A method for culturing a monkey gastric organoid

Abstract

The application belongs to the field of organoid culture, and specifically discloses a culture method of monkey gastric organoids, and discloses a culture medium for culturing monkey gastric organoids, wherein the culture medium comprises (1) an expansion culture medium and (2) a complete culture medium; the application screens key regulation factors and appropriate concentrations for the growth of monkey gastric organoids according to the biological characteristics and growth characteristics of monkey gastric organs, optimizes the culture method, maintains the activity of monkey gastric organoids in vitro for a long time, and can be stably passaged, frozen, recovered, and cultured on a large scale for functional experiments, so as to provide a primate in-vitro model for the research on the mechanism of gastric diseases, toxicity testing, drug research and development, and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of organoid culture, and specifically discloses a culture method of monkey gastric organoids and a culture medium for culturing monkey gastric organoids. BACKGROUND

[0002] As the latest in vitro model, organoids have similar structure and functional characteristics to the original tissue compared with cell lines and traditional primary cells. Organoids have wide application value in tissue organ physiological function, disease mechanism research, drug development, toxicity testing, etc.

[0003] At present, gastric organoid methods of different species such as humans and mice have been established. However, normal human samples are difficult to obtain, and there are great differences between mouse organoids and humans in genetic background and disease models, such as Helicobacter pylori infection and other gastric diseases that are difficult to reproduce in mouse models. Monkeys have a similar genetic background to humans and have greater advantages in simulating human diseases. Therefore, the construction of monkey-derived normal organoids can make up for the problem of limited human samples.

[0004] Due to the difference in genetic background, the composition, ratio, and concentration of each component of the organoid culture medium have a great influence on the success rate and long-term culture of different species of organoids. The organoid culture system of the same tissue source needs to be further optimized in different species. Although the composition of the gastric organoid culture medium of humans, mice, and sheep has been reported, there is no report on the construction method and medium composition of monkey gastric organoids, and the construction process and culture conditions need to be screened and optimized. SUMMARY

[0005] Based on the deficiencies of the prior art, the application discloses a culture method of monkey gastric organoids and a culture medium for culturing monkey gastric organoids.

[0006] The application includes the following technical solutions:

[0007] A culture medium for culturing monkey gastric organoids, the culture medium comprising (1) an expansion culture medium and (2) a complete culture medium;

[0008] The expansion culture medium (1) comprises the following components:

[0009] Advance DMEM / F12, 90-95%; HEPES, 5-10 mM; Glutamax, 1-2X; B27, 1-2X; N-acetyl-L-cysteine, 1-10 mM; Nicotinamide, 2-10 mM; EGF, 20-100 ng / mL; FGF-10, 10-100 ng / mL; FGF-2, 20-100 ng / mL; IGF, 20-200 ng / mL; HGF, 5-50 ng / mL; MSP, 1-10 ng / mL; R-spondin 1, 100-500 ng / mL; Wnt3a, 100-500 ng / mL; Noggin, 20-300 ng / mL; A83-01, 0.1-1 µM; SB202190, 1-10 µM; CHIR99021, 1-10 µM; Gastrin, 1-20 nM; Prostaglandin E2, 100-1000 nM; primocin, 50-200 µg / mL; penicillin-streptomycin, 1%-3%;

[0010] (2) Complete medium: add Y27632, 1-20 µM to the expansion medium to obtain the complete medium.

[0011] Further, the application discloses application of the culture medium in culturing monkey gastric organoids.

[0012] Further, the application discloses a culture method of monkey gastric organoids, which uses the culture medium to culture, the expansion medium is used in a monkey gastric organoid maintenance expansion culture process, and the complete medium is used in a primary separation, subculture and resuscitation process.

[0013] Further, the culture method of the monkey gastric organoids has the advantages that the culture method is simple, the culture medium is simple, and the culture method is suitable for culturing monkey gastric organoids.

[0014] Further, the culture method of the monkey gastric organoids has the advantages that the culture method is simple, the culture medium is simple, and the culture method is suitable for culturing monkey gastric organoids.

[0015] 1) Ex vivo monkey gastric low-temperature transportation and preservation, and ex vivo time control is within 6 hours;

[0016] 2) placing a fresh monkey stomach in a sterile culture dish on ice, cutting the stomach bag, adding tissue preservation solution to wash the stomach inner wall, using sterile scissors and tweezers to cut the stomach body and antrum, removing the outer wall muscle, leaving about one-third of the inner wall tissue, transferring the stomach body and antrum tissue to a new culture dish, adding tissue preservation solution, and cutting into small tissue blocks; collecting and centrifuging the tissue blocks; after removing the supernatant, adding enzyme digestion solution to digest the tissue;

[0017] 3) After enzymatic digestion, add organoid washing solution to terminate; centrifugal collection of cell clusters; after removing the supernatant, add organoid washing solution to resuspend; resuspend the suspension through the cell sieve to collect the enzymatically digested cells; add red blood cell lysate to remove red blood cells; add organoid washing solution to terminate; centrifugal collection of cell clusters, resuspend after adding complete medium and place on ice; cell counting of viable cell clusters; add pre-cooled extracellular matrix glue; inoculate cells and matrix glue into the well plate, and solidify in the incubator;

[0018] 4) After solidification, add complete medium and culture in a conventional cell incubator for a period of time; then replace the expansion medium; change the medium every other day and use the expansion medium for culture.

[0019] Further, the above-mentioned culture method of a monkey stomach organoid comprises the following steps:

[0020] 1) Ex vivo monkey stomach low-temperature transportation and preservation, with an ex vivo time controlled within 6 hours,

[0021] 2) Place the fresh monkey stomach in a sterile culture dish on ice; cut open the stomach sac and add 5 mL of tissue preservation solution to thoroughly wash the stomach inner wall, repeat the washing for 3 times; use sterile scissors and tweezers to cut the stomach body and antrum, remove the outer wall muscle, and leave the inner third of the tissue; transfer the stomach body and antrum tissue to a new culture dish, add 2 mL of tissue preservation solution, cut into about 1 mm 3 tissue blocks; centrifugal collection of tissue blocks at 1000 rpm; after removing the supernatant, add enzymatic solution to digest the tissue, the enzymatic solution comprises mixed collagenase and DNAse I, digest for 30-60 min at 37 degrees, and terminate under a microscope to collect the gland-like structure;

[0022] 3) After enzymatic digestion, add organoid washing solution to terminate, the organoid washing solution comprises DMEM / DF12, 1% bovine serum albumin, and 3% penicillin-streptomycin; centrifugal collection of cell clusters at 1000 rpm; after removing the supernatant, add 10 mL of organoid washing solution to resuspend; resuspend the suspension through a 100-mesh cell sieve to remove the incompletely digested tissue on the sieve, collect the filtered suspension; centrifugal collection of cell clusters at 1000 rpm; remove the supernatant, add 2 mL of red blood cell lysate for 3 minutes at room temperature, remove the red blood cells; add 10 mL of organoid washing solution to terminate; centrifugal collection of cell clusters; resuspend after adding 0.3 mL of complete medium and place on ice; cell counting of viable cell clusters; add pre-cooled extracellular matrix glue according to a 1:1 ratio, the extracellular matrix glue is selected from Matrigel or BME glue; inoculate cells and matrix glue into a low-adhesion 24-well plate, 50 μL of glue droplet per well, the cell cluster content is 200 per glue droplet, and solidify in a 37-degree incubator for 20 min;

[0023] 4) After solidification, add complete medium 0.5 mL, and culture in a conventional cell culture box at 37℃ / 5% carbon dioxide / 95% humidity for 24 hours; after 24 hours, add expansion medium 0.5 mL, and change the liquid every other day, and use 0.5 mL expansion medium for culture.

[0024] Further, the above-mentioned culture method of the monkey gastric organoid, the tissue preservation solution comprises the following components: Advance DMEM / F12, 90%; 3% bovine serum albumin; 3% penicillin; primocin, 100 μg / m; Y27632, 10 μM.

[0025] The application further discloses a kit for culturing a monkey gastric organoid, comprising the culture medium.

[0026] The application further discloses a monkey gastric organoid, which is obtained by the above-mentioned method.

[0027] Compared with the prior art, the application has the following outstanding beneficial effects:

[0028] The application discloses a culture method of a monkey gastric organoid and a culture medium for culturing a monkey gastric organoid. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 , enzymatic separation of monkey gastric tissue to obtain gland cell clusters;

[0030] Figure 2 , monkey gastric organoids cultured for different days;

[0031] Figure 3 , monkey gastric organoids of different generations;

[0032] Figure 4 , comparison of different medium addition methods;

[0033] Figure 5 , screening of medium addition components. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work are within the protection scope of the present application.

[0035] Embodiment 1

[0036] A culture medium for monkey gastric organoid culture, the culture medium comprising (1) expansion medium and (2) complete medium;

[0037] The expansion medium components and concentrations are as follows: Advance DMEM / F12, 90%; HEPES, 10 mM; Glutamax, 1X; B27, 1.5X; N-acetyl-L-cysteine, 2 mM; Nicotinamide, 10 mM; EGF, 50 ng / mL; FGF-10, 50 ng / mL; FGF-2, 20 ng / mL; IGF, 150 ng / mL; HGF, 25 ng / mL; MSP, 5 ng / mL; R-spondin 1, 500 ng / mL; Wnt3a, 100 ng / mL; Noggin, 120 ng / mL; A83-01, 0.5 μM; SB202190, 12 μM; CHIR99021, 3.5 μM; Gastrin, 10 nM; Prostaglandin E2, 500 nM; primocin, 100 μg / m; penicillin, 1%; The expansion medium is mainly used for gastric organ maintenance and expansion culture process;

[0038] The complete medium components are as follows: The complete medium is based on the expansion medium and adds Y27632, 10 μM, which is mainly used for primary separation, subculture and recovery process;

[0039] Material specification:

[0040] Advance DMEM / F12: purchased from Thermofisher

[0041] HEPES: purchased from Sigma

[0042] Glutamax: purchased from Thermofisher

[0043] B27: purchased from Thermofisher

[0044] N-acetyl-L-cysteine: purchased from Sigma

[0045] Nicotinamide: purchased from Sigma

[0046] EGF: purchased from Pepro Tech

[0047] FGF-10: purchased from Pepro Tech

[0048] FGF-2: purchased from Pepro Tech

[0049] IGF: purchased from Pepro Tech

[0050] HGF: purchased from Pepro Tech

[0051] MSP: purchased from R&D

[0052] R-spondin 1: purchased from Pepro Tech

[0053] Wnt3a: purchased from Pepro Tech

[0054] Noggin: purchased from Pepro Tech

[0055] A83-01: purchased from Sigma

[0056] SB202190: purchased from Sigma

[0057] CHIR99021: purchased from Selleck

[0058] Gastrin: purchased from Tocris

[0059] Prostaglandin E2: purchased from Sigma

[0060] Primocin: purchased from invivoGen;

[0061] Y27632: purchased from Selleck

[0062] Cyanomycin: purchased from Biyun Tian.

[0063] Example 2

[0064] Monkey gastric tissue was enzymatically separated to obtain gland cell clusters.

[0065] Ex vivo monkey gastric low-temperature transportation and preservation, the ex vivo time was controlled within 6 hours, the fresh monkey stomach was placed in a sterile culture dish on ice; the stomach sac was cut open, 5 mL of tissue preservation solution was added to wash the stomach wall thoroughly, and the washing was repeated 3 times; using sterile scissors and tweezers, the stomach body and antrum were cut, the outer wall muscle was removed, and the inner third of the tissue was left Figure 1) ; transfer the body and antrum tissue to a new dish, add 2 mL of tissue preservation solution, cut into about 1 mm 3 tissue pieces; centrifuge the tissue pieces at 1000 rpm to collect them; after removing the supernatant, add enzyme solution to digest the tissue, the enzyme solution includes mixed collagenase and DNase I (0.06 mg / mL), digest at 37 degrees for 30-60 minutes, and stop under a microscope when the gland-like structure is collected Figure 1 ) ; after enzyme digestion of the tissue, add organoid washing solution to stop, the organoid washing solution includes DMEM / DF12, 1% bovine serum albumin, and 3% penicillin-streptomycin; centrifuge the cell mass at 1000 rpm to collect it; after removing the supernatant, resuspend it by adding 10 mL of organoid washing solution; pass the resuspension through a 100-mesh cell sieve to remove the tissue that has not been completely digested on the sieve, and collect the filtered suspension; centrifuge the cell mass at 1000 rpm to collect it; remove the supernatant, add 2 mL of red blood cell lysis solution, and remove the red blood cells at room temperature for 3 minutes; add 10 mL of organoid washing solution to stop; centrifuge the cell mass to collect it; resuspend it by adding 0.3 mL of complete medium and place it on ice; count the number of viable cell masses; add pre-cooled extracellular matrix gel according to a 1:1 ratio, and the extracellular matrix gel can be Matrigel or BME gel; inoculate the cells and the matrix gel into a low-adhesion 24-well plate, 50 μL of gel droplet per well, and the cell mass content is 200 cells / gel droplet, 50 μL / gel droplet, solidify for 20 minutes in a 37-degree incubator; after solidification, add 0.5 mL of complete medium, and culture in a conventional cell culture incubator at 37 degrees / 5% carbon dioxide / 95% humidity for 24 hours; replace the organoid expansion medium with 0.5 mL after 24 hours; observe the formation of organoids at different times during the culture process Figure 2 ).

[0066] Example 3

[0067] Primary and subculture culture.

[0068] According to the growth of the organoids, the primary culture is performed for 7-15 days, and the subculture culture is performed after the second generation or more is cultured for 5-7 days. Figure 3 Different generations of monkey gastric organoids.

[0069] Example 4

[0070] Comparison of expansion medium and complete medium culture methods

[0071] Example Medium Formula-1 (Expansion Medium): Advance DMEM / F12, 90%; HEPES, 10 mM; Glutamax, IX; B27, 1.5X; N-acetyl-L-cysteine, 2 mM; Nicotinamide, 10 mM; EGF, 50 ng / mL; FGF-10, 50 ng / mL; FGF-2, 20 ng / mL; IGF, 150 ng / mL; HGF, 25 ng / mL; MSP, 5 ng / mL; R-spondin 1, 500 ng / mL; Wnt3a, 100 ng / mL; Noggin, 120 ng / mL; A83-01, 0.5 mM; SB202190, 12 mM; CHIR99021, 3.5 mM; Gastrin, 10 nM; Prostaglandin E2, 500 nM; primocin, 100 pg / m; penicillin-streptomycin, 1%;

[0072] Example Medium Formula-2 (Complete Medium): Example Medium Formula-1 + 10 mM Y27632

[0073] Example Medium Formula-1 and Example Medium Formula-2 and culture method comparison: In the method established in Example 2 of the present application, the primary cultured organoids were added to Example Medium Formula-2 (Complete Medium) for 1 day, and then Example Medium Formula-1 (Expansion Medium) was used for continuous culture for 5 days. In the comparative example method, Example Medium Formula-2 (Complete Medium containing Y27632) was used for continuous culture for 6 days, and both groups were changed every 2 days. The results showed that the continuous use of complete medium caused apoptosis of the organoids, central necrosis, and a significant decrease in the number of living organoids. This indicates that Y27632 has an inhibitory effect on the long-term culture of organoids Figure 4 A);

[0074] Further comparison, in the method established in Example 2 of the present application, the primary cultured organoids were added to Example Medium Formula-2 (Complete Medium containing Y27632) for 1 day, and then Example Medium Formula-1 (Expansion Medium) was used for continuous culture for 5 days. In the comparative example method, the step of adding Example Medium Formula-2 (Complete Medium containing Y27632) for 1 day was omitted, and Example Medium Formula-1 (Expansion Medium) was used for continuous culture for 6 days, and both groups were changed every 2 days. The results showed that the direct use of Example Medium Formula-1 (Expansion Medium) for primary culture significantly reduced the number of organoids. This indicates that Y27632 inhibits apoptosis and promotes the survival of organoids in the short term Figure 4 B).

[0075] The replacement of the above-mentioned medium and the adding method of Y27632 play a very important role in constructing organoids and long-term culture.

[0076] Example 5

[0077] Example medium and comparative medium screening:

[0078] Example medium formula-1 (expansion medium): Advance DMEM / F12, 90%; HEPES, 10 mM; Glutamax, 1X; B27, 1.5X; N-acetyl-L-cysteine, 2 mM; Nicotinamide, 10 mM; EGF, 50 ng / mL; FGF-10, 50 ng / mL; FGF-2, 20 ng / mL; IGF, 150 ng / mL; HGF, 25 ng / mL; MSP, 5 ng / mL; R-spondin 1, 500 ng / mL; Wnt3a, 100 ng / mL; Noggin, 120 ng / mL; A83-01, 0.5 μM; SB202190, 12 μM; CHIR99021, 3.5 μM; Gastrin, 10 nM; Prostaglandin E2, 500 nM; primocin, 100 μg / m; penicillin, 1%;

[0079] Comparative medium 1: Example medium formula-1 - 20 ng / mL FGF-2;

[0080] Comparative medium 2: Example medium formula-1 - 25 ng / mL HGF;

[0081] Comparative medium 3: Example medium formula-1 - 5 ng / mL MSP;

[0082] Comparative medium 4: Example medium formula-1 - 10 nM Gastrin;

[0083] Comparative medium 5: Example medium formula-1 - 500 nM Prostaglandin E2;

[0084] Embodiment: Refer to the monkey gastric organoid construction method of Example 2, the organoid culture was carried out using the above-mentioned culture medium and comparative examples. The first generation of monkey gastric organoids was digested into single cells or small cell clusters, and the suspension was mixed with Matrigel at a volume ratio of 1:1, then inoculated on a 96-well plate, 10 μL (about 1000 single cells or small cell clusters) per well, and incubated in a conventional cell culture incubator at 37℃ / 5% carbon dioxide / 95% humidity for 20 min to allow the Matrigel to fully solidify, then 100 μL of organoid complete culture medium was added to each well, and the culture medium of Example 1 (expansion culture medium) and comparative examples was replaced after 24 h, and the medium was replaced every 2 days. After 10 days of culture, the number of organoids was counted, and the formation rate was calculated: organoid formation rate = (number of organoids per well / 1000) x 100%.

[0085] Conclusion: Compared with the culture medium of Example (expansion culture medium), the number of organoids formed was significantly reduced when the single components FGF-2, HGF, MSP, Gastrin, and Prostaglandin E2 were removed from the expansion culture medium, and the organoids were generally small. This indicates that the simultaneous addition of these five factors can improve the success rate of organoid culture and maintain long-term culture.

[0086] In summary, the culture medium of the monkey gastric tissue organoid of the present application is optimized for the growth characteristics of cells derived from monkey tissues, and optimizes a variety of cell factors and small molecules, as well as the concentrations of different components. The expansion culture and subculture, recovery culture medium are separated, the addition of Y27632 is controlled, the monkey gastric organoid can be successfully constructed in a short period of time, the success rate is as high as 90%, the cell viability of subculture and recovery is maintained above 80%; and the expansion culture can make the organoid rapidly expand to the required number for research and development, which creates an efficient method for the culture of monkey gastric organoids.

[0087] The above are only a few preferred embodiments of the present application, which are described in more detail and in detail, but should not be construed as limiting the scope of the patent of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application.

Claims

1. A culture medium for monkey gastric organoid culture, characterized by, The culture medium comprises (1) an expansion culture medium and (2) a complete culture medium; The (1) expansion culture medium is composed of the following components: Advance DMEM / F12, 90-95%; HEPES, 5-10 mM; Glutamax, 1-2X; B27, 1-2X; N-acetyl-L-cysteine, 1-10 mM; Nicotinamide, 2-10 mM; EGF, 20-100 ng / mL; FGF-10, 10-100 ng / mL; FGF-2, 20-100 ng / mL; IGF, 20-200 ng / mL; HGF, 5-50 ng / mL; MSP, 1-10 ng / mL; R-spondin 1, 100-500 ng / mL; Wnt3a, 100-500 ng / mL; Noggin, 20-300 ng / mL; A83-01, 0.1-1 μM; SB202190, 1-10 μM; CHIR99021, 1-10 μM; Gastrin, 1-20 nM; Prostaglandin E2, 100-1000 nM; primocin, 50-200 μg / mL; penicillin, 1%-3%. The (2) complete culture medium is obtained by adding Y27632, 1-20 μM to the expansion culture medium.

2. The culture medium of claim 1 is used for culturing monkey gastric organoids.

3. A method for culturing a monkey gastric organoid, characterized by, The expansion culture medium is used for the maintenance and expansion culture of the monkey gastric organoids, and the complete culture medium is used for the primary separation, subculture and resuscitation.

4. The method for culturing a monkey gastric organoid according to claim 3, characterized in that, The cells of the monkey gastric organoids are derived from the body or antrum of the stomach of a monkey.

5. The method for culturing a monkey gastric organoid according to claim 3, characterized in that, The method comprises the following steps: 1) Ex vivo low-temperature transportation and preservation of the monkey stomach, with the ex vivo time controlled within 6 hours; 2) The fresh monkey stomach is placed in a sterile culture dish on ice; the stomach sac is cut open, and tissue preservation solution is added to clean the inner wall of the stomach; sterile scissors and forceps are used to cut the body and antrum of the stomach, remove the outer wall muscle, and leave about one-third of the inner wall tissue; the body and antrum of the stomach are transferred to a new culture dish, tissue preservation solution is added, and the tissue is cut into small pieces; The tissue pieces are collected and centrifuged; After removing the supernatant, enzyme digestion solution is added to digest the tissue; 3) After the tissue is digested, organoid washing solution is added to terminate the reaction; The cell clusters are collected by centrifugation; After removing the supernatant, organoid washing solution is added to resuspend the cell clusters; The resuspended solution is collected by a cell strainer to collect the cells after enzyme digestion; Red blood cell lysis solution is added to remove the red blood cells; organoid washing solution is added to terminate the reaction; the cell clusters are collected by centrifugation, resuspended in complete culture medium, and placed on ice; The number of viable cell clusters is counted; pre-cooled extracellular matrix glue is added; the cells and the matrix glue are inoculated into a well plate and solidified in a culture box; 4) After solidification, complete culture medium is added, and the cells are cultured in a conventional cell culture box for a period of time; The expansion culture medium is replaced after a period of time; the medium is replaced every other day, and the cells are cultured in the expansion culture medium.

6. The method of claim 3, wherein the monkey gastric organoid is cultured in a medium comprising a combination of a Wnt agonist and a Notch agonist. The method comprises the following steps: 1) Ex vivo monkey stomach low temperature transportation and preservation, ex vivo time control within 6h, 2) Place fresh monkey stomach in a sterile petri dish on ice; cut open the stomach sac and wash the interior walls of the stomach thoroughly with 5 mL of tissue preservation solution, repeating the wash 3 times; using sterile scissors and forceps, cut out the body and antrum of the stomach, removing the outer muscle layer, leaving the inner third of the tissue; transfer the body and antrum tissue to a new petri dish, add 2 mL of tissue preservation solution, and cut into approximately 1 mm 3 tissue pieces; centrifuge the tissue pieces to collect; After removing the supernatant, add enzyme solution to digest the tissue, the enzyme solution includes mixed collagenase, DNase I, 37 degrees digestion for 30-60 min, and collect the gland-like structure under a microscope to terminate; 3) After tissue enzymolysis, add organoid washing solution to terminate, the organoid washing solution includes DMEM / DF12, 1% bovine serum albumin, 3% penicillin-streptomycin; centrifugal collection of cell clusters at 1000 rpm; after removing the supernatant, add 10 mL of organoid washing solution to resuspend; the resuspension passes through a 100-mesh cell sieve to remove the tissue that has not been completely digested on the sieve, and the filtered suspension is collected; centrifugal collection of cell clusters at 1000 rpm; remove the supernatant, add 2 mL of red blood cell lysis solution, and remove the red blood cells at room temperature for 3 minutes; add 10 mL of organoid washing solution to terminate; centrifugal collection of cell clusters; add 0.3 mL of complete culture medium to resuspend and place on ice; cell counting of viable cell clusters; add pre-cooled extracellular matrix glue according to a 1:1 ratio, and the extracellular matrix glue is selected from Matrigel or BME glue; inoculate the cells and the matrix glue into a low-adhesion 24-well plate, 50 μL of glue droplet per well, and the cell cluster content is 200 per glue droplet, and solidify for 20 min in a 37°C incubator; 4) After solidification, add 0.5 mL of complete culture medium, and culture in a conventional cell culture incubator at 37°C / 5% carbon dioxide / 95% humidity for 24 h; add 0.5 mL of expansion culture medium after 24 h, and change the medium every other day, and use 0.5 mL of expansion culture medium for culture.

7. The method for culturing a monkey gastric organoid according to claim 6, characterized in that, The tissue preservation solution includes the following components: Advance DMEM / F12, 90%; 3% bovine serum albumin; 3% penicillin-streptomycin; primocin, 100 μg / m; Y27632, 10 μM.

8. A kit for culturing monkey gastric organoids, characterized by, The culture medium as claimed in claim 1.

9. A monkey gastric organoid, characterized in that, Obtained by the method as claimed in any one of claims 3-7.

Images