requirement and extracellular matrix composition all show species specificity, and the established human or mouse culture system is directly sleeved on large animals such as pigs, so that organoid formation efficiency is low, growth stagnation and even rapid apoptosis are often caused, and long-term stable amplification and function maintenance cannot be realized. The current situation severely restricts the progress of fields such as cross-species comparative biological research, animal disease model construction, zoonotic mechanism analysis and the like. Therefore, there is a need to develop a gastric three-dimensional organoid culture medium which has definite components, can be standardized and can meet the species-specific requirements, and establish corresponding preparation and culture methods, so as to fill the blank of pigs and other economic animals and biomedical models in the research field of gastric organoids, and provide an efficient and reliable technical platform for animal nutrition regulation, drug target verification, regenerative medicine and accurate livestock. Disclosure of Invention The invention aims to solve the problems that the existing organoid culture technology is difficult to effectively maintain and expand the gastric organoid with functional acid secretion cells, and the lineage is unstable and the structure is incomplete. The technical proposal adopted for solving the technical problems is that the three-dimensional gastric organoid culture medium comprises a basic culture medium and a gastric 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. The invention adopts the technical scheme that the invention effectively simulates the proliferation promoting and differentiation inhibiting balance of the gastric stem cell niche by simultaneously introducing the Wnt signal channel agonist and the TGF-beta signal channel inhibitor into the culture medium, and solves the problem that organoids cannot form or rapid