Culture system and culture method for intestinal organs of euryhaline fish

Abstract

The invention discloses a culture system and a culture method for intestinal organs of euryhaline fish, and belongs to the technical field of bioengineering. The culture system is composed of a basic culture medium and a complete culture medium, wherein the basic culture medium is Advanced DMEM / F12, and is supplemented with a GlutaMax, Hepes and mycillin double-antibody solution; and on the basis of the complete culture medium, N-2, B-27, N-acetylcysteine, EGF (Epidermal Growth Factor), Noggin, R-Spondin 1 and other key cell factor combinations are further added. By optimizing the tissue pretreatment and digestion process, the efficient fish intestinal tract organoid culture and passage method is established, the organoid model with the crypt-villus-like structure, the cell heterogeneity and the intestinal tract barrier function is successfully constructed, and the model provides a highly bionic in-vitro research tool for fish physiology and toxicology research.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a culture system and method for intestinal organoids of euryhaline fish. Background Technology

[0002] Currently, research on the intestinal physiology of euryhaline fish mainly relies on in vivo experiments, which presents several key problems: 1) High cost: The breeding and rearing of experimental fish, as well as the construction and maintenance of the rearing environment (especially the alternating seawater / freshwater acclimatization system), are expensive, consuming a large amount of human and material resources; 2) Long cycle: Fish grow slowly, and the experimental cycle usually takes several months (such as observing the effect of salinity adaptation on intestinal structure); 3) Difficulty in dynamic monitoring: In vivo experimental models are limited by the accessibility of imaging observation, the presence of confounding variables, and limited throughput, making it impossible to observe changes at the intestinal cell level in real time (such as ion transport and crypt proliferation), and only static data can be obtained through dissection; 4) Ethical controversy: In vivo experiments face animal welfare pressures, and the EU REACH regulation has gradually restricted fish toxicity testing.

[0003] Existing alternative models (such as 2D cell lines) also have many shortcomings. The structure of 2D cell lines is simplified. Traditional fish intestinal cell lines (such as ZF4 and RTG-2) can only grow in a single layer and cannot simulate the three-dimensional structure of crypts and villi, which leads to the distortion of research data on drug absorption and pathogen infection. In addition, 2D cultured cells have some functional deficiencies, such as loss of polarization characteristics (such as the apical membrane-basement membrane material transport function) and do not express key salinity regulating proteins (such as Na⁺ / K⁺-ATPase and CFTR).

[0004] Organoid technology, which involves the self-assembly of stem cells to form organ-specific three-dimensional structures, has been widely applied in disease modeling, drug screening, and other fields. While mammalian organoid technology (such as human / mouse intestinal organoids) is mature, direct transplantation into fish faces species barriers. Currently, the construction technology for intestinal organoids in euryhaline fish is almost nonexistent, mainly due to the lack of fish-specific culture systems. Fish and mammals exhibit significant differences in intestinal developmental regulatory pathways (such as Wnt / β-catenin signaling), and key mammalian growth factors (such as Wnt3a and R-spondin1) may have a weak effect on promoting the proliferation of fish intestinal stem cells. Furthermore, there are currently no standardized procedures for culturing fish intestinal organoids (such as digestive enzyme types and passage methods), making it difficult to compare data from different laboratories. In recent years, fish organoid research has largely focused on organs such as the liver and retina, while the construction of intestinal organoids still lacks systematic exploration, necessitating the optimization of culture systems specifically for the characteristics of euryhaline fish.

[0005] The golden shad (Scatophagus argus) is an economically farmed species widely distributed along the southeastern coast of my country, valued for both its ornamental and edible qualities. In recent years, intensive farming of golden shad has led to frequent outbreaks of bacterial enteritis and parasitic infections, yet targeted prevention and control measures remain lacking. The fish gut is a core organ for nutrient absorption, immune regulation, and host-microbe interactions, directly impacting fish growth performance and health. Developing fish intestinal organoid models can accurately simulate intestinal barrier function, immune responses, and pathogen interactions, providing a platform for disease mechanism analysis and drug target screening. Furthermore, organoid technology offers high-throughput and high-biomimicry advantages in the toxicity assessment of environmental pollutants (such as microplastics and heavy metals), potentially promoting the sustainable development of aquaculture. However, current methods for constructing intestinal organoids in euryhaline fish, such as the golden shad, are still lacking, with a deficiency in species-specific culture medium design and functional validation systems, severely limiting their potential application in microbe-host interaction mechanism research, toxicity assessment, and drug screening. Summary of the Invention

[0006] To address the aforementioned problems in the prior art, the main objective of this invention is to provide a culture system for intestinal organoids of euryhaline fish. By designing a culture system adapted to the characteristics of fish intestinal stem cells, this invention solves the problems of low intestinal crypt survival rate and difficulty in forming three-dimensional organoid structures in the prior art.

[0007] Another objective of this invention is to provide a method for culturing and passaged intestinal organoids of euryhaline fish, optimizing tissue processing and digestion conditions, and constructing organoid models with crypt-villi-like structures, cellular heterogeneity, and intestinal barrier function, thus providing an in vitro model for fish physiological and toxicological studies.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a culture system for intestinal organoids of euryhaline fish, comprising a basal culture medium and a complete culture medium; wherein the basal culture medium comprises Advanced DMEM / F12 medium, GlutaMax additive, Hepes buffer, and penicillin-streptomycin solution; and the complete culture medium is prepared by further supplementing the basal culture medium with N-2 additive, B-27 additive, N-acetylcysteine, EGF, Noggin, R-Spondin 1, Gastrino, A83-01, Y-27632, and CHIR99021 factor.

[0010] Preferably, the composition of the basal culture medium is shown in the table below:

[0011] .

[0012] More preferably, the concentrations of penicillin and streptomycin in the penicillin-streptomycin dual antibody solution are 400 U / mL and 400 μg / mL, respectively.

[0013] Preferably, the composition of the complete culture medium is shown in the table below:

[0014] .

[0015] Secondly, a method for culturing intestinal organoids of euryhaline fish is provided, comprising the following steps:

[0016] S1. Blood was drawn from 3-month-old golden coin fish, the entire intestine was removed, the mesentery and fat were quickly removed, and the fish was placed in a pre-cooled DPBS solution containing 1% penicillin and streptomycin at 4°C.

[0017] S2. Remove the intestinal segment 1 cm from the stomach and the hindgut, longitudinally cut open the anterior and middle guts, and rinse 3 times with pre-cooled DPBS solution in a sterile culture dish.

[0018] S3. Cut the intestinal tissue transversely into 4 cm segments, transfer to 15 mL centrifuge tubes, add 5 mL of pre-cooled DPBS, gently pipette 15 times, let stand for 30 s and discard the supernatant, repeat 5-6 times until the supernatant is clear.

[0019] S4. Add 8 mL of 5 mM EDTA digestion solution to the sample, then transfer it to an ice bath shaker and shake continuously at 200 rpm for 15 min.

[0020] S5. Let stand for 30 seconds, aspirate the supernatant, add 5 mL of pre-cooled DPBS, gently aspirate and discard the supernatant, repeat twice to completely remove residual digestive fluid.

[0021] S6. Spread the digested intestinal tissue onto a sterile culture dish, fix one end with a coverslip, scrape the intestinal villi and crypts with another coverslip, rinse with DPBS and collect into a new centrifuge tube.

[0022] S7. Hold the centrifuge tube and shake it vigorously up and down 2-3 times;

[0023] S8. The above suspension is filtered through a 70-mesh filter and the filtrate is collected.

[0024] S9. Centrifuge the filtrate at 70 ×g for 5 min at 4℃.

[0025] S10. Discard the supernatant and resuspend the precipitate in Advanced DMEM / F12 medium, adjusting the crypt density to 10 crypts / μL.

[0026] S11. Mix the crypt suspension with the matrix gel at a volume ratio of 1:3, and the matrix gel concentration shall not be less than 70%.

[0027] S12. Using a pre-cooled pipette tip, draw 50 μL of the matrix gel resuspension and drop it into the center of a preheated 24-well plate. Place the plate in an incubator containing 5% CO2 and cure at 28°C for 1 h.

[0028] S13. After the colloid stabilizes, add 600 μL of complete culture medium to each well and continue culturing at 28℃ until budding structures appear, thus obtaining the target organoid.

[0029] It should be noted that in step S4, the tissue digestion time should not exceed 20 minutes. Digestion can be stopped when the tissue edges gradually loosen and the number of single cells in the digestive fluid increases during microscopic examination. Excessive digestion time will affect cell viability, reducing crypt clumping and budding success rates. In step S8, a 70-mesh filter is used, which can trap most of the nearly 300 μm intestinal villi in fish and reduce damage to crypts. In step S9, the centrifugation rate of the crypt suspension is 70 × g; the rate should not be too high, as the resulting centrifugal shear force will damage the crypts. In step S13, the culture time at 28℃ is 5-6 days, until the edges of the euryhaline fish intestinal organoids are clearly diffused and budding stops, at which point subsequent passage operations can be performed.

[0030] Thirdly, a method for passage of intestinal organoids in euryhaline fish is provided, including:

[0031] A1. Remove the culture medium from the well plate, add 500 μL of cell dissociation solution to each well, and gently blow to loosen the colloid and detach the organoid from the matrix gel.

[0032] A2. Digest at room temperature for 5 min, transfer the liquid to a 15 mL centrifuge tube, add an equal volume of Ad DMEM / F12 medium, and centrifuge at 4℃ and 70 ×g for 3 min.

[0033] A3. Discard the supernatant, resuspend the precipitate in Advanced DMEM / F12 medium, mix it with matrix gel at a volume ratio of 1:1, and control the passage density at 1:2 to 1:3.

[0034] Add 50 μL of matrix gel resuspension to each well of A4 and 24-well plates, place them in an incubator containing 5% CO2, and cure at 28°C for 1 hour;

[0035] A5. Add 600 μL of complete culture medium to each well and incubate in a 28℃ cell culture incubator for long-term culture.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] I. The culture system for euryhaline fish intestinal organoids in this invention, by adjusting the proportions of its components, promotes organoid growth, budding, and provides nutrition. The cultured euryhaline fish intestinal organoids exhibit biological structures similar to intestinal tissue. First, the main component of the basal culture medium is Advanced DMEM / F12 medium, which is superior to high-glucose DMEM medium, avoiding high-glucose-induced metabolic stress and ROS accumulation, making it more suitable for fish intestinal metabolism. It is also superior to DMEM / F12 medium, containing essential trace elements for stem cells, supporting the self-renewal and antioxidant properties of intestinal crypt stem cells, and significantly improving the survival rate and stability of the euryhaline fish intestinal organoids. Second, the addition of penicillin and streptomycin dual antibiotic solution to the basal culture medium, with penicillin and streptomycin concentrations of 400 U / mL and 400 μg / mL respectively, effectively inhibits microbial contamination in the culture system, improving the success rate of in vitro construction of fish intestinal organoids and the culture stability. Finally, CHIR99021 was added to the complete culture medium of the present invention to replace Wnt3a. As a small molecule compound, CHIR99021 has significantly better chemical stability and duration of action than Wnt3a. CHIR99021 bypasses the membrane receptor-mediated process dependent on Wnt3a and directly targets intracellular GSK3, thereby providing a more stable and precise Wnt / β-catenin signal in long-term organoid culture.

[0038] II. This invention addresses the problems of existing fish intestinal research models, such as model deficiency, poor adaptability, and a lack of organoid technology, by providing a method for culturing euryhaline fish intestinal organoids. Through extensive experiments and successful formulation of the components (Advanced DMEM / F12 basal medium, GlutaMax, Hepes buffer, penicillin-streptomycin antibiotic solution, and supplementary factors), this mixed system successfully maintains the stemness of crypt cells, providing sufficient and comprehensive nutrition for organoid growth and budding, resulting in organoids with biological structures similar to intestinal tissue. Furthermore, the organoid passage method is highly feasible, maintaining proliferative capacity even after 15 consecutive passages, making it suitable for long-term experimental research.

[0039] Third, the in vivo experiments using euryhaline fish intestinal organoids as a substitute for live in vitro experiments of this invention comply with the EU's 3R principles (replace, reduce, optimize), significantly reducing R&D costs. The cost of a single organoid experiment is approximately 1 / 5 of that of live in vitro experiments. It also shortens the R&D cycle; fish intestinal organoid culture only requires 1-2 weeks for experimental use, which is more than 6 times faster than live culture (3-6 months). Furthermore, it supplements the fish intestinal organoid database, promoting precision aquaculture and rapidly advancing research on feed optimization and disease control for euryhaline fish. Attached Figure Description

[0040] Figure 1This is a flowchart illustrating the construction process of intestinal organoids from euryhaline fish in this embodiment.

[0041] Figure 2 The images show the morphology of fish intestinal organoids at different culture times in the examples, corresponding to culture times of 0 h, 20 h, 40 h, and 70 h. The image scale bar is 20 μm.

[0042] Figure 3 The image shows a comparison of different centrifugation speeds for the crypts in the example, with corresponding centrifugation speeds of 200 ×g and 70 ×g. The image scale bar is 10 μm.

[0043] Figure 4 The images show the morphology of fish intestinal organoids cultured in different basal culture media in the examples. The corresponding culture media are high-glucose DMEM medium and Advanced DMEM / F12 medium, respectively. The image scale bar is 20 μm.

[0044] Figure 5 The images show the morphology of fish intestinal organoids cultured with different growth factors in the examples. The corresponding growth factors are Wnt3a and CHIR99021. The image scale bar is 20 μm.

[0045] Figure 6 The images show the morphology of fish intestinal organoids cultured with different concentrations of penicillin and streptomycin in the examples. The corresponding concentrations of the penicillin and streptomycin were 100 U / mL penicillin + 100 μg / mL streptomycin (1×) and 400 U / mL penicillin + 400 μg / mL streptomycin (4×). The image scale bar is 20 μm.

[0046] Figure 7 The images show the morphology of fish intestinal organoids at different culture times in the examples, corresponding to culture times of 20 h, 40 h, 140 h, and 160 h. The image scale bar is 20 μm. Detailed Implementation

[0047] To more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that other embodiments obtained by those skilled in the art without departing from the concept of the present invention are all within the protection scope of the present invention.

[0048] Example 1

[0049] This embodiment proposes a culture system for euryhaline fish intestinal organoids, comprising the basal culture medium AdvancedDMEM / F12, GlutaMax additive, Hepes buffer, penicillin-streptomycin solution, and supplementary factors; the supplementary factors are N-2 additive, B-27 additive, N-acetylcysteine, EGF, Noggin, R-Spondin 1, Gastrino, A83-01, Y-27632, and CHIR99021. Through systematic optimization of the concentration and ratio of each component, the developed fish intestinal organoid culture medium possesses the following characteristics: it effectively promotes organoid proliferation and budding morphology, continuously provides key nutritional support, and guides organoid formation to achieve a biomimetic structure that closely resembles the in vivo natural intestinal tissue in terms of both tissue structure and cellular composition.

[0050] The culture system for the intestinal organoids of the above-mentioned euryhaline fish consisted of two types: basal medium and complete medium. The components, concentrations, and amounts of these media are shown in Tables 1 and 2. Advanced DMEM / F12 medium was purchased from Gibco, GlutaMax™ additive was purchased from Therno, Hepes buffer was purchased from Macklin, N-2 additive (100×) was purchased from Biosensors, B-27 additive was purchased from Absin, A83-01 was purchased from InviviChem, Y-27632 was purchased from GPL, CHIR99021 was purchased from Sigma-Aldrich, N-acetylcysteine ​​was purchased from Wako, and Recombinant Mouse EGF (C-6His), Recombinant Mouse Noggin (C-6His), Recombinant Human RSPO1 (C-6His), and Recombinant Human ProGRP were all purchased from novoprotein (hereinafter referred to as EGF, Noggin, R-Spondin 1, and Gastrino).

[0051] Table 1: Composition of the basal culture medium for intestinal organoids of euryhaline fish

[0052]

[0053] Table 2: Composition of complete culture medium for intestinal organoids of euryhaline fish

[0054]

[0055] The main component of the basal medium in the above-mentioned euryhaline fish intestinal organoid culture medium is Advanced DMEM / F12 medium, which is superior to high-glucose DMEM medium. It avoids high-glucose-induced metabolic stress and ROS accumulation, and is more suitable for fish intestinal metabolism. It is also superior to DMEM / F12 medium because it contains trace elements necessary for stem cells, supporting the self-renewal and antioxidant properties of intestinal crypt stem cells.

[0056] The addition of penicillin and streptomycin antibiotics to the above-mentioned euryhaline fish intestinal organoid culture medium (400 U / mL and 400 μg / mL, respectively) effectively inhibited microbial contamination in the culture system, improving the success rate and culture stability of in vitro fish intestinal organoid construction. Furthermore, Wnt3a was not used in the complete culture medium; CHIR99021 was added as a preferred alternative. As a small molecule compound, CHIR99021 exhibits significantly superior chemical stability and duration of action compared to Wnt3a. CHIR99021 bypasses the Wnt3a-dependent membrane receptor-mediated process, directly targeting intracellular GSK3, thereby providing a more stable and precise Wnt / β-catenin signaling during long-term organoid culture.

[0057] Example 2

[0058] This embodiment follows the following... Figure 1 The procedure for constructing intestinal organoids from euryhaline fishes is shown below. The steps for culturing intestinal organoids from euryhaline fishes are as follows:

[0059] (1) Blood was drawn from 3-month-old golden coin fish, the entire intestine was removed, the mesentery and fat were quickly removed, and the fish was placed in a pre-cooled DPBS solution containing 1% penicillin and streptomycin at 4°C.

[0060] (2) Remove the intestinal segment 1 cm from the stomach end and the hindgut, and longitudinally cut open the anterior and middle guts. Rinse the intestinal segment 3 times with pre-cooled DPBS solution in a sterile culture dish.

[0061] (3) Cut the intestinal tissue transversely into 4 cm segments, transfer them to 15 mL centrifuge tubes, add 5 mL of pre-cooled DPBS, gently pipette 15 times, let stand for 30 s and discard the supernatant, repeat 5-6 times until the supernatant is clear.

[0062] (4) Add 8 mL of 5 mM EDTA digestion solution to the sample, then transfer it to an ice bath shaker and shake continuously at 200 rpm for 15 min.

[0063] (5) Let stand for 30 seconds, aspirate the supernatant, add 5 mL of pre-cooled DPBS, gently blow and aspirate, then discard the supernatant. Repeat twice to completely remove residual digestive fluid.

[0064] (6) Spread the digested intestinal tissue on a sterile culture dish, fix one end with a coverslip, scrape the intestinal villi and crypts with another coverslip, rinse with DPBS and collect into a new centrifuge tube.

[0065] (7) Hold the centrifuge tube and shake it up and down vigorously 2-3 times.

[0066] (8) The above suspension was filtered through a 70-mesh filter and the filtrate was collected.

[0067] (9) Centrifuge the filtrate at 70 × g for 5 min at 4℃.

[0068] (10) Discard the supernatant and resuspend the precipitate in Advanced DMEM / F12 medium, adjusting the crypt density to 10 crypts / μL.

[0069] (11) Mix the crypt suspension with the matrix gel at a volume ratio of 1:3, and the matrix gel concentration shall not be less than 70%.

[0070] (12) Use a pre-cooled pipette tip to draw 50 μL of matrix gel resuspension and drop it into the center of a preheated 24-well plate. Place the plate in an incubator containing 5% CO2 and cure at 28°C for 1 h.

[0071] (13) After the colloid stabilizes, add 600 μL of complete culture medium to each well and continue culturing at 28℃ until budding structures appear, thus obtaining the target organoid.

[0072] Example 3

[0073] This embodiment proposes a method for passage of intestinal organoids from euryhaline fish. When the intestinal organoids of euryhaline fish have grown for 5-6 days, and the edges of the organoids are clearly diffused and budding has stopped, passage can be performed. The steps are as follows:

[0074] (1) Remove the culture medium from the well plate.

[0075] (2) Add 500 μL of cell dissociation solution to each well and gently blow to loosen the colloid and allow the organoid to detach from the matrix gel.

[0076] (3) Digest at room temperature for 5 min, transfer the liquid to a 15 mL centrifuge tube, add an equal volume of Ad DMEM / F12 medium, and centrifuge at 4℃ and 70 g for 3 min.

[0077] (4) Discard the supernatant, resuspend the precipitate in Advanced DMEM / F12 medium, mix it with matrix gel at a volume ratio of 1:1, and the passage density is 1:2~1:3.

[0078] (6) Add 50 μL of matrix gel resuspension to each well of the 24-well plate.

[0079] (7) Place the well plate in an incubator containing 5% CO2 at 28°C for 1 h to solidify. Add 600 μL of complete culture medium to each well and incubate in a cell culture incubator at 28°C for long-term culture.

[0080] (8) After observing the organoid status, it can be placed in a 28℃ cell culture incubator for long-term culture.

[0081] like Figure 2 As shown, the morphology of fish intestinal organoids cultured for different times reveals that the crypts of freshly isolated intestinal organoids are rod-shaped, transforming into sac-like structures within 20 hours and exhibiting a tendency to sprout; at 40 hours, the organoid volume increases and the internal structure thickens significantly, with budding on the outer side; at 70 hours, the number of budding organoids increases. The fish intestinal organoids constructed in the examples show good growth.

[0082] like Figure 3 As shown, a comparison of centrifugation speeds for crypts revealed that at a centrifugation speed of 70 × g, the obtained intestinal organoid crypts exhibited intact morphology and almost no single cells in the background, demonstrating superior crypt separation performance compared to centrifugation speeds of 200 × g.

[0083] like Figure 4 As shown, the morphology of fish intestinal organoids cultured in different basal media revealed that organoids cultured in high-glucose DMEM medium were less likely to survive and had a looser morphology; organoids cultured in Advanced DMEM / F12 medium had a higher survival rate, with more complete and solid morphology, which is more conducive to the in vitro culture of intestinal organoids of euryhaline fish.

[0084] like Figure 5 As shown, the morphology of fish intestinal organoids cultured with different growth factors shows that, compared with Wnt3a, fish intestinal organoids cultured with CHIR99021 survive and can form sac-like structures.

[0085] like Figure 6 As shown, the morphology of fish intestinal organoids cultured with different concentrations of penicillin and streptomycin showed that fish intestinal organoids could be successfully cultured using a medium supplemented with 4× penicillin and streptomycin, while cultured with 1× penicillin and streptomycin could not effectively inhibit microbial contamination and the organoids died.

[0086] like Figure 7 As shown, the morphology of fish intestinal organoids cultured for different times shows that after passage, the organoid fragments increase in size through continuous division, showing a budding trend at 140 h and obvious budding at 160 h.

[0087] In summary, this invention provides a culture system, culture method, and passage method for intestinal organoids of euryhaline fish. First, an optimized Advanced DMEM / F12 medium is used to replace the traditional high-glucose DMEM and DMEM / F12 media, and CHIR99021 is selected as a preferred alternative to Wnt3a, significantly improving the survival rate and stability of the intestinal organoids of the euryhaline fish *Cyprinus fulvidraco*. Second, adding an appropriate concentration of penicillin-streptomycin antibiotics to the organoid basal medium effectively inhibits microbial contamination in the culture system, improving the success rate of in vitro construction of fish intestinal organoids and the stability of the culture system.

[0088] This invention, through extensive experimentation and successful formulation of the components (basal culture medium: Advanced DMEM / F12 medium, GlutaMax additive, Hepes buffer, penicillin-streptomycin antibiotic solution, and supplementary factors), demonstrates that this mixed system successfully maintains the stemness of crypt cells, providing sufficient and comprehensive nutrition for organoid growth and budding, resulting in organoids with a biological structure similar to intestinal tissue. The passage method for intestinal organoids in euryhaline fishes is highly feasible; after 15 consecutive passages, the organoids still retain their proliferative capacity. Based on this, it will promote precision aquaculture and rapidly advance research on feed optimization and disease control in euryhaline fishes.

[0089] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A culture system of a euryhaline fish intestinal organoid, characterized by, The basic medium and the complete medium; The basic medium comprises an Advanced DMEM / F12 medium, a GlutaMax additive, a Hepes buffer and a penicillin-streptomycin double-antibiotic solution. The complete medium is further supplemented with an N-2 additive, a B-27 additive, N-acetyl cysteine, EGF, Noggin, R-Spondin 1, Gastrin, A83-01, Y-27632 and CHIR99021 factors on the basis of the basic medium.

2. The culture system of halophilic fish enteroid according to claim 1, characterized in that, The composition of the basic medium is as follows: 。 3. The culture system of halophilic fish enteroid according to claim 2, characterized in that, The concentrations of penicillin and streptomycin in the penicillin-streptomycin double-antibiotic solution are 400 U / mL and 400 μg / mL respectively.

4. The culture system of halophilic fish enteroid according to claim 1, wherein, The composition of the complete medium is shown in the following table: 。 5. A method of culturing a euryhaline fish intestinal enteroid, characterized by, The method comprises the following steps: S1. Blood of 3-month-old goldfish is extracted, the entire intestinal tract is removed, the mesentery and fat are quickly removed, and the intestinal tract is placed in a pre-cooled DPBS solution containing 1% penicillin-streptomycin double-antibiotic at 4°C; S2. The intestinal tract is cut to remove a 1 cm section near the stomach end and the posterior intestine, and the anterior and middle intestines are longitudinally cut open and washed with a pre-cooled DPBS solution in a sterile culture dish for 3 times; S3. The intestinal tissue is cut into 4 cm small sections transversely, transferred to a 15 mL centrifuge tube, 5 mL of pre-cooled DPBS is added, and the mixture is gently blown for 15 times, and then the supernatant is discarded after standing for 30 s, and the operation is repeated for 5-6 times until the supernatant is clear; S4. 8 mL of 5 mM EDTA digestion solution is added to the sample, and then the sample is transferred to an ice bath shaker and continuously shaken at a speed of 200 rpm for 15 min; S5. After standing for 30 s, the supernatant is discarded, 5 mL of pre-cooled DPBS is added, and the mixture is gently blown and then the supernatant is discarded, and the operation is repeated for 2 times to completely remove the residual digestion solution; S6. The digested intestinal tissue is spread on a sterile culture dish, one cover glass is used to fix one end, and the other cover glass is used to scrape the intestinal villi and crypts, and the mixture is collected in a new centrifuge tube by washing with DPBS; S7. The centrifuge tube is shaken up and down violently for 2-3 times by hand; S8. The above suspension is filtered through a 70-mesh filter screen, and the filtrate is collected; S9. The filtrate is centrifuged at 70 × g for 5 min at 4°C; S10. The supernatant is discarded, and the precipitate is resuspended in an Advanced DMEM / F12 medium, and the crypt density is adjusted to 10 / μL; S11. The crypt suspension is mixed with the matrix glue at a volume ratio of 1:3, and the concentration of the matrix glue is not less than 70%; S12. 50 μL of the matrix glue resuspension is sucked by a pre-cooled gun head, and is dropped into the center of a preheated 24-well plate, and is placed in a culture box containing 5% CO2, and is solidified at 28°C for 1 h; S13. After the glue is stable, the complete medium is added to each well, and the culture is continued at 28°C until the budding structure appears, and the target organoids are obtained.

6. A method of passaging a euryhaline fish gut enteroid, the method comprising, The method comprises the following steps: A1. The medium in the well plate is removed, 500 μL of cell dissociation solution is added to each well, the glue is loosened by gentle blowing, and the organoids are separated from the matrix glue; A2. The digestion is performed at room temperature for 5 min, the liquid is transferred to a 15 mL centrifuge tube, an equal volume of Ad DMEM / F12 medium is added, and the mixture is centrifuged at 4°C and 70 × g for 3 min; A3, discard the supernatant, resuspend the precipitate with Advanced DMEM / F12 medium, mix with Matrigel according to the volume ratio of 1:1, and the passage density is controlled at 1:2~1:3; A4, add 50 μL Matrigel resuspension solution to each well of the 24-well plate, and place it in a 5% CO2 incubator at 28°C for 1 h; A5, add 600 μL complete medium to each well, and place it in a 28°C cell incubator for long-term culture.

Images